Vaidlustuse esitamise teade - Riigi Tugiteenuste Keskus

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Saatja: Riina Loorpuu </O=RMV/OU=EXCHANGE ADMINISTRATIVE GROUP (FYDIBOHF23SPDLT)/CN=RECIPIENTS/CN=56B65E4A68034D718AE6CBC5064A1483-RIINA LOORPUU> Saaja: Katharina Kalavus Teema: FW: Täiendavad seisukohad Hei! Annan teada, et vastus on teele läinud. Palun pane see ka Deltasse vaidlustuse juurde 😊 Tervitades Riina From: Riina Loorpuu Sent: Tuesday, March 19, 2024 4:22 PM To: RAM RHVAKO Vaidlustuskomisjon <[email protected]>; '[email protected]' <[email protected]>; '[email protected]' <[email protected]> Subject: Täiendavad seisukohad Tere! Edastan Riigi Tugiteenuste Keskuse vastuse vaidlustaja täiendavatele seisukohtadele vaidlustuse osas riigihankes „Kapillaarsekvenaatorite ja qPCR seadmete kuluvahendid (EKEI)“ (viitenumber 272907). Lugupidamisega Riina Loorpuu Talituse juhataja Mob: +372 569 75666 Telefon: +372 663 8213 www.rtk.ee <http://www.rtk.ee> [email protected] <mailto:[email protected]> <https://www.rtk.ee/sites/default/files/RTK-mailijalus-173x71.jpg> -866140 -147320 0 0 Lp Angelika Timusk Riigihangete vaidlustuskomisjon [email protected] Hankija: Riigi Tugiteenuste Keskus registrikood 70007340 [email protected] Esindaja : Riina Loorpuu Talituse juhataja tel +372 569 75666 e-post: [email protected] Teenuse tellija: Eesti Kohtuekspertiisi Instituut (EKEI) registrikood 70003572 Vaidlustaja: LanLab OÜ registrikood 10590451 Vaidlustaja esindaja: Martin Jürgenson Juhatuse liige e-post [email protected] VASTUS VAIDLUST AJA TÄIENDAVATELE SEISUKOHTADELE Riigihankes „ Kapillaarsekvenaatorite ja qPCR seadmete kuluvahendid (EKEI) “ (viitenumber 272907 ) ASJAOLUD Riigihangete vaidlustuskomisjon (VAKO) edastas hankijale 05 . 03 .202 4 teate esitatud vaidlustusest riigihanke s „ Kapillaarsekvenaatorite ja qPCR seadmete kuluvahendid (EKEI) “ (viitenumber 272907 ) (edaspidi riigihange) vastu võetud otsusele kvalifitseerida Lanmer OÜ (edaspidi kolmas isik) . VAKO palus hankijal hiljemalt 08 . 03 .202 4 esitada VAKO-le oma seisukoht vaidlustuse kohta. Hankija esitas oma vastuväited vaidlustusele 08.03. Vaidlustaja esitas 14.03 täiendavad seisukohad. ÕIGUSLIKUD PÕHJENDUSED Vaidlustaja on oma 14.03 esitatud täiendavates seisukohtades leidnud, et hankija on enda poolt kehtestatud kvalifitseerimistingimust tõlgendanud valesti ning leiab, et kolmas isik ei ole täitnud kehtestatud kvalifitseerimistingimust. H ankija jääb kõikide oma 08.03 esitatud seisukohtade juurde ning esitab järgnevaga täiendavad vastuväited vaidlustaja 14.03 seisukohtadele. Hankija on teostanud hanke käigus põhjaliku kvalifikatsiooni kontrolli ning tuvastanud, et kolmanda isiku esitatud referentsid vastavad hankes nõutule. Hankija ei nõustu vaidlustaja väitega selle kohta, et hankija on tõlgendanud kvalifitseerimistingimust selliselt, et pakkujad võivad olla hanketeate avaldamise kuupäevale eelneva 36 kuu jooksul teostanud vähemalt ühe samalaadse lepingu, mille objektiks on kuluvahendite tarne kohtuekspertiisi valdkonnas kasutatavatele kapillaarsekvenaatoritele või qPCR seadmetele maksumusega vähemalt 50 000 aastas (käibemaksuta)“. Vastupidi, hankija on tuvastanud, et kolmas isik on esitatud referentslepingute alusel tarninud kuluvahendeid nii kapillaarsekvenaatoritele kui qPCR seadmetele. Meelevaldne on vaidlustaja väide, et hankija vastuse kohaselt on kolmas isik müünud vaid järgmist kaupa: „ Thermo Scientific ™ 339650, Nunc ™ 15mL, Conical Sterile Polypropylene Centrifuge Tubes “ n i n g vaidlustaja väidab, et nimetatute puhul ei ole tegemist kuluvahendi tega , mida saab kasutada nii kapillaarsekvenaatoritel kui ka qPCR seadmetel. Hankija juhib tähelepanu, et nimetatud toodete puhul on tegemist hankija vastuses vaidlustusele välja toodud näitlike toodetega. Hankija ei nõustu ka väitega, et nimetatud toodete puhul ei ole tegemist kapillaarsekvenaatorite ja qPCR seadmete kuluvahenditega. T oetudes tootjapoolsetele kasutusjuhendile, on plastiktuubid vajalikud kuluvahendid nii qPCR kui ka kapil l aarelektroforeesi töövoos. Too me välja mõned näited tootja kasutusjuhendist, mis annavad ülevaate, millistes etappides nimetatud plastiktuube kasutakse : 2.8.1. Kapillaarelektroforeesi seadme (AB 3500 ja 3500xl) tootjapoolne kasutusjuhend (lisatud vastusele „ 3500/3500xL Genetic Analyzer user guide ; publication number 100079380, revision E “ ) annab juhiseid seadme regulaarseks hooldamiseks (tuleb puhastada autosampler , drip tray , puhastada masina pinnad kuivanud jääkidest, mustusest, lk 257). Hooldamiseks vajalike reaktiive (vesi, etanool) hoitakse plastiktuubides. Lisaks näeb tootjapoolne kasutusjuhend ette (lk 267), et kui kasutatud kapillaare mõnda aega ei kasutata, siis tuleks neid säilitada vees (selleks, et ennetada polümeeri kuivamist kapliaarides ) ning igakuiselt tuleb pumpa (pump trap ) veega pesta (lk 269). Vett hoitakse laboris plastiktuubides. 2.8.2. Kapillaarelektroforeesi seadme (AB 3500 ja 3500xl) tootjapoolne kasutusjuhend (lisatud vastusele „ 3500/3500xL Genetic Analyzer user guide ; publication number 100079380, revision E “) annab juhiseid vajadusel proovide lahjendamiseks ( lk 292) ning selleks kasutatakse deioniseeritud vett, mida hoitakse plastiktuubides. Proove on vaja lahjendada, kui need sisaldavad näiteks liiga suures koguses DNA-d. Seega, toetudes tootjapoolsele seadme kasutusjuhendile, on plastiktuubid vajalikud kuluvahendid kapilaarelektroforeesi töövoos. 2.8.3. Kapillaarelektroforees on üks osa STR-testsüsteemiga DNA tüpiseerimise metoodikast. See on vajalik analüüsitud DNA visualiseerimiseks. Proovide ettevalmistamiseks on vaja kokku segada reaktsioonisegu, milleks tootja näeb ette, et tuleb kasutada 1,5 ml, 2 ml või mõne muu suurusega sobivaid plastiktuube (tootjapoolne kasutusjuhend lk 131 , lisatud vas t usele „ NGM Detect ™ PCR Amplification Kit user guide ; publication number 100044085, revision D “ ). Kui korraga analüüsida suuremas koguses proove, siis kasutatakse reaktsioonisegu kokkusegamiseks 15 ml tuube. 2.8.4. qPCR seadme kasutamisel näeb tootjapoolne kasutusjuhend ette (lisatud manusesse „ QuantStudio ™ 5 Real-Time PCR Instrument ( for Human Identification ) user guide ; publication number MAN0017162, eevision B.0 “ ), et tausta kontrollimiseks võib kasutada ka ise ettevalmistatud plaati, mille tegemiseks on vaja deioniseeritud vett (lk 50). Vett hoitakse 15 või 50 ml plastiktuubides. Samuti näeb tootjapoolne kasutusjuhend ette, et dekontamineerimiseks tuleb puhastada seadme blokk ning selleks tuleb kasutada vett, etanooli ja naatrium hüpokloriidi lahuseid (lk 61-63) . Lahuseid hoitakse plastiktuubides. Nimetatud lahused on väljatoodud ka tootjapoolses juhised lk 72, kus on loetletud seadme kasutamiseks vajalikud kuluvahendid. Seega, toetudes tootjapoolsele seadme kasutusjuhendile, on plastiktuubid vajalikud kuluvahendid qPCR töövoos. 2.8.5. On tegemist üldlevinud praktika ga , et vee, etanooli ja hüpokloriidi jm lahuste hoidmiseks vajalikke plastiktuube ei tooda tootjapoolses kasutusjuhendis välja, sest see on kasutaja enda otsustada, millise mahuga ja mis firma plastiktuube ta soovib kasutada. Lisaks on üldlevinud praktika, et tootjapoolses kasutusjuhendis tuuakse välja ainult need tooted, mida tootja ise pakub ning ei tooda välja tooteid, mida tuleb osta mujalt. Enamasti tuuakse tootjapoolses kasutusjuhendis välja kriitilisema tähtsusega kuluvahendid, mida tootja tungivalt soovitab tema käest osta ning ei tooda välja vähem kriitilisemaid kuluvahendeid, mida on samuti igapäevaselt vaja (näiteks pipeti otsikud ja plastiktuubid), kuid mille puhul kasutaja ise valib, mis firma tooteid ta soovib kasutada. Juhime tähelepanu, et a ntud hange ei sisaldanud ainult neid kuluvahendeid, mida on vaja otseselt proovide analüüsimiseks konkreetsete seadmetega. Hanke käigus han gitakse ka näiteks plastikut, mida on vaja proovide ettevalmistamiseks (nt lahjendamiseks) või reaktsioonisegu kokku segamiseks (1,5 ja 2 ml reaktsioonituubid; tehnilise kirjelduse punktid 1.22 ja 1.23). Samuti han gitakse kuluvahendeid, mis on vajalikud seadme ettevalmistamiseks proovide analüüsimiseks (näiteks spektraalse kalibreerimise komplektid; tehnilise kirjelduse punktid 1.9, 1.10, 1.19, 1.21) ja seadme töökorras oleku kontrollimiseks (verifikatsiooni plaadid, tehnilise kirjelduse punkt 1.20). Vaidlustaja 13.03 vastuses punkt is 2.7 viidatud kuluvahendid ei sisalda kõiki E KEI kasutuse s oleva kapillaarelektroforeesi instrumendi kuluvahendeid. Antud loetelust on puudu mitmeid kuluvahendeid, näiteks erineva mahuga plastiktuubid, plastikpudelid, plaadid, pipetiotsikud. Hankija sätestatud kvalifitseerimistingimus on teadlikult koostatud selliselt, et tagada konkurents ja mitte piirata pakkujate ringi sellega, et teostatud lepingu raames pakutavad tooted peavad sobituma täpselt EKEI kasutuses olevate seadmetega, vaid eesmärk on kehtestatud tingimusega veenduda, et pakkuja on suuteline analoogseid lepinguid analoogses mahus täitma. EKEI- le hanke tulemusel sõlmitud lepingu alusel tarnitavate toodete sobivus on tagatud pakutavate toodete kontrollimisega vastavustingimuste all. Kuna hankija on kolmanda isiku kvalifitseerimistingimuse täitmiseks esitatud referentside osas teostanud sisulise kontrolli ja veendunud, et kolmanda isiku poolt esitatud leping sisaldab nii kapillaarsekvenaatorite kui ka qPCR seadmete ga töötamisel kasutata vate kuluvahend ite tarnimist , siis puudub hankijal igasugune alus pakkuja kvalifitseerimata jätmiseks. HANKIJA TAOTLUSED: Jätta vaidlustaja esitatud vaidlustus rahuldamata. Jätta kõik menetluskulud vaidlustaja kanda. Lugupidamisega (allkirjastatud digitaalselt) Riina Loorpuu Talituse juhataja KÄSKKIRI 30.03.2023 nr 1-2/23/22 Riigi Tugiteenuste Keskuse riigihangete korraldamise ja hankelepingute sõlmimise korra kinnitamine Riigihalduse ministri 14.11.2017. a määruse nr 84 „Riigi Tugiteenuste Keskuse põhimäärus“ § 20 lg 1 p 7 alusel: 1. kinnitan Riigi Tugiteenuste Keskuse riigihangete korraldamise ja lepingute sõlmimise korra. 2. tunnistan kehtetuks Riigi Tugiteenuste Keskuse peadirektori 21.01.2022. a käskkirja nr 1-2 / 006 „Riigi Tugiteenuste Keskuse riigihangete korraldamise ja lepingute sõlmimise korra kinnitamine“. 3. Käskkiri jõustub selle allkirjastamise hetkest. (allkirjastatud digitaalselt) Pärt-Eo Rannap peadirektor Teatavaks teha: RTK töötajad Koostaja: Helina Kudu Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 1 / 12 Riigihangete korraldamise teenus (K14) Protsess: Riigihangete korraldamine Protsessi omanik: RHO juhataja Versioon: 9 Kinnitatud: kuupäev digiallkirjas Riigihangete korraldamise ja hankelepingute sõlmimise kord sisaldab: 1. ÜLDPÕHIMÕTTED .................................................................................................................. 1 2. MÕISTED JA LÜHENDID ........................................................................................................ 2 3. HANKEPLAANI KOOSTAMINE ............................................................................................ 3 4. RIIGIHANGETE ETTEVALMISTAMINE JA KORRALDAMINE RHO POOLT ................ 5 5. VÄIKEOSTUDE LÄBIVIIMINE .............................................................................................. 8 6. RAAM- VÕI HANKELEPINGU SÕLMIMINE ....................................................................... 9 7. RAKENDUSSÄTTED ............................................................................................................. 11 1. ÜLDPÕHIMÕTTED 1.1. Riigi Tugiteenuste Keskuse (edaspidi RTK) riigihangete korraldamise ja hankelepingute sõlmimise korras (edaspidi kord) kehtestatakse ühtsed reeglid RTK ja RTKga riigihangete tugiteenuse osutamise kokkuleppe (edaspidi kokkulepe) sõlminud asutuste (edaspidi asutus) riigihangete planeerimisel, korraldamisel ning raam- või hankelepingute sõlmimisel. 1.2. Korras toodud nõuetest kinnipidamise eest vastutavad kõik RTK töötajad ja korrast lähtuvate asutuste töötajad ning RTKga kokkuleppe sõlminud asutuste töötajad kokkuleppes sätestatud ulatuses. 1.3. RTK korraldab riigihankeid ning sõlmib raam- või hankelepinguid RTK ning RTKga kokkuleppe sõlminud asutuste vajadustest lähtuvalt. 1.4. RTK osutab asutusele riigihangete korraldamise tugiteenust kokkuleppes fikseeritud tingimustel ja ulatuses ning RTK töötajatele vastavalt korras sätestatule. RTKs vastutab riigihangete korraldamise ja tugiteenuse osutamise eest riigihangete osakond (edaspidi RHO). Riigihangete seaduses sätestatud hankija õigusi ja kohustusi täidavad RTK ja asutus ühiselt, võttes aluseks kokkuleppes määratud pädevuste jaotuse. 1.5. RTK ei korralda riigihankeid, mille korraldamiseks on Vabariigi Valitsus määranud kohustusliku keskse hankija. 1.6. Asutusel on võimalik lähtuda korrast ulatuses, milles asutus ei ole kehtestanud eraldi hankekorda. Kokkuleppes võivad RTK ja asutus täpsustada hankekorra kohaldamise tingimusi. 1 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 2 / 12 1.7. Asjade ostmisel, teenuste ja ehitustööde tellimisel ning muude riigihangete seaduse kohaselt riigihankeks kvalifitseeruvate menetluste puhul lähtutakse riigihangete seadusest, muudest õigusaktidest ja korrast. RHO tagab riigihangete õiguspärase korraldamise ja riigihangete tugiteenuse korrektse ning kvaliteetse osutamise. 1.8. Riigihankega seotud teabevahetus ja pakkumuste või taotluste esitamine ning menetlemine toimub elektrooniliselt, välja arvatud riigihangete seaduses sätestatud juhtudel. 1.9. Riigihangete planeerimisel ja korraldamisel arvestatakse võimalusel sotsiaalsete kaalutluste, innovatsiooni rakendamise ja keskkonnasäästlike lahendustega. 1.10. Asutustele ja RTK töötajatele riigihangete valdkonna nõustamisteenuse osutamisel annab RHO nõu kõigis riigihankeid ja RTK poolt läbi viidud riigihangete tulemusel sõlmitud lepinguid puudutavates küsimustes. 1.11. Korruptsioonivastasest seadusest tulenevad toimingupiirangud kohalduvad:  lähteülesande koostamise ja esitamise eest vastutavale isikule;  RHO töötajale;  raam- või hankelepingu sõlmimist korraldavale isikule;  raam- või hankelepingu täitmise eest vastutavale isikule;  väikeostu korraldavale isikule;  väikeostu lepingu täitmise eest vastutavale isikule. 1.12. Toimingupiirangu kohaldamise kord ja tingimused RTKs sätestatakse RTK töökorralduse reeglites. Asutuse töötaja toimingupiirangust kinnipidamise, teavitamise ja taandamise eest vastutab asutus vastavalt asutuse sätestatud korrale. 1.13. Korra lisad Korra lisad (vormid ja juhised) on töödokumendid, mida ei kinnitata korra koosseisus käskkirjaga ja mille muutmise, täiendamise, täpsustamise õigus on protsessi omanikul. Vormi või juhise muutmisel tagab protsessi omanik, et muudetud vormid ja juhised oleksid kättesaadavad ja avaldatud sise- ja/või välisveebis ning muutusest on vormide ja juhiste kasutajaid teavitatud.  K14 L1 – väikeostu dokumendi vorm;  K14 L2 – lähteülesande vorm;  K14 L3 – hankeplaani sisendi vorm;  K14 L4 – raam- või hankelepingu lõppemise teate vorm;  K14 L5 – hindamiskomisjoni liikme huvide konflikti puudumise kinnituse vorm. 2. MÕISTED JA LÜHENDID  Dokumendihaldussüsteem (DHS) – RTK-s kasutatav elektrooniline dokumendihaldussüsteem, mida kasutatakse dokumentide loomiseks, haldamiseks, menetlemiseks ja hoidmiseks.  Keskne riigihange – RTK kui keskse hankija ettevalmistatav ja läbiviidav riigihange, mis hõlmab ühe või mitme asutuse hankevajadusi ning mille tulemusena sõlmib rahalist kohustust mittesisaldava raamlepingu RTK või rahalist kohustust sisaldava raamlepingu või hankelepingu asutus. 2 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 3 / 12  Kulujuht – RTK peadirektori või asutuse juhi poolt määratud töötaja, kes vastutab kulutuste tegemisel konkreetse kuluüksuse eelarve piisavuse eest.  Lähteülesanne – sisendi andja poolt RHOle lähteülesande vormil esitatav sisend riigihanke korraldamiseks.  Otsustusõiguslik isik – isik, kellel on RTKs või asutuses õigus otsustada rahaliste vahendite kasutamise üle.  Riigihangete register – töökeskkond hankijatele riigihangete korraldamiseks ja haldamiseks ning pakkujatele riigihangetel osalemiseks https://riigihanked.riik.ee/rhr- web/#/.  Riigihangete tugiteenus – riigihangete korraldamise teenus, sh nõustamine ning riigihanke ettevalmistamine, korraldamine ja toimingute tegemine asutuse nimel.  RHO – Riigi Tugiteenuste Keskuse riigihangete osakond.  Sisendi andja – RTK töötaja või RTKga kokkuleppe sõlminud asutus.  Töötaja – RTK või asutusega avalik-õiguslikus teenistus- ja usaldussuhtes olev ametnik või eraõiguslikus töösuhtes olev töölepinguline töötaja.  Vastutav isik – RHO töötaja, kes vastutab riigihanke nõuetekohase ja eesmärgipärase korraldamise eest.  Väikeost – asjad, teenused ja ehitustööd, mis hangitakse korra alusel ja mille käibemaksuta maksumus on väiksem kui korra punktis 3.3.3 nimetatud summad.  Väikeostu dokument – väikeostu eesmärgi, tulemuste, planeeritud eelarve ja finantseerimisallikate kirjeldus.  Ühishange – RTK läbiviidav riigihange, mis hõlmab rohkem kui ühe asutuse hankevajadusi ja kus üheks sisendi andjaks on RTK.  Üleriigiline keskne riigihange – keskne riigihange või ühishange, millega võivad hankijatena liituda nii kokkuleppe sõlminud kui ka kokkulepet mittesõlminud asutused. 3. HANKEPLAANI KOOSTAMINE 3.1. Tegevuses osalejad ja vastutajad on RHO töötajad, RTK juhtkond ja RTK korrast lähtuvate asutuste töötajad. 3.2. Sisendi kogumine hankeplaani koostamiseks 3.2.1 RHO esitab igal aastal hiljemalt detsembris hankeplaani koostamiseks vormikohase (K14 L3) sisendi kogumise päringu RTK juhtkonna liikmetele ning RTKga kokkuleppe sõlminud asutustele järgmise aasta hankevajaduste kohta. 3.2.2. Esialgse hankeplaani sisendi küsimiseks järgmise aasta I kvartaliks esitab RHO päringu igal aastal hiljemalt septembris. 3.2.3. Sisendi andja esitab RHOle vormikohase hankeplaani sisendi, päringus märgitud tähtajaks. 3.2.4. Hankeplaani sisend peab kajastama kõiki sisendi andja planeeritavast eelarvest või muudest alustest tulenevaid vajadusi riigihangeteks, mille eeldatav maksumus käibemaksuta on vähemalt 30 000 eurot (sh summeritavad hanked) või mis riigihangete seaduse või 3 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 4 / 12 riigihanke rahastaja finantseerimistingimuste kohaselt nõuavad toiminguid riigihangete registris. Juhul, kui sisendi andja soovib RHOlt taotleda madalama eeldatava maksumusega riigihanke korraldamist, esitab sisendi andja taotletava riigihanke kohta nõuetekohased andmed hankeplaani sisendis. 3.2.5 Riigihanke eeldatava maksumuse määramisel tuleb sisendi andjal lisaks riigihangete seaduses sätestatule arvestada järgmisi põhimõtteid:  riigihankeid ei tohi kunstlikult osadeks jagada eesmärgiga vältida riigihangete seadusest tulenevaid kohustuslikke menetlusreegleid;  kui riigihanke objekti hooldust ja tarvikuid on võimalik hankida vaid toote pakkujalt, arvestatakse riigihanke eeldatava maksumuse hulka ka hoolduse ja tarvikute eeldatav kulu;  asja ostmisega kaasnev väljaõppekulu arvestatakse riigihanke eeldatava maksumuse sisse. 3.3. Hankeplaani koostamise põhimõtted 3.3.1. Hankeplaanis fikseeritakse RTK ja RTKga kokkuleppe sõlminud asutuste riigihangete vajadustest lähtuvalt ühte kalendriaastasse planeeritud RHO poolt korraldatavad riigihanked. 3.3.2. Hankeplaani koostab RHO ja hankeplaani kantakse riigihanked, mille korraldamisel on riigihangete seaduse kohaselt nõutud toimingute tegemine riigihangete registris. RHO võib hankeplaani lisada ka need riigihanked, mille korraldamisel ei ole nõutud riigihangete seaduse kohaselt toimingute tegemine riigihangete registris, kuid mida RTK või asutus soovib läbi viia riigihangete registris lähtuvalt hankelepingu esemest või rahastaja tingimustest või muu põhjendatud vajaduse tõttu. 3.3.3. Riigihangete seaduse kohaselt on riigihangete registris kohustus teha toiminguid alljärgnevate piirmääradega riigihangete osas:  asjade ostmine ja teenuste tellimine eeldatava käibemaksuta maksumusega alates 30 000 eurost;  eriteenuste tellimine, teenuste kontsessioonilepingu sõlmimine, sh eriteenuste kontsessioonilepingu sõlmimine ja ideekonkursi korraldamine eeldatava käibemaksuta maksumusega alates 60 000 eurost;  ehitustööde tellimine eeldatava käibemaksuta maksumusega alates 60 000 eurost;  ehitustööde kontsessioonilepingu sõlmimine, sotsiaalteenuste tellimine, sh sotsiaalteenuste kontsessioonilepingute sõlmimine eeldatava käibemaksuta maksumusega alates 300 000 eurost. 3.3.4. Hankeplaanis kajastatakse iga planeeritava riigihanke kohta vähemalt:  sisendi andja lähteülesande esitamise eest vastutav isik;  riigihanke nimetus ja objekti lühikirjeldus;  lähteülesande RHOle esitamise tähtpäev;  hanke- või raamlepingu kestvus ja eeldatav sõlmimise tähtpäev;  hanke- või raamlepingu eeldatav maksumus; 4 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 5 / 12  riigihanke korraldamine üleriigilise keskse riigihankena, mitme asutusega keskse riigihankena või ühishankena. 3.3.5. Mitme asutusega keskse riigihanke või ühishanke korraldamise otsustab kokkuleppes fikseeritud erisusi arvestades õiguslikel ja otstarbekuse kaalutlustel RHO koostöös asutustega. 3.3.6. Üleriigiliste kesksete riigihangete korraldamise otsustab RTK eelkõige ressursi saadavusest lähtuvalt. Vajadusel võib RTK määrata üleriigilise keskse riigihankega ühinemise kokkuleppe tingimused, sh esinduse ulatuse, kulude hüvitamise või tasu maksmise, avaldades need RTK veebilehel. 3.4. Hankeplaani kinnitamine ja avalikustamine 3.4.1. Enne hankeplaani kinnitamist kooskõlastab RHO kokkuleppe tingimustest tulenevalt hankeplaani kõigi sisendi andjatega vähemalt sisendi andja poolt antud sisendi ulatuses. 3.4.2. RTK peadirektor kinnitab jooksva aasta hankeplaani hiljemalt kümne tööpäeva jooksul pärast hankeplaani kooskõlastamist sisendi andjate poolt. RHO korraldab hankeplaani avaldamise RTK välisveebis ning teavitab sisendi andjaid hankeplaani kinnitamisest. 3.4.4. Asutused, kellele RTK osutab kokkuleppe kohaselt riigihangete tugiteenust ning kelle riigihankevajadused on hankeplaanis kajastatud, eraldiseisvat hankeplaani kinnitama ei pea ja asutuse hankeplaaniks loetakse RTK koostatud ja kinnitatud hankeplaan. 3.5. Hankeplaani muutmine ja hankeplaanivälise riigihanke korraldamine 3.5.1. Hankeplaan kinnitatakse üks kord aastas ja hankeplaanivälise riigihanke korraldamine või hankeplaanikohase riigihanke hankevajaduse muutumine ei too kaasa hankeplaani uut kinnitamist. 3.5.2. Hankeplaanis kinnitatud riigihanke korraldamiseks hankeplaanis sätestatust erineval ajal või riigihanke vajaduse muul viisil muutumisest teavitab sisendi andja RHOd esimesel võimalusel. 3.6. Hankeplaani täitmise aruandlus 3.6.1. RHO esitab RTKle ja asutustele hankeplaani täitmise aruande üks kord kvartalis eelmise kvartali kohta kokkuleppes määratud aja jooksul pärast kvartali lõppu ning kokkuleppe alusel hankeplaani täitmise analüüsi kord aastas enne uue hankeplaani kinnitamist. 4. RIIGIHANGETE ETTEVALMISTAMINE JA KORRALDAMINE RHO POOLT 4.1. Tegevuses osalejad ja vastutus 4.1.1. Tegevuses osalejad ja vastutajad on RTK töötajad ja RTK korrast lähtuvate asutuste töötajad ning RTKga kokkuleppe sõlminud asutuste töötajad kokkuleppes sätestatud ulatuses. 5 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 6 / 12 4.1.2. RHO vastutab riigihanke korraldamisel riigihangete seaduse ja teiste õigusaktide korrektse järgimise ning kokkuleppe tingimuste täitmise eest. RHO korraldatavate riigihangete puhul suhtleb menetluse käigus riigihankel osalemisest huvitatud ettevõtjate, taotlejate ja pakkujatega RHO. Sisendi andja menetluse käigus riigihankel osalemisest huvitatud ettevõtjate, taotlejate ja pakkujatega otse ei suhtle ning riigihanke menetluse kohta kolmandatele isikutele informatsiooni ei avalda, välja arvatud RHOga eelnevalt kooskõlastatud juhtudel. 4.1.3. Hankelepingu eseme tehnilise kirjelduse kvaliteedi tõstmiseks või keskmisele turuhinnale vastava hinnataseme määramiseks viib turu-uuringu läbi sisendi andja, RHOl on nõustav roll, sh annab RHO vajadusel nõu kuidas turu-uuringut läbi viia. Info läbi viidud turu-uuringu kohta esitatakse RHOle lähteülesandes. 4.2. Lähteülesande koostamine 4.2.1. RHO töötaja edastab riigihanke ettevalmistamiseks hankeplaanis märgitud lähteülesande esitamise eest vastutavale isikule meeldetuletuse lähteülesande esitamise kohta üks kuu enne lähteülesande esitamise tähtpäeva. Meeldetuletust ei edastata juhul, kui lähteülesande valmistab ette RHO. 4.2.2. Lähteülesande esitamise eest vastutav isik edastab korraldatava riigihanke vormikohase lähteülesande (K14 L2) RHOle aadressile [email protected] hankeplaanis sätestatud kuu esimeseks tööpäevaks. Lähteülesande esitamise eest vastutav isik vastutab lähteülesandes sisalduva informatsiooni õigsuse, sh riigihanke eeldatava maksumuse määramise ning olemasolevate rahaliste vahendite piisavuse eest lähteülesandekohase teenuse tellimise või asja ostmise jaoks. 4.2.3. Hankeplaanis mittekajastatud riigihanke korraldamiseks esitab sisendi andja põhjendatud taotluse RHOle tuues välja korra punktis 3.3.4 kirjeldatud andmed ja hankeplaani lisamata jätmise põhjendused. Täiendava riigihanke korraldamise otsustamine toimub kokkuleppes sätestatud tingimustel RHO ja asutuse koostöös. RTKs tuleb täiendava riigihanke korraldamise vajadus kooskõlastada peadirektoriga enne RHOle lähteülesande esitamist. 4.3. Riigihanke alusdokumentide koostamine ja kooskõlastamine 4.3.1. Igale korraldatavale riigihankele määrab RHO juhataja või tema poolt määratud isik vastutava isiku. Vastutav isik peab kooskõlastama riigihanke alusdokumendid, alusdokumentide muudatused ja vastuvõetavad otsused osakonna juhataja või tema poolt määratud isikuga. Vastutav isik võib kooskõlastada riigihanke menetluse käigus taotlejale või pakkujale esitatavad selgitused ning küsimused osakonna juhataja või tema poolt määratud isikuga. 4.3.2. Sisendi andjaga kooskõlastatakse riigihanke alusdokumendid ja nende muudatused, juhul kui muudatus on sisuline (nt muudetakse tehnilist kirjeldust) ja muudatuse tõttu peab pikendama pakkumuste esitamise tähtaega, ning vastuvõetavad otsused. 6 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 7 / 12 4.3.3. Riigihanke alustamise eelduseks on sisendi andja otsustusõigusliku isiku poolt riigihanke alusdokumentide kooskõlastamine kokkuleppes sätestatud ulatuses. RTKs kooskõlastab riigihanke alusdokumendid kulujuht, kes kinnitab kooskõlastuse andmisega rahaliste vahendite olemasolu ja riigihanke tehnilise kirjelduse sobivust. Kooskõlastus edastatakse vastutavale isikule kirjalikku taasesitamist võimaldavas vormis. 4.3.4. Keskse hanke puhul koostab riigihanke alusdokumendid (sh tehnilise kirjelduse) RHO. RTKga hanketeenuse osutamiseks teenusleppe sõlminud asutused edastavad otsustusõigusliku isiku kinnituse hankes osalemise soovi ja hankelepingu alusel tasu maksmiseks vajalike rahaliste vahendite olemasolu kohta. Asutused, kes ei ole hanketeenuse osutamiseks teenuslepet sõlminud, annavad RHOle riigihanke läbiviimiseks volituse. Volituse andmisega kooskõlastavad nad riigihanke alusdokumendid. 4.4. Riigihanke korraldamine/läbiviimine 4.4.1. Vastutava isiku ülesanded riigihanke korraldamisel on muuhulgas:  lähteülesande täpsustamine koostöös lähteülesande esitamise eest vastutava isikuga (sh vajadusel eeldatava ajakava esitamine ja võimalikuks kohtumiseks ettepaneku tegemine);  keskse hanke puhul tehnilise kirjelduse koostamine ja hanke jaoks vajaliku sisendi kogumine;  hankemenetluse liigi valimine;  riigihanke alusdokumentide koostamine, sh raamlepingu ja hankelepingu projektide koostamine koostöös lähteülesande esitamise eest vastutava isikuga;  nõuetekohane kooskõlastamine;  riigihanke läbiviimine;  pakkujatega läbirääkimiste korraldamine ja läbirääkimistel osalemine;  selgituste andmine riigihankel osalemisest huvitatud ettevõtjatele, taotlejatele ja pakkujatele vajadusel koostöös lähteülesande esitamise eest vastutava isikuga;  vajadusel riigihanke otsuste kohta seisukoha küsimine lähteülesande esitamise eest vastutavalt isikult;  riigihanke otsuste tegemine;  taotlejate, pakkujate ja lähteülesande esitamise eest vastutava isiku teavitamine tehtud otsustest;  ametijuhendist või asutusega sõlmitud kokkuleppest tulenevate muude kohustuste täitmine. 4.5. Pakkumuste hindamine 4.5.1. Vajadusel moodustab sisendi andja pakkumuste hindamiseks hindamiskomisjoni, kuhu kuuluvad sisendi andja määratud liikmed, kes omavad riigihanke objektile vastavas valdkonnas eriteadmisi. RTKs või RTK korrast lähtuvas asutuses moodustab hindamiskomisjoni ja määrab selle liikmed riigihanke sisendi andmise eest vastutav struktuuriüksuse juht või tema poolt määratud isik. Hindamiskomisjoni moodustamine ja liikmete määramine toimub kirjalikku taasesitamist võimaldavas vormis. 7 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 8 / 12 4.5.2. Sisendi andja otsustab hindamiskomisjoni liikmete arvu ning tagab liikmete sõltumatuse ja komisjoni töös osalemise. RTKs ja RTK korrast lähtuvas asutuses moodustatud hindamiskomisjonis peab olema vähemalt kaks liiget ning hindamiskomisjoni töös osalemine on hindamiskomisjoni liikmetele kohustuslik. 4.5.3. Hindamiskomisjoni liikmete ülesandeks on pakkumuste hindamine, lähtudes riigihanke alusdokumentides sätestatud hindamiskriteeriumidest. 4.5.4. Hindamiskomisjoni liikmete poolt pakkumustele antud hindamispunktid koos põhjendustega esitatakse vastutavale isikule hindamiskomisjoni protokollis, mis on digitaalselt allkirjastatud hindamiskomisjoni kõigi hindamisel osalenud liikmete poolt. Hindamiskomisjoni iga liige esitab vastutavale isikule digitaalselt allkirjastatud vormikohase (K14 L5) kinnituse huvide konflikti puudumise kohta. 4.6. Riigihanke vaidlustamine 4.6.1. Riigihangete seaduse kohase vaidlustuse esitamisel esindab RTKd, kui riigihanke korraldajat, ja asutust riigihangete vaidlustuskomisjonis ning kohtus, RHO töötaja. Vajaduse korral kaasatakse vaidlustusmenetlusse lähteülesande esitamise eest vastutav isik või sisendi andja poolt määratud isik. Otsuse vaidlemise otstarbekuse ja edasiste tegevuste osas teeb RHO koostöös sisendi andjaga. 5. VÄIKEOSTUDE LÄBIVIIMINE 5.1. Tegevuses osalejad ja vastutajad on RTK töötajad ja RTK korrast lähtuvate asutuste töötajad. 5.2. Väikeostu vajadus kooskõlastatakse enne väikeostu korraldamist kulujuhiga. 5.3. Väikeostude läbiviimisel arvestatakse võimalusel keskkonnahoidlike lahendustega. 5.4. Väikeostude, mille eeldatav käibemaksuta maksumus on alla 5000 euro, läbiviimisel tagatakse rahaliste vahendite säästlik ja otstarbekas kasutamine. Kirjalikult võrreldavaid pakkumusi ei pea võtma, kui riigihanke eset arvestades ei oleks see proportsionaalne või otstarbekas. Võrreldavaid pakkumusi ei pea võtma ka juhul, kui mittevõtmise lubatavus tuleneb rahastaja tingimustest. Põhjendus pakkumuse valiku kohta lisatakse arvete keskkonda ning sellega loetakse riigihangete seaduse kohane riigihangete korraldamise üldpõhimõtete järgimise kohustus täidetuks. 5.5. Väikeostude, mille eeldatav käibemaksuta maksumus on alates 5000 eurost, läbiviimisel lähtutakse järgmistest nõuetest:  võrreldakse soovitud asja või teenuse kohta avalikult kättesaadavat infot (sh maksumust) või koostatakse ostetava asja või tellitava teenuse tehniline kirjeldus, et tagada võrreldavate pakkumuste saamine ja määratakse eduka pakkumuse valimise kriteeriumid;  pakkumust küsitakse võimalusel vähemalt kolmelt pakkujalt kirjalikku taasesitamist võimaldavas vormis või võrreldakse võimalusel vähemalt kolme pakkuja avalikult kättesaadavat infot. Pakkujale saadetud ja pakkujalt saadud ning menetluse käigus koostatud dokumendid tuleb registreerida dokumendihaldussüsteemis (edaspidi DHS), välja arvatud juhul, kui väikeost viiakse läbi riigihangete registris; 8 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 9 / 12  koostatakse vormikohane (K14 L1) väikeostu dokument, mis sisaldab eduka pakkumuse valimise kriteeriume või põhjendust võrreldavate pakkumuste mittevõtmise kohta. Eraldi vormikohast väikeostu dokumenti ei pea koostama, kui eelpool kirjeldatud informatsioon on dokumenteeritud mõnes muus dokumendis. Väikeostu dokumendile lisatakse asja või teenuse kirjeldus ja laekunud pakkumused ning edastatakse DHSi kaudu kulujuhile kooskõlastamiseks. 6. RAAM- VÕI HANKELEPINGU SÕLMIMINE 6.1. Tegevuses osalejad ja vastutajad on RHO töötajad, RTK juhtkond ja RTK korrast lähtuvate asutuste töötajad ning RTKga kokkuleppe sõlminud asutuste töötajad. 6.2. Tegevuse põhimõtted ja tähtaeg 6.2.1. Raam- või hankelepingu sõlmimiseks edastab vastutav isik sisendi andjale raam- või hankelepingu projekti, riigihanke läbiviimise kohta koostatud memo ning vajadusel edukaks tunnistatud pakkumuse, kui kokkuleppes ei ole sätestatud teisiti. 6.2.2. Riigihanke tingimuste kohase raam- või hankelepingu sõlmimise korraldab lähteülesande esitamise eest vastutav isik või sisendi andja poolt määratud sisendi andjaga seotud muu isik. 6.2.3. Riigihanke tulemusena edukaks tunnistatud pakkumuse esitanud pakkujaga sõlmib raam- või hankelepingu sõltuvalt sisendi andjast kas RTK või asutus. 6.2.4. RTKs kirjaliku raam- või hankelepingu sõlmimisel kooskõlastab sisendi andja lepingu DHSis kulujuhiga ja juhul, kui hanke on läbi viinud RHO, siis ka RHO juhataja või tema poolt määratud isikuga. Väikeostu puhul lisatakse lepingu juurde väikeostu dokument. 6.2.5. RTKs saadab sisendi andja kirjaliku raamlepingu ja hankelepingu allkirjastamiseks RTK peadirektorile või peadirektori vastutusvaldkonda kuuluva hankelepingu peadirektori asetäitjale. 6.2.6. RHO poolt korraldatud riigihanke tulemusel raam- või hankelepingu sõlmimisel, esitab lepingu sõlmimist korraldav isik vastutavale isikule sõlmitud lepingu kümne tööpäeva jooksul lepingu sõlmimisest arvates. Vastutav isik esitab lepingu andmete alusel riigihangete registrile hankelepingu sõlmimise teate. 6.2.7. Raam- või hankelepingu täitmise eest vastutavaks isikuks RTKs on lepingus määratud töötaja. Asutuses määratakse lepingu täitmise eest vastutav isik raam- või hankelepingus või vastavalt asutuse töökorraldusele. 6.3. Raam- või hankelepingu täitmise eest vastutava isiku ülesanded:  vastutab raam- või hankelepingu täitmise, õiguskaitsevahendite kohaldamise ning lõpetamise õiguspärasuse eest;  kooskõlastab raam- või hankelepingu muutmise vajaduse tekkimisel lepingu muutmise RHOga enne muudatuse vormistamist. Kui vastavalt riigihangete seadusele on muudatuste kohta vaja esitada registrile raam- või hankelepingu muutmise teade, esitab täitmise eest vastutav isik viie tööpäeva jooksul muudatuse tegemisest arvates RHOle asjakohase teabe. 9 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 10 / 12 RHO esitab teabe alusel riigihangete registrile teate raam- või hankelepingu muutmise kohta;  esitab raamlepingu korral RHOle andmed raamlepingu alusel kaheteistkümne kuu jooksul sõlmitud hankelepingute sõlmimise aja ja maksumuse kohta kümne päeva jooksul pärast iga kaheteistkümne kuu möödumist raamlepingu sõlmimisest arvates. RHO esitab teate alusel riigihangete registrile teabe raamlepingu alusel sõlmitud hankelepingute kohta;  esitab RHOle vormikohase (K14 L4) teate RHO poolt korraldatud riigihanke tulemusel raam- või hankelepingu lõppemise kohta kümne tööpäeva jooksul raam- või hankelepingu lõppemisest arvates. RHO esitab teate alusel riigihangete registrile teabe raam- või hankelepingu lõppemise kohta. 6.4. Lepingu, mida soovitakse sõlmida riigihangete seaduses sätestatud erandit kohaldades ja menetlusreegleid järgimata ning mille eeldatav maksumus käibemaksuta on alates 20 000 eurost, sõlmimise kavatsuse võib lepingu sõlmimist korraldav isik enne tehingu ettevalmistamist RHOga kooskõlastada. RTKs peab lepingu, mida soovitakse riigihangete seaduses sätestatud erandit kohaldades ja menetlusreegleid järgimata sõlmida ja mille eeldatav maksumus käibemaksuta on alates 20 000 eurost, sõlmimise kavatsuse kooskõlastama RHOga. RHO ei kooskõlasta töölepingu ja kinnisasja omandamiseks, üürimiseks või rentimiseks sõlmitava lepingu sõlmimise kavatsust. 6.5. Üleriigilise keskse riigihanke, keskse riigihanke või ühishanke tulemusena sõlmitava rahalist kohustust mittesisaldava raamlepingu sõlmimise õigus RTK ja asutuste nimel on RTKl ning raamlepingu täitmise eest vastutavaks isikuks on RHO juhataja või RHO juhataja poolt määratud RHO töötaja. 6.6. Üleriigilises keskses riigihankes, keskses riigihankes või ühishankes osalenud asutused edastavad iga kaheteistkümne kuu möödudes raamlepingu sõlmimisest ja raamlepingu lõppedes RHOle info raamlepingu alusel sõlmitud hankelepingute sõlmimise aja ja maksumuse kohta, kui selline info ei ole RHOle kättesaadav riigihangete registrist või vastavalt riigihanke alusdokumentidele edastatav pakkuja(te) poolt. 6.7. RTK vastutab üleriigilise keskse riigihanke, keskse riigihanke ja ühishanke korraldamise ning rahalist kohustust mittesisaldava raamlepingu sõlmimise, täitmise ning lõpetamise õiguspärasuse eest, riigihankes osalenud asutused vastutavad raamlepingu alusel hankelepingute sõlmimise, täitmise ning lõpetamise õiguspärasuse eest. 6.8. Lepingud, mille eeldatav käibemaksuta maksumus on alates 20 000 eurost, sõlmitakse kirjalikus või digitaalselt allkirjastatud vormis (edaspidi kirjalik vorm). 6.9. Olenemata lepingu eeldatavast maksumusest peavad kirjalikus vormis olema:  füüsiliste isikutega sõlmitavad lepingud;  autoriõigusi kajastavad lepingud;  rahalist kohustust mittesisaldavad koostöölepingud;  lepingud, mille puhul tuleb kirjaliku vormi nõue õigusaktidest. 6.10. Lepingu täitmise eest vastutavaks isikuks on lepingus määratud töötaja. 10 Protsessi omanik: RHO juhataja Tähis: K14 Riigi Tugiteenuste Keskus Kinnitamise kuupäev: digiallkirjas Versioon nr: 9 Avaldatud: RTK sise- ja välisveebis Lk. 11 / 12 6.11. Lepingu täitmise eest vastutav isik on kohustatud kontrollima lepingu täitmist, vajadusel kohaldama või tegema ettepaneku õiguskaitsevahendite kohaldamiseks ning väljastama lepingu täitmise kohta tõendeid või muid hinnanguid. 7. RAKENDUSSÄTTED 7.1. RHO korraldab asutuste riigihankeid kokkuleppes sätestatud tähtpäevast arvates. 7.2. RHO korraldab RTKga kalendriaasta jooksul liituvate uute asutuste riigihankeid vastavalt asutuste kinnitatud hankeplaanidele ning nende hangete osas RTK peadirektori kinnitatud hankeplaani ei täiendata. 7.3. Enne riigihangete korraldamise tugiteenuse osutamise alguse tähtpäeva asutuse poolt alustatud riigihanked viiakse RHO poolt lõpuni kokkuleppes fikseeritud tingimuste kohaselt. 7.4. Korra loomise ja ajakohasena hoidmise ning korrast lähtuvate asutuste teavitamise eest hankekorda tehtud muudatustest vastutab RHO juhataja või tema poolt määratud isik. 11 3500/3500xL Genetic Analyzer USER GUIDE 3500 Series Data Collection Software v3.3 Windows™ 10 Operating System Catalog Numbers 4405186, 4405186R, 4405187, 4405187R Publication Number 100079380 Revision E For Research Use Only. Not for use in diagnostic procedures. Life Technologies Holdings Pte Ltd | Legal manufacturer of these products: Block 33 | Marsiling Industrial Estate Road 3 | • Applied Biosystems™ 3500 Genetic Analyzer #07-06, Singapore 739256 • Applied Biosystems™ 3500xL Genetic Analyzer Life Technologies Corporation | Legal manufacturer of these products: 6055 Sunol Blvd | Pleasanton, California 94566 USA • Applied Biosystems™ 3500 Genetic Analyzer (refurbished) • Applied Biosystems™ 3500xL Genetic Analyzer (refurbished) Revision history: 100079380 E (English) Revision Date Description E 1 December 2023 • Refurbished instruments were added to the list of supported instruments (4405186R and 4405187R). • Connect cloud-based platform was updated to Thermo Fisher™ Connect Platform. • The instructions to install the Cathode Buffer Container (CBC) septa were updated for clarification. • The environmental design standards were updated to include Commission Delegated Directive 2015/863. D 3 November 2021 • The IMPORTANT text instructing the user to log in to the computer with the same Windows™ Administrator account was added in “Start the computer and instrument” on page 33 and “Restart the instrument and the computer” on page 279. • The following figures were updated: pump image in “Check for leaks and spills” on page 38; capillary to plate mapping image in “Capillary-to-plate mapping” on page 67. • The 384-well plate mapping image figure was updated to indicate the plates are for use supported on 24-capillary instruments. • The CRL definition was updated to the latest approved wording in “Review sequence quality” on page 104, “Pass/fail criteria for the sequencing install check” on page 150, and “Basecalling protocol—QV settings” on page 205. • Dye set Z was added to “Condition Number” on page 136. • The information for AnyDye was updated by request from Tech Support, “Create a new dye set using the AnyDye template” on page 195. • The RFID read/write unit model and manufacturer was updated in “Specifications” on page 327 • The safety compliance standards for 60825 were updated to 2014 in “Safety (compliance)” on page 338. • Radio compliance information was added in “Radio compliance” on page 340. C 4 May 2020 • Product information: Add new status for blinking amber front panel indicator. Add network configuration and security information. Update formamide storage conditions. Update information for antivirus software. • Spectral calibration: Add information to spectral calibration and the grayed out Cancel button. Add example spectral calibration result for dye set E • Install check fragment only: Change from POP-6™ to POP-7™ Polymer. Update capillary failure information to indicate that you can deselect 1 failed capillary for 8‑capillary instruments or 2 failed capillaries for 24‑capillary instruments. • Instrument specifications: Add note about using computers that have been validated by Thermo Fisher Scientific. • Change CutePDF to PDF printer. • Troubleshooting: Add camera error to Error message and Run troubleshooting. • View and print an install check report. Add note: The Date Performed field reflects the date that the install check was accepted, not the date it was run. • Restart instrument and computer: Update the list of reasons to use the procedure, add information about using only one Windows™ administrator account to log in to Windows™ on the instrument computer. • Environmental requirements: Add Electromagnetic interference to update information for CISPR 11 Class B TO A. Update electrical ratings. Add warnings. • Run modules: Update fragment analysis long run modules. Update FragmentAnalysis50_POP7: 40 to 45; Update LongFragAnalysi s50_POP7 125 to 130; To run times for these modules, add fn to runtime - Throughput is an estimate extrapolated from average run time. • RFID appendix: Update distances to 10 cm. Revision Date Description C 4 May 2020 • RFID Specifications: Table 12 Minimum separation distance of the instrument from external RFID read/write (contin units: Change 3 feet to 10 cm; Add row: "Minimum separation distance of the instrument from other lab ued) equipment (e.g. centrifuge, thermal cycler)" - "1 m" • Safety: Update safety labels on the instrument. • Safety compliance: Update EU Directive standard. • EMC Compliance: Update standards. • Connect cloud-based platform: – For non-UI elements, changed name from 'Thermo Fisher Connect'. – Updated website from thermofisher.com/cloud to thermofisher.com/connect. B 29 January 2019 Fix minor errors. In Chapter 3, change "Disconnect individual users" to "Unlink individual users" and update information. A 30 October 2018 New document for v3.3 features. • Preferences for reagent to run past on-instrument time and expiry. • Signal optimization in Spatial Calibration. • Size Standard Normalization Factor and Avg Normalization PH displayed in results. • Export Consumables log. • EPT plots available for terminated runs if plate is still linked. Flexible plate loading – pause a run, load a plate, then run new plate. • Export injection list • Connect cloud-based platform function. • Remove license manager. The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Dell and OptiPlex are trademarks of Dell Inc. Microsoft, Windows, and Word are trademarks of Microsoft Corporation. ©2018-2023 Thermo Fisher Scientific Inc. All rights reserved. Contents ■ CHAPTER 1 Instrument and software description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Instrument and software description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Precautions for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Instrument interior components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Instrument parts and functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Instrument front panel indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Instrument and computer requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Windows™ software requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Antivirus software requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Other software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Instrument firmware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Theory of operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Preparing samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Preparing the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 During a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Materials for routine operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Instrument consumables handling, usage limits, and expiration . . . . . . . . . . . . . . . . . . . . . . 22 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Conditioning reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Capillary arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Hi‑Di™ Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Important notice regarding use of consumables that exceed supported limits . . . . . 25 Overview of the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 About the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Dashboard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Maintenance workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Library workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Thermo Fisher Connect menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 SAE menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Tools menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Manage menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Preferences menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Contents Help menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Use the software without an instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 ■ CHAPTER 2 Start the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Start the computer and instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Start the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Step one: Start the Server Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Step two: Start the 3500 Series Data Collection Software v3.3 . . . . . . . . . . . . . . . . . . . 34 Step three: Log in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Check system status in the Dashboard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Check calendar reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Check consumables status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 How the polymer sample and injection counters calculate usage . . . . . . . . . . . . . . . . 38 Check for leaks and spills . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Check buffer fill levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Replenish consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Ensure proper installation of CBC septa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Set preferences (optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 System preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 User preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 ■ CHAPTER 3 Use the instrument with the Thermo Fisher™ Connect Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Thermo Fisher™ Connect Platform features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Register and obtain a Thermo Fisher™ Connect Platform account . . . . . . . . . . . . . . . . . . . . 47 Connect the instrument to your Thermo Fisher™ Connect Platform account . . . . . . . . . . . 47 Set up the data storage location and email notifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 View your upload history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Monitor a run from InstrumentConnect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Monitor a run from a mobile device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 View notifications from the instrument on your Thermo Fisher™ Connect Platform account . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 For more information on using InstrumentConnect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Thermo Fisher™ Connect Platform administrators for an instrument . . . . . . . . . . . . . . . . . . 53 First user who links is assigned administrator role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Instrument administrator functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Manage the users and administrators of your instrument . . . . . . . . . . . . . . . . . . . . . . . . 54 Unlink individual users from an instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 5 Contents ■ CHAPTER 4 Set up and run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Setup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Prepare the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Create or import a plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 About plate templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Create a plate from a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Import a plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Assign plate contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Access the Assign Plate Contents screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Name samples and assign sample types in the plate view . . . . . . . . . . . . . . . . . . . . . . 63 Assign assay, file name convention, and results group in the plate view . . . . . . . . . . 65 How file location in file name conventions and results groups work . . . . . . . . . . . . . . 66 Print the plate layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Prepare and assemble sample plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Capillary-to-plate mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Allelic ladder run requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Results group for one allelic ladder per run folder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Prepare sample plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Prepare the plate assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Load the plate in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Link the plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Quick Start a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Load plates for run and create the injection list . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Link a plate (if a plate is not linked) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Link a plate from the Recent Plates or Recent Runs tab . . . . . . . . . . . . . . . . . . . . . . . . 77 Review, modify, or export the injection list in Preview Run . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Start the run from Preview Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Export the injection list from Preview Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Monitor the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Pause a run and load a new plate (flexible plate loading) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Check sequence or sample quality and re-inject samples . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Check sequence or sample quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Re‑inject samples from the Monitor Run screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 If you select a protocol other than the original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 How re-injections are displayed in the plate view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Re-inject HID allelic ladder samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Review completed injections in Review Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Pause, resume, or stop a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Pause and resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Abort or terminate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 6 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Contents More features in Assign Plate Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Name samples in the plate view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Customize the plate view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 View the capillary-to-plate map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Use the table view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Sort by one or multiple columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Customize a table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Add assays, file name conventions, and results groups to a plate . . . . . . . . . . . . . . . . 95 Create a plate import template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Create a plate import file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Edit a plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Import and export a plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Create a plate template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Specify the default plate type for the Open Plate dialog box . . . . . . . . . . . . . . . . . . . . 98 Save electronic version of reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 More features in Monitor Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Review the Instrument Run views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Array view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Sample view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 EPT view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ■ CHAPTER 5 Review sequencing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Access the View Sequencing Results screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Review results for the currently running sequencing plate . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Review previously run sequencing samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Review sequence quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Review traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Display thumbnails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Understand Quality Values (QVs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Quality value ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Pure base versus mixed base QVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Quality values (QV) and probability of error (Pe) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Re‑inject samples from Review sequencing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 View, print, and save (PDF) trace quality reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 View Trace Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Report options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Export sequencing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Modify sequence data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 7 Contents ■ CHAPTER 6 Review fragment/HID analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Access the View Fragment/HID Results screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Export the injection list from Samples view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Review results for the currently running fragment/HID analysis plate . . . . . . . . . . . . . . . . . 114 Review previously run fragment analysis/HID samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Review sample quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Review normalized data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 How normalization is applied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Normalization factor in secondary analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Review plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Zoom on data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Change plot settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Overlay samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Label peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 View thumbnails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Rename samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Sort by one or multiple columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Review sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Set up the sizing table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Examine the size standard plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Overlay the sizing curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Re-inject samples from Review fragment results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 View, print, and save (PDF) sample quality reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Report options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Export sizing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Modify fragment analysis or HID data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 ■ CHAPTER 7 Run calibrations and install checks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Section 7.1 Run spatial and spectral calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Run a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Spatial calibration overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 When to perform a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Perform a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Evaluate the spatial calibration profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Example spatial profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Export spatial calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 8 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Contents View and print a calibration report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Save historical reports (PDF) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Run a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Spectral calibration overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 When to perform a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Estimated run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Prepare for spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Perform a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Spectral Quality Values and Condition Numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Evaluate the spectral calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 What you see during a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Capillary information sharing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Example spectral calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Export spectral calibration results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 View and print a calibration report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Save historical reports (PDF) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 View the spectral calibration history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Section 7.2 Run an install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Run a Sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 When to perform a sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Estimated run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Prepare for the sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Perform a sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 What you see during a sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Pass/fail criteria for the optional spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Pass/fail criteria for the sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 Evaluate sequencing install standard data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Example sequencing install check results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 View previously run install standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 View and print an install check report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Save historical reports (PDF) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Run a fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 When to perform a fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Estimated run time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Prepare for the fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Perform the fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 What you see during a fragment install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Pass/fail criteria for the fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Evaluate fragment install standard data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Example fragment install standard results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Example HID install standard results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 View previously run install standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 View and print an install check report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Save historical reports (PDF) for record keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 9 Contents ■ CHAPTER 8 Manage library resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Overview of libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Factory-provided template and locked items . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 General library procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Access libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Create a new entry from a factory-provided template or locked entry . . . . . . . . . . . . 164 Delete a library entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 Edit a library entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Import and export a library entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 View audit and e‑signature histories for library entries . . . . . . . . . . . . . . . . . . . . . . . . . 165 Sort and search library entries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Customize a table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Plates library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Plate overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Create a new plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Define plate properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Assays library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Assay overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Create a new assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Assay settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 File Name Conventions library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 File name convention overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Create a new file name convention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 File name convention settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Results Group library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Results Group overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Allelic ladder location (HID analysis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Create a new Results Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Results group settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 Results group example 1: store files by plate name . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Results Group example 2: store re-injections in separate folders . . . . . . . . . . . . . . . . 185 Results Group example 3: store one allelic ladder per run folder (8‑capillary instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 Instrument protocol library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Instrument protocol overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Create a new instrument protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Instrument protocol settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Dye sets library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 Dye set overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 Create a new dye set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Dye set settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Create a new dye set using the AnyDye template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 10 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Contents Size standards library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Size standard overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Normalization size standards provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Create a new size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Modify a factory-provided normalization size standard . . . . . . . . . . . . . . . . . . . . . . . . 200 Basecalling protocols library (primary analysis—sequencing) . . . . . . . . . . . . . . . . . . . . . . . 201 Basecalling protocol overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Create a new basecalling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Basecalling protocol—Analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Basecalling protocol—QV settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Sizecalling protocols library (primary analysis—fragment) . . . . . . . . . . . . . . . . . . . . . . . . . . 206 Sizecalling protocol overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 Create a new sizecalling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 Sizecalling protocol—Analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 Sizecalling protocol—QC settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 QC protocols library (primary analysis—HID) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 QC protocol overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Create a new QC protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 QC protocol—Analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 QC protocol—QC settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 ■ CHAPTER 9 Use Security, Audit, and E-Sig functions (SAE Module) . . . . . . . 218 Administrators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Administrators overview of system security auditing and electronic signature . . . . . 218 Configure the security system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Manage user accounts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Manage auditing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Generate audit reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Manage electronic signature (E-sig) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 Export and import user accounts security audit and electronic signature settings 244 Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Users overview of System Security Audit Trail and E-Signature . . . . . . . . . . . . . . . . . 246 Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Audit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Electronic signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 ■ CHAPTER 10 Maintain the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Maintenance schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Review calendar reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Daily instrument maintenance tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 Weekly instrument maintenance tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Monthly instrument maintenance tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Quarterly maintenance tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 11 Contents Annual planned maintenance tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 As-Needed instrument maintenance tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 Use the maintenance calendar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Create calendar entries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 View the Planned Maintenance Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Review the Notifications Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Export the consumables log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Clean the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Install buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Prepare the Anode Buffer Container (ABC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 Prepare the Cathode Buffer Container (CBC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Replenish, change, flush, and store polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 Precautions for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Replenish polymer or change polymer type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Store partially used polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 Fill capillary array with fresh polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Change and store a capillary array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Install or change the capillary array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 Store a capillary array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Maintain the pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Avoiding damage to the pump assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Remove bubbles from the polymer pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Wash the pump chamber and channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Flush the water trap (pump trap) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Shutdown move and reactivate the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Shutdown the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Move and level the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 Reactivate the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Maintain the computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Back up the datastore during software uninstall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Archive, purge, and restore data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 Monitor disk space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 Service Log and Usage Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 ■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 Restart the instrument and the computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Instrument components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Instrument troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 RFID troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 Error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Dashboard troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Software troubleshooting — general . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Run, re-run, or re-inject troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 12 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Contents Data/electropherogram troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 Review Results troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Link/load plate troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 Assign Plate Contents troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 Spatial calibration troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 Spectral calibration troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Sequencing install standard troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Fragment/HID install standard troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Monitor Run troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 Audit troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Electronic signature troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Manual commands troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 Troubleshooting procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 Run Troubleshooting Utility.bat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 View the log files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 View instrument sensor details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Review error message details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 Reset the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 ■ APPENDIX B Run modules and dye sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Run modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Dye sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Sequencing analysis dye sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Fragment analysis dye sets for all applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 HID analysis dye sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 ■ APPENDIX C Instrument specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Instrument specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Environmental requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316 Power and communication connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 ■ APPENDIX D Catalog numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Plates bases retainers and septa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Instrument consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Sequencing analysis reagents and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Fragment and HID analysis reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 13 Contents ■ APPENDIX E Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 Instrument and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Calibration and install checks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Security Audit and E-sig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 ■ APPENDIX F Radio Frequency Identification (RFID) technology . . . . . . . . . . . . 325 Precautions for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Locations of RFID read/write units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 ■ APPENDIX G Signal optimization and size standard normalization . . . . . . . . . 329 Signal optimization feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Spatial calibration-dependent signal optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Run module-dependent signal optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 Size standard normalization feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Overview of the normalization feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 When to use the normalization feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 ■ APPENDIX H Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 Conformity symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Safety alerts on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 Location of safety labels on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Physical injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 Electrical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 Cleaning and decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . 338 Safety (compliance) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 EMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 Environmental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 Radio compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 14 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Contents Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 ■ APPENDIX I Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 15 1 Instrument and software description ■ Instrument and software description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 ■ Instrument and computer requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Theory of operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ■ Materials for routine operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 ■ Instrument consumables handling, usage limits, and expiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 ■ Overview of the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Instrument and software description Overview The Applied Biosystems™ 3500 Genetic Analyzer and 3500xL Genetic Analyzer are fluorescence-based DNA analysis instruments using capillary electrophoresis technology with 8 or 24 capillaries. The 8-capillary model (Cat. No. 4405186 and Cat. No. 4405186R) and the 24-capillary model (Cat. No. 4405187 and Cat. No. 4405187R) include the following components: • 8-capillary or 24-capillary array and POP™ polymer • Reagents and consumables for your application and for system qualification • Computer workstation and monitor • Integrated software for instrument control, data collection, quality control, basecalling, and sizecalling of samples 16 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Instrument and software description 1 IMPORTANT! The protection provided by the equipment may be impaired if the instrument is operated outside the environment and use specifications, the user provides inadequate maintenance, or the equipment is used in a manner not specified by the manufacturer (Thermo Fisher Scientific). IMPORTANT! Observe current good laboratory practices when using this instrument. Precautions for use WARNING! Radio frequency identification (RFID) could possibly disrupt the operation of patient- worn and/or implanted active medical devices. To minimize such effects, do not come within 10 cm of this instrument if you have a patient-worn and/or implanted active medical device. Instrument interior components 1 6 7 2 8 3 4 9 5 Figure 1 Instrument interior 1 Detection cell heater block 2 Polymer delivery pump (PDP) 3 Anode buffer container (ABC) 4 Polymer or conditioning pouch 5 Cathode buffer container (CBC) 6 Oven door 7 Capillary array 8 Oven condensation reservoir 9 Autosampler 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 17 Chapter 1 Instrument and software description 1 Instrument and software description Instrument parts and functions Part Function Anode buffer container Contains 1X running buffer to support all electrophoresis applications on the (ABC) instrument. Has a built-in overflow chamber to maintain constant fluid height. Autosampler Holds the sample plates and cathode buffer container (CBC) and moves to align the plates and CBC with the capillaries. Capillary array Enables the separation of the fluorescent-labeled DNA fragments by electrophoresis. It is a replaceable unit composed of 8 or 24 capillaries. Cathode buffer container Contains 1X running buffer to support all electrophoresis applications on the (CBC) instrument. Detection cell heater Holds the detection cell in place for laser detection and maintains the detection cell block temperature of 50°C. Oven/oven door Maintains uniform capillary array temperature. Oven condensation Collects condensation from the oven. reservoir Polymer delivery pump Pumps polymer into the array and allows for automated maintenance procedures. (PDP) Includes the displacement pump chamber, polymer chambers, piston water seal, capillary array port, check valve fitting, water trap waste container, buffer valve, anode electrode, buffer gasket, and holds the anode buffer container. Polymer pouch or • Polymer pouch—Supplies polymer to the polymer delivery pump. conditioning reagent • Conditioning reagent pouch—Used for priming the polymer pump, washing the pouch polymer pump between polymer type changes, and during instrument shut down. Has adequate volume for a one-time use. Radio frequency RFID tags on the following primary instrument consumable labels are detected by identification (RFID) (for read/write units in the instrument interior: more information, see • Capillary array Appendix F, “Radio • Cathode buffer container (CBC) Frequency Identification (RFID) technology”). • POP™ polymer • Anode buffer container (ABC) The instrument reads and tracks the following information: • Lot numbers • Serial numbers • Dates (expiration) • Capacity (usage) RFID tags are read and written in response to a user action (for example, running a wizard or starting a run). All dashboard values are updated when RFID tags are read and written. The days on Instrument is also updated automatically every 6 minutes. 18 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Instrument and computer requirements 1 Instrument front panel indicators Indicator Status All lights off Instrument off Green light Idle Green light (blinking) Run is in progress Note: You can only abort an injection when the green light is flashing, not when it is solid green. Amber light (blinking) Power-up self-test is in progress Instrument has paused. If the door is open, close it. If the amber light is still blinking, restart the instrument and computer (see “Restart the instrument and the computer” on page 279). Communication issue between instrument and software. Restart the instrument and computer (see “Restart the instrument and the computer” on page 279). Amber light Standby Red light Self-test failed or instrument failure. Restart the instrument and computer (see “Restart the instrument and the computer” on page 279). Instrument and computer requirements IMPORTANT! Do not modify the instrument hardware or software without notifying Thermo Fisher Scientific. Any modifications must be made by Thermo Fisher Scientific under change control. For minimum computer requirements, see “Instrument specifications” on page 315. Windows™ software requirements 3500 Series Data Collection Software v3.3 runs on the Windows™ 10, 64-bit operating system (IOT Enterprise). The computer provided with the instrument contains validated software and settings. Do not update the Windows™ operating system or firewall settings. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 19 Chapter 1 Instrument and software description 1 Theory of operation Antivirus software requirements The computer provided with the instrument does not include antivirus software because customer preferences and network requirements vary. The 3500 Series Data Collection Software v3.3 has been tested with these antivirus software applications: • Symantec Endpoint Protection 12 • McAfee Endpoint Security version 10.5 IMPORTANT! McAfee Endpoint Security can block services that are needed to start the Data Collection software. If you observe this issue, disable the firewall from McAfee Endpoint Security Settings or create a rule to allow traffic for the IP address 192.168.0.1 on the local network. Other software CAUTION! Do not install additional software on the computer other than antivirus software. Changes to the configured software could void the instrument warranty and cause the instrument software to be non-operational. IMPORTANT! Do not rename the computer after the software is installed. The instrument computer has been assigned a unique name. Changing the name may cause the software to malfunction. Instrument firmware Instrument firmware is to be updated only by a Thermo Fisher Scientific representative. Theory of operation Preparing samples When DNA samples are prepared for sequencing and fragment analysis on the instrument, fluorescent dyes are attached to the DNA. Preparing the instrument Two calibrations are required to prepare the instrument for sample runs: • Spatial calibration—Determines the position of the image from each capillary on the CCD array. For more information, refer to page 125. • Spectral calibration—Generates a matrix for each capillary that compensates for dye overlap and is used to convert the 20-bin data into 4-, 5-, or 6-dye data. For more information, refer to “Perform a spectral calibration” on page 133. 20 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Materials for routine operation 1 During a run During a run, the instrument: • Prepares the capillaries by pumping fresh polymer solution under high pressure from the polymer delivery pump to the waste position in the cathode buffer container (CBC). • Electrokinetically injects the sample into the capillaries by briefly applying a low voltage. • Washes the capillary tips in the rinse position of the CBC, then returns the capillary to the buffer position of the CBC. • Ramps the voltage up to a constant level. A high electric field is created between the ground end of the anode buffer container (ABC) and the negative voltage applied to the load header of the capillary array. This field pulls the negatively charged DNA through the separation polymer. The smaller fragments migrate faster than the larger fragments and reach the detector first. To ensure optimal separation and maintain denaturation of the DNA, the capillaries are thermally controlled in the oven and in the detection cell. The oven has a Peltier heat unit and fan-circulated air. In the detection cell, the dyes attached to DNA are excited by a narrow beam of laser light. The laser light is directed into the plane of the capillaries from both the bottom and top. A small amount of laser light is absorbed by the dyes and emitted as longer wavelength light in all directions. • Captures the fluorescent light on the instrument optics while blocking the laser light. The light passes through a transmission grating, which spreads the light out. The light is imaged onto a cooled CCD array. For each capillary, 20 zones on the CCD are collected to provide 20-bin data for each capillary. • Converts the 20-bin data into multi-dye data for the entire run. For sequencing applications, 4 different dyes are used to determine the 4 bases A, G, C and T. For fragment analysis applications, up to 6 dyes can be used in a single run for higher throughput. Results The software generates an electropherogram (intensity plot) for each dye based on the migration of DNA fragments over the run and generates primary analysis results: • For sequencing applications, the electropherogram is adjusted to compensate for slight mobility differences due to the dyes, then basecalling is performed and quality values are assigned. • For fragment and Human Identification (HID) analysis, the software uses the internal size standard to assign a fragment size and a sizing quality value to each peak. Materials for routine operation All materials for routine operation are provided when the instrument is installed. For more information: • See Appendix D, “Catalog numbers” • Contact your Thermo Fisher Scientific representative 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 21 Chapter 1 Instrument and software description 1 Instrument consumables handling, usage limits, and expiration Instrument consumables handling, usage limits, and expiration IMPORTANT! Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see Appendix I, “Documentation and support”. Containers and pouches are ready-to-use. Labels include a radio frequency identification (RFID) tag that the instrument uses to track usage and expiration date. Buffers Cat. No. Description Storage 4393927 Anode Buffer Container (ABC) 2–8°C 1X running buffer, 4 containers The 1X running buffer has been qualified to ship at ambient conditions. 4408256 Cathode Buffer Container (CBC) See the expiration date on the label. Do not use expired product. 1X running buffer, 4 containers On-instrument supported Instrument limits Guidelines Lower of: 8-capillary 14 days, 240 injections, or The buffer has been verified for use for up to 14 days on the instrument. expiry date The software displays a warning message when a usage limit is met and 24-capillary 14 days, 100 injections, or allows you to continue running. Before doing so, see “Important notice expiry date regarding use of consumables that exceed supported limits” on page 25. Polymer Cat. No. Description Storage A26070 POP-4™ Polymer (96‑sample)[1] 2–8°C 4393715 POP-4™ Polymer (384‑sample)[1] 4393710 POP-4™ Polymer (960‑sample)[1] A26071 POP-6™ Polymer (96-sample) 4393717 POP-6™ Polymer (384-sample) 4393712 POP-6™ Polymer (960-sample) A26073 POP-7™ Polymer (96‑sample) 22 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Instrument consumables handling, usage limits, and expiration 1 (continued) Cat. No. Description Storage 4393708 POP-7™ (384) Performance Optimized Polymer 2–8°C 4393714 POP-7™ (960) Performance Optimized Polymer [1] The polymer has been validated for HID applications. IMPORTANT! For the POP-4™ Polymer and POP-7™ Polymer (Cat. Nos. A26070, 4393715, 4393710, A26073, 4393708, and 4393714), the on‑instrument supported limit is 14 days only when the instrument operating temperature is 15–25°C. When the instrument operating temperature is > 25°C, the supported limit is 7 days. For the POP-6™ Polymer (Cat. Nos. A26071, 4393717, and 4393712), the on‑instrument supported limit is 14 days when the instrument operating temperature is 15–30°C. On-instrument Pouch size Instrument supported limits[1] Guidelines Lower of: 96 8-capillary 14 days, 96 samples, The polymer has been verified for use for up to 14 days on the samples 12 injections, or expiry instrument. date The software displays a warning message when a usage limit 24-capillary 14 days, 96 samples, is met and allows you to continue running. Before doing so, 5 injections, or expiry see “Important notice regarding use of consumables that exceed date supported limits” on page 25. 384 8-capillary 14 days, 384 samples, samples 60 injections, or expiry date 24-capillary 14 days, 384 samples, 20 injections, or expiry date 960 8-capillary 14 days, 960 samples, samples 120 injections, or expiry date 24-capillary 14 days, 960 samples, 50 injections, or expiry date [1] The pouch has adequate polymer to support the stated number of samples or injections, plus additional volume to accommodate installation and wizard operations. Multiple pouch installations and/or excessive use of wizards reduce the number of remaining samples and injections. For example, if you run the total bubble remove option in the Remove Bubbles wizard more than four times, the number of remaining samples and injections is reduced. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 23 Chapter 1 Instrument and software description 1 Instrument consumables handling, usage limits, and expiration Conditioning reagent Cat. No. Description Storage 4393718 Conditioning reagent , 1 pouch 2–8°C After removing from storage, use the pouch within 24 hours. On-instrument supported limits Guidelines After it is installed on the instrument, the pouch is See the expiration date on the label. See “Important notice good for a one-time use. regarding use of consumables that exceed supported limits” on page 25. Capillary arrays WARNING! SHARP. The electrodes on the load-end of the capillary array have small, blunt ends that can lead to piercing injury. Cat. No. Description Storage 4404685 8-Capillary, 50 cm Room temperature 4404689 24-Capillary, 50 cm 4404683 8-Capillary, 36 cm 4404687 24-Capillary, 36 cm On-instrument limits Guidelines 160 injections when used Capillary arrays have been verified for use for 160 injections when used with with Thermo Fisher Scientific Thermo Fisher Scientific reagents. reagents, or expiration date The software displays a warning message when a usage limit is met and allows listed on packaging and RFID you to continue running. Before doing so, see “Important notice regarding use of label consumables that exceed supported limits” on page 25. Store capillary arrays with the loading-end of the capillary array in distilled water to prevent the polymer from drying in the capillaries. 24 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Instrument consumables handling, usage limits, and expiration 1 Hi‑Di™ Formamide Formamide is used to prepare samples, it is not installed on the instrument as are the other consumables listed in this section. It does not include an RFID tag on the label. Material Cat. No. Storage Hi‑Di™ 4440753 –25°C to –15°C Formamide If frequent sampling is required, dispense and freeze small aliquots of Hi‑Di™ (4 × 5‑mL Formamide into smaller tubes. Minimize freeze-thaw cycles and exposure to air and bottles) room temperature because the quality of the material may decrease when exposed to Hi‑Di™ 4311320 air. Formamide (25‑mL bottle) Important notice regarding use of consumables that exceed supported limits BEFORE DISMISSING THE WARNING THAT THE CONSUMABLES HAVE REACHED SUPPORTED LIMITS AND CONTINUING WITH OPERATION OF THE INSTRUMENT, PLEASE READ AND UNDERSTAND THE FOLLOWING IMPORTANT NOTICE AND INFORMATION: Thermo Fisher Scientific does not recommend the use of consumables that exceed supported limits. The recommended limits are designed to promote the production of high-quality data and minimize instrument downtime. Reagent and consumable lifetime minimum performance are based on testing and studies that use reagents and consumables that have not exceeded supported limits. The use of consumables beyond the supported limits may impact data quality or cause damage to the instrument or capillary array. The cost of repairing such damage is NOT covered by any Thermo Fisher Scientific product warranty or service plan. Customer use of expired consumables is at customer's own risk and without recourse to Thermo Fisher Scientific. For example, product warranties do not apply to defects resulting from or repairs required due to misuse, neglect, or accident including, without limitation, operation outside of the environmental or use specifications or not in conformance with Thermo Fisher Scientific instructions for the instrument system, software, or accessories. Please see your specific service contract or limited product warranty for exact language regarding coverage and ask your Thermo Fisher Scientific representative if you have further questions. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 25 Chapter 1 Instrument and software description 1 Overview of the software Overview of the software About the software During a run, the software: • Controls the instrument and generates sample data files: – Sequencing (AB1) – Fragment analysis (FSA) – HID analysis (HID) • Performs primary analysis: – For sequencing applications: Basecalling – For fragment analysis and HID applications: Sizecalling Dashboard You can access the Dashboard from any screen by clicking the Dashboard tab. Figure 2 Dashboard overview 26 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Overview of the software 1 The Dashboard gives you quick access to the information and tasks you need to set up and run: • Workflow, Maintenance, and Library tabs—Advances to the screens to set up, load, run, and review plates, maintenance wizards, and library items. • Menu bar—Accesses administrative and tools functions. • Common operations—Allows you to quick-start (load a plate that is set up), create or edit plates, view results, and access the Maintenance workflow. • Quick view—Displays gauges that show the remaining usage of consumables and gives the status of instrument conditions. Consumable usage is automatically tracked by the instrument by RFID tags. • Consumables information—Gives details for the installed consumables and indicates if any consumable is about to expire based on RFID tags. • Calendar reminders—Displays the tasks listed in the schedule. • Help icon —Displays a help topic specific to a screen or an area of the screen. All screens include icons. Workflow Click the Workflow tab at the top left of the screen to access the Workflow screen. The Workflow tab contains the screens where you set up, load, and run plates, and view results. Select a task in the navigation pane to access each screen. The Workflow navigation pane is designed as a task workflow. Each screen contains a button that you can click to advance to the next screen in the workflow. You can click Dashboard or any other tab item at any time to advance from the Workflow. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 27 Chapter 1 Instrument and software description 1 Overview of the software Maintenance workflow Select Maintenance in the menu bar to access the Maintenance workflow. The Maintenance workflow contains the screens where you calibrate, run install checks, run maintenance procedures, and access records about instrument maintenance and service. You can click Main Workflow or Dashboard or select any other menu item at any time to advance from the Maintenance workflow. The Maintenance workflow is described in Chapter 10, “Maintain the instrument”. Library workflow Click the Library tab at the top left of the screen to access the Library. The Library contains items that you manage plates, assays, file name conventions, and results groups that you use to acquire and process data. You can click Workflow or select Dashboard or any other menu item at any time to advance from the Library workflow. 28 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Overview of the software 1 Thermo Fisher Connect menu This option is not available if your system includes a license for the Security, Audit, and E-signature modules. Select Thermo Fisher Connect in the menu bar to access: • The screen that allows you to link the instrument to your Thermo Fisher™ Connect Platform account • An upload log for a list of all sample files uploaded to your Thermo Fisher™ Connect Platform account SAE menu This option is available if your system includes a license for the Security, Audit, and E-Signature module. Select SAE in the menu bar to access: • Security, Audit, and E-signature modules • Change Password function Tools menu Select Tools in the menu bar to access: • View Run Logs for reports of instrument runs • Manual Commands to troubleshoot instrument performance 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 29 Chapter 1 Instrument and software description 1 Overview of the software Manage menu Select Manage in the menu bar to access Archive, Restore, and Purge functions. Preferences menu Select Preferences in the menu bar to set default parameters. Preferences allow you to set system and user defaults for settings such as the date format, sample data file storage location, export file formats for sequencing data, and a variety of sequencing-specific settings. Help menu Select Help in the menu bar to access software help. The Help menu provides quick access to brief information about how to perform tasks on a screen. For details about tasks and other information, refer to the chapters in this user guide. Use the software without an instrument You can install the software on a computer that is not connected to an instrument. You can use this stand-alone version of the software to: • Create plates, protocols, and other library items, then import them into a version of the software that is installed on an instrument computer • Review completed results 30 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 1 Instrument and software description Overview of the software 1 Do not select instrument-related functions in the stand-alone version of the software. IMPORTANT! Do not rename the computer after the software is installed. The instrument computer has been assigned a unique name. Changing the name may cause the software to malfunction. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 31 2 Start the system ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 ■ Start the computer and instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 ■ Start the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 ■ Check system status in the Dashboard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 ■ Set preferences (optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Workflow Start the system Power on the computer (do not log in), then power on the instrument (page 33) When the green front panel indicator stops blinking, log in to Windows™ operating system, then start the software (page 34) Check calendar reminders (page 36) Check consumables status (page 37) Replenish consumables (page 39) 32 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Start the computer and instrument 2 Start the computer and instrument IMPORTANT! The order in which you turn on the computer and instrument is critical for proper communication between the instrument and the computer. Follow the sequence of steps given in this section (power on computer but do not log in, power on instrument, log in to Windows™ operating system). 1. Power on the computer and monitor, but do not log in to the Windows™ operating system. 2. Verify that the instrument is connected to the appropriate power supply. CAUTION! Do not unpack or plug in any components until a service representative has configured the system for the proper operating voltage. IMPORTANT! Do not rename the computer after the software is installed. The instrument computer has been assigned a unique name. Changing the name may cause the software to malfunction. 3. Inspect the instrument interior. Ensure that: a. The oven door is closed. b. No objects are left inside the instrument. IMPORTANT! Misplaced objects left inside the instrument can cause damage. 4. Close the instrument door. 5. Power on the instrument: Power button Tray button Light button a. Press the power on/off button on the front of the instrument and wait for the green status light to turn on. Note: If the door is open during power on, the yellow light will continue to flash until you close the door. See indicator descriptions in “Instrument front panel indicators” on page 19. b. If desired, press the Light button to turn on the interior light. c. Check the instrument status. Ensure the green status light is on and not flashing before proceeding. See indicator descriptions in “Instrument front panel indicators” on page 19. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 33 Chapter 2 Start the system 2 Start the software 6. Log on to the Windows™ operating system. IMPORTANT! Use the same administrator Windows™ user account to log in to the instrument computer. Use of a non-administrator Windows™ user account, or use of different administrator Windows™ user accounts can interfere with software operation. Start the software Step one: Start the Server Monitor 1. After you log on to the Windows™ operating system, wait ~1 to 2 minutes. As the server monitor starts, the list of services is displayed at the bottom of the desktop. 2. Check the status of the server monitor (a background application that the software requires). In the Windows™ taskbar at the bottom right of the desktop, click the arrow to display the running tasks. If the Server Monitor icon is displayed, go to “Step two: Start the 3500 Series Data Collection Software v3.3” on page 34. 3. Select 4Applied Biosystems4Server Monitor. The Server Monitor icon is displayed in the taskbar, then a status bubble is displayed. It takes ~1 minute for the Server Monitor to start up. When the Server Monitor icon is displayed, go to “Step two: Start the 3500 Series Data Collection Software v3.3” on page 34 below. IMPORTANT! If the Server Monitor icon does not change to , you cannot start the software. See “Step one: Start the Server Monitor” on page 34 for more information. Step two: Start the 3500 Series Data Collection Software v3.3 Select Start4Applied Biosystems43500. The 3500 Series Data Collection Software v3.3 splash screen is displayed for a few seconds, then the 3500 Series Data Collection Software v3.3 Login dialog box opens. 34 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Start the software 2 Step three: Log in The Login dialog box is displayed if your system includes a license for the Security, Audit, and E‑signature module. In the Login dialog box: Enter your User Name and Password, then click OK. See your 3500 system administrator for login information. Note: For information on creating user accounts, see Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)”. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 35 Chapter 2 Start the system 2 Check system status in the Dashboard Check system status in the Dashboard Check calendar reminders and consumables status in the Dashboard. Gauges Instrument information Consumables Calendar reminders Figure 3 Dashboard Check calendar reminders The Calendar Reminders section displays reminders for the tasks listed in the schedule (see “As- Needed instrument maintenance tasks” on page 254). You can set the time to trigger calendar reminders in Preferences. 1. Review the Calendar Reminders pane. 2. Perform any scheduled tasks, then click to mark it as complete, (or click to mark it as dismissed if you do not perform the task). Actions are recorded in the Notifications Log (for more information, see “Review the Notifications Log” on page 256. 3. Perform any daily, monthly, or quarterly tasks that are not listed in the Calendar Reminders pane (see “Maintenance schedule” on page 251). 36 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Check system status in the Dashboard 2 Check consumables status 1. Click Refresh to update consumable status. The Consumables pane displays expiration dates and lot numbers (determined from the RFID tags on the consumable containers). Note: The Expiration Date for consumables is displayed in red if the consumable is within the following days of expiration: Pouch 7 days, Buffers 7 days, Capillary array 1 day. 2. Check the consumables gauges for the number of injections, samples, or days remaining for a consumable. See “Instrument consumables handling, usage limits, and expiration” on page 22 for information. When <10% of the allowed use of the consumable remains, the gauge moves into the red warning range. The consumable also displays in red in the Consumables pane. IMPORTANT! We recommend that you add a calendar reminder to the schedule for polymer and buffer replacement. Set the notification to display 2 days before the polymer should be replaced. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 37 Chapter 2 Start the system 2 Check system status in the Dashboard How the polymer sample and injection counters calculate usage The polymer sample counter decrements only for wells that contain sample, but the polymer injection counter decrements for each injection, regardless of whether all wells contain sample. The sample limit and the corresponding injection limit may not coincide. The first limit that is reached depends on whether you perform partial or full injections. Example: Instrument configuration: 24-capillary, 960 sample polymer pouch Partial injection example (not 1 injection with 24 samples + The 50 injection count limit is reached all wells contain sample) before the 960 sample count limit. 49 injections with 1 sample = 73 samples, 50 injections Full injection example (all wells 40 injections with 24 samples = The 960 sample count limit is reached contain sample) before the 50 injection count limit. 960 samples, 40 injections Check for leaks and spills 1. Open the front doors. 2. Inspect the instrument interior. 3. Wipe any spills. 38 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Check system status in the Dashboard 2 4. Check for leaks around the buffer-pin valve, check valve, and array port lock. 3 1 2 1 Buffer-pin valve 2 CV (check valve) fitting 3 Array port lock 5. Remove dried residue and ensure that the array port lock is securely tightened. Check buffer fill levels Check the fill levels on buffers. Verify that the buffer level is at the top of the fill line and check that the seal is intact. The meniscus must line up at or above the fill line. Ensure that the septa on the CBC are properly seated. IMPORTANT! Replace the buffer if the buffer level is too low. Replenish consumables If any consumables are expired or if buffer fill level is too low, replenish the consumables as described in: • “Replenish polymer or change polymer type” on page 263 IMPORTANT! Wear gloves while handling polymer, the capillary array, septa, ABC, or CBC. • “Prepare the Anode Buffer Container (ABC)” on page 258 • “Prepare the Cathode Buffer Container (CBC)” on page 259 • “Fill capillary array with fresh polymer” on page 265 • “Install or change the capillary array” on page 266 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 39 Chapter 2 Start the system 2 Check system status in the Dashboard Ensure proper installation of CBC septa When you install the CBC buffer septa, press firmly to seat the septa. IMPORTANT! Look at the CBC from the side and ensure that there is no gap between the container and the lip of the septum. IMPORTANT! Ensure the septa are securely seated to prevent displacement of the septa during operation. Inspect the underside of the CBC lid to ensure that all capillary washing septum blades are fully engaged and unobstructed. IMPORTANT! A correctly inserted capillary washing septum shows a clear, linear slit in all wells. To easily view the slits, illuminate the CBC from the bottom, such as with an LED screen, and observe the CBC from above. If any holes do not show a clear, linear slit, firmly press down on those specific holes to ensure that the septum blades are visually engaged and the slit of light is unobstructed. 1 2 Figure 4 Illuminated CBC with the capillary washing septum correctly in place 1 Starter tab positioned in alignment hole 2 Linear slit visible For more information about installing the septa, see “Prepare the Cathode Buffer Container (CBC)” on page 259. 40 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Set preferences (optional) 2 Set preferences (optional) Overview To access the Preferences dialog box, select Preferences in the toolbar. You have the option to set any or all preferences. Note: The type filter text field at the top of the dialog box is not used. These system settings apply to all users: • Date Format • Instrument Settings (instrument name, reagent use, message boxes, and run logs) • Scheduler Preference (trigger time for calendar reminders) • Sequencing Export Settings • Spectral Calibration (number of allowed borrowing events) 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 41 Chapter 2 Start the system 2 Set preferences (optional) These user preferences are saved individually for each user. • Library filtering • Plate Setup • Reports Settings • Run Setup • Sequencing Settings (review and report settings) • Warning dialogs Note: Users can also save user preferences while viewing tables. See “Customize a table” on page 94. System preferences In the Preferences dialog box, click a system preference, select a setting, then click Apply to save the preference. System Sets preference Date Format Date and time format for the software. Instrument • Instrument name (appears in the Dashboard, reports, file name conventions, instrument Settings sensor details, view sequencing results.) Note: If you have multiple instruments, you can assign each instrument a unique instrument name. • Suppress the messages that are displayed when at the start of a run that indicate the number of days left before a consumable expires or should be replaced. • Number of runs to preserve in the Run Log (accessed by selecting Tools4View Run Logs). 42 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Set preferences (optional) 2 (continued) System Sets preference Instrument • Allow runs with reagents that exceed limits. Settings for – By default, the software allows use of reagents that are expired or exceed on- reagent use instrument limits. This setting allows a user to dismiss expiration or limit warnings, then continue the run. An Administrator can prevent the use of reagents that are expired or exceed on- instrument limits by deselecting reagents in the Instrument Settings screen. By default, all reagents are selected, which means that a user can dismiss a reagent usage limits and/or expiration warning and continue to run. Deselect the reagents that will not allow runs unless they are within on-instrument limits (to set a "hard stop"). If you do not have Administrator role, these options are not active. • Enable warning messages. – You can control the display and timing of warnings that are related to reagent usage limits and expiration. – Expiration warnings are displayed when a reagent is expired or exceeds on-instrument limits. – Pre-expiration warnings are displayed the following number of days before a reagent is expired: – ABC and CBC: 7 days – Array and polymer: 14 days Scheduler Time for calendar reminders to be displayed in the Dashboard (see “Check calendar reminders” Preference on page 36). Spectral Number of allowed borrowing events for spectral calibration (see “What you see during a Calibration spectral calibration” on page 138). Sequencing Default file types for files exported during a sequencing run. Exported files are stored in the Settings Export same directory as the .ab1 files: • *.annotation.txt — Information from the Annotation tab in the sequencing trace view such as data collection time, run time start finish • *.phd.1, *.scf — Sequencing files • *.fsta, *.qual, *.seq — Reference files - specify Entire Sequence or Post-trim Sequence Only 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 43 Chapter 2 Start the system 2 Set preferences (optional) User preferences In the Preferences dialog box, click a user preference, select a setting, then click Apply to save the preference. User preference Sets IVD Setup Not supported. Workflow Library Filtering Default filter for items displayed in the Open Plate from Library dialog box, the Plates library, and the Assays library. You can set the filter to include only one application type, 8- and/or 24‑capillary-specific items, or items that contain in their names the text you specify. Use an asterisk (*) wildcard character to indicate that text may precede or follow the text you specify. Excluded text is not case-sensitive. Example: To exclude only items named "ABC", enter ABC . To exclude items named "ABCDE", enter ABC* . To exclude items named "123ABC", enter *ABC . You can disable filters in each location to display all items. Plate setup Default settings for plate name. Reports Settings Default font and text size and custom logo in reports. Note: You can override this setting in each report view. Run Setup • Default storage location for data files in file name conventions and results groups. Note: You can override this setting in file name conventions and results groups. • Pause After Last Injection—When enabled, allows reinjection of the last injection by pausing after the last injection is complete (before completing the run). Sequencing Default settings for color representation of nucleotide and quality value bars in the Trace View Settings Trace in View Sequencing Results: • NT (nucleotide) Base Color—Click an NT or mixed base Foreground or Background color block, then select a color for the letter annotation or the highlight color for the letter annotation. • Pure Base and Mixed Base QV Colors—Sets the colors and ranges for pure and mixed base QVs (quality values) displayed in the Trace View: a. Click a pure base or mixed base color bar to select a new color. b. Place the mouse pointer over a slider, then drag to set a new range. We recommend that you set the following ranges for QVs: – Pure bases: Low QV < 15, Medium QV = 15 to 19, High QV = 20+ (default). – Mixed bases: Low QV < 5, Medium QV = 5 to 10, High QV > 10 (investigate to determine the best range for your application). 44 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 2 Start the system Set preferences (optional) 2 (continued) User preference Sets Sequencing Settings for sequencing trace reports: Type of trace data, specific print settings, and Y-Scale. Settings Trace Print Sequencing Quality ranges for: Settings Trace • QC report—Trace Score and CRL Quality • Plate report—Trace Score Set colors and ranges: a. Click a color bar to select a new color. b. Place the mouse pointer over a slider, then drag to set a new range. Sequencing Content and formatting used in QC, Plate, Trace Score, CRL, QV20+, and Signal Strength Settings Trace reports: Quality Report • Sort data—Sort data in Trace Score, CRL, QV20+, and Signal Strength Reports based on Run Name or Capillary Number. • Signal based on—Base signal in QC and Signal Strength Reports based on Average Raw Signal Intensity or Average Raw Signal to Noise Ratio. • Display well image by—Specify the thumbnail option for Plate Reports: Wider thumbnail without file name or Smaller thumbnail without file name. Warning Dialogs Suppress warning messages for deleting an injection or exporting a library item. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 45 3 Use the instrument with the Thermo Fisher™ Connect Platform ■ Thermo Fisher™ Connect Platform features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 ■ Register and obtain a Thermo Fisher™ Connect Platform account . . . . . . . . . . . . . . . . . . . . . . . . . . 47 ■ Connect the instrument to your Thermo Fisher™ Connect Platform account . . . . . . . . . . . . . . . . . 47 ■ Set up the data storage location and email notifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 ■ View your upload history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 ■ Monitor a run from InstrumentConnect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 ■ Monitor a run from a mobile device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 ■ View notifications from the instrument on your Thermo Fisher™ Connect Platform account . . . . 52 ■ For more information on using InstrumentConnect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 ■ Thermo Fisher™ Connect Platform administrators for an instrument . . . . . . . . . . . . . . . . . . . . . . . . 53 Thermo Fisher™ Connect Platform features This option is not available if your system includes a license for the Security, Audit, and E-signature module. If the instrument is connected to a network, you can use the Thermo Fisher™ Connect Platform feature. The Thermo Fisher™ Connect Platform provides the following functions: • Automatically transfer data files from the instrument to your Thermo Fisher™ Connect Platform account. • Receive instrument status email notifications. • View instrument status on InstrumentConnect or a mobile device. IMPORTANT! The instrument communicates with the computer by Ethernet connection. Do not make any changes to the computer ethernet/internet connections during a run or during calibration. 46 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform Register and obtain a Thermo Fisher™ Connect Platform account 3 Register and obtain a Thermo Fisher™ Connect Platform account 1. Go to www.thermofisher.com. 2. On the home page, select Sign In4 Register. 3. Fill in all information, then click Create account. Connect the instrument to your Thermo Fisher™ Connect Platform account This option is not available if your system includes a license for the Security, Audit, and E-Signature module. The Thermo Fisher Connect menu is not active. 1. In any screen, select Thermo Fisher Connect4Thermo Fisher Connect. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 47 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform 3 Set up the data storage location and email notifications 2. Enter the User ID and Password for your Thermo Fisher™ Connect Platform account, then click Link Account. An email is sent to the email address associated with your account, and the instrument is listed in the screen on the Thermo Fisher™ Connect Platform (see “Monitor a run from InstrumentConnect” on page 51). Set up the data storage location and email notifications When an instrument is linked to your Thermo Fisher™ Connect Platform account, you can store run data in your Thermo Fisher™ Connect Platform account. You can also have email notifications sent to your Thermo Fisher™ Connect Platform account email address. 1. In any screen, select Thermo Fisher Connect4Thermo Fisher Connect. 48 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform Set up the data storage location and email notifications 3 2. In the middle of the screen, click your User ID to connect the instrument to your Thermo Fisher™ Connect Platform account. 3. Select the Enable Destination checkbox, then select the storage location in your Thermo Fisher™ Connect Platform account. When a run is complete, the data is stored in the results group folder that is specified in the plate record. The data will be automatically uploaded to your account whenever your User ID is the active ID. Results are saved in your account folders named by plate and injection, not in results group folders. 4. Select the Receive Notifications checkbox. Run status emails will be sent to the email address associated with your Thermo Fisher™ Connect Platform account. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 49 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform 3 View your upload history View your upload history To view a list of injections (which includes a set of sample files) that have been uploaded to your Thermo Fisher™ Connect Platform account, select Thermo Fisher Connect4Upload History. Sample file names are retained in the list for 30 days. Possible upload status conditions are listed below. Upload Status Description Pending Files are waiting to be uploaded. Processing Upload is in process. Completed Upload is complete. Failed Files were not uploaded. Click Retry Upload. 50 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform Monitor a run from InstrumentConnect 3 Monitor a run from InstrumentConnect 1. Sign in to thermofisher.com/connect. 2. In the left panel, then click to access InstrumentConnect. Note: The Add an Instrument function is not supported for the instrument. See “Connect the instrument to your Thermo Fisher™ Connect Platform account” on page 47. 3. Click the instrument to display instrument status. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 51 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform 3 Monitor a run from a mobile device Monitor a run from a mobile device The instrument must be connected to your Thermo Fisher™ Connect Platform account before you can monitor it. See the “Connect the instrument to your Thermo Fisher™ Connect Platform account” on page 47. 1. On your mobile device, download the InstrumentConnect app from the Apple Store or from Google™ Play. 2. On your mobile device, launch InstrumentConnect. 3. Touch the instrument to monitor. View notifications from the instrument on your Thermo Fisher™ Connect Platform account 1. In any screen in the Thermo Fisher™ Connect Platform, click . 2. Click a notification, then click Dismiss or Dismiss all to dismiss the notification. 52 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform For more information on using InstrumentConnect 3 For more information on using InstrumentConnect In the top left of any screen in InstrumentConnect, click , then select Help guide. Thermo Fisher™ Connect Platform administrators for an instrument First user who links is assigned administrator role The first user who links the instrument to their Thermo Fisher™ Connect Platform account is assigned administrator role for the instrument. Additional instrument administrators can be assigned, and user roles can be changed after linking. Instrument administrator functions An administrator can perform the following tasks from InstrumentConnect. • Access the Manage users function to see a list of all accounts that are linked to the instrument. • Assign administrator role to one or more users. • Remove an account from an instrument. • Disconnect the instrument from InstrumentConnect. • Change the instrument name. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 53 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform 3 Thermo Fisher™ Connect Platform administrators for an instrument Manage the users and administrators of your instrument Any user with administrator role can manage users for an instrument or disconnect an instrument from InstrumentConnect. If an administrator... The software... Assigns Administrator role to a Allows the user to perform all administrator functions (see “Instrument user administrator functions” on page 53). Removes a user Unlinks the instrument from their Thermo Fisher™ Connect Platform account. Disconnects the instrument • Unlinks the instrument from all Thermo Fisher™ Connect Platform accounts. • Removes the instrument from InstrumentConnect. 1. Sign in to thermofisher.com/connect. 2. In the left panel, click to access InstrumentConnect. 3. Select the instrument. Note: The Manage users and other administrator functions are not enabled until you select an instrument. 4. To assign the Administrator role to a user or to remove a user, click Manage users, then perform the following tasks as needed. To... Do this... Assign the Administrator role to an additional user Select the Admin checkbox, then click Close. Remove a user Click , then click Confirm. 54 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 3 Use the instrument with the Thermo Fisher™ Connect Platform Thermo Fisher™ Connect Platform administrators for an instrument 3 Unlink individual users from an instrument You cannot unlink individual users from an instrument in InstrumentConnect. To unlink a user in the data collection software, select Thermo Fisher Connect4Thermo Fisher Connect, select the User ID, then click Unlink Account. You can disconnect the instrument from InstrumentConnect. However, doing so unlinks all accounts and removes the instrument, and all user data for that instrument, from InstrumentConnect. You can remove a user from the instrument. However, doing so deletes the user data from the instrument. For more information, see “Manage the users and administrators of your instrument” on page 54. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 55 4 Set up and run ■ Setup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 ■ Prepare the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 ■ Create or import a plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 ■ Assign plate contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 ■ Prepare and assemble sample plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 ■ Quick Start a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 ■ Load plates for run and create the injection list . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 ■ Review, modify, or export the injection list in Preview Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 ■ Start the run from Preview Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 ■ Export the injection list from Preview Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 ■ Monitor the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 ■ Pause a run and load a new plate (flexible plate loading) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 ■ Check sequence or sample quality and re-inject samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 ■ Review completed injections in Review Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 ■ Pause, resume, or stop a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 ■ More features in Assign Plate Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 ■ More features in Monitor Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 56 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Setup workflow 4 Setup workflow Workflow Prepare and check the instrument, sample plates, and dashboard system 1. “Prepare the instrument” on page 57 2. “Prepare sample plates” on page 69 and “Load the plate in the instrument” on page 72 3. “Check system status in the Dashboard” on page 36 Plate Settings Select a workflow for plate settings: • Create and assign new plate settings. a. “Create or import a plate” on page 58 b. “Assign plate contents” on page 61 c. “Print the plate layout” on page 66 d. “Link the plate” on page 72 • Start a run with preprepared plate settings. “Quick Start a run” on page 73 Set up and run 1. “Load plates for run and create the injection list” on page 74 2. “Review, modify, or export the injection list in Preview Run” on page 78 3. “Start the run from Preview Run” on page 79 4. “Check sequence or sample quality and re-inject samples” on page 83 Prepare the instrument 1. In the Dashboard, check consumables status (see “Check consumables status” on page 37). Ensure that: • Consumables are not expired • Adequate injections remain for consumables 2. Ensure that buffer levels are at the fill lines (see “Check buffer fill levels” on page 39). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 57 Chapter 4 Set up and run 4 Create or import a plate 3. Set the oven temperature, then click Start Pre-heat. Pre-heat the oven and detection cell while you prepare for a run (detection cell temperature is set by the software). Pre-heating helps mitigate subtle first-run migration rate effects. The pre-heat function automatically turns off after 2 hours. We recommend that you pre-heat the oven for at least 30 minutes before you start a run if the instrument is cold. Temperatures are displayed in red as they warm to the set-points. When temperatures are at the set-point they are displayed in green. Temperatures may fluctuate slightly when they reach the set-point as they stabilize. 4. Check the pump assembly for bubbles and run the Remove Bubble wizard if needed (see “Remove bubbles from the polymer pump” on page 268). Create or import a plate Note: If you are running a stand-alone version of the software (a version that is not installed on the instrument computer), you can create plates, then export them for use on the instrument computer. About plate templates The software includes plate templates that you can use as a starting point to create a plate. Plate template names reflect the run module associated with the plate. The run module contains data collection settings. Example sequencing plate templates are shown below. For a list of the run time and size range collected for each run module, see Appendix B, “Run modules and dye sets”. You can also create your own templates. In addition to defining plate parameters, a plate template can also contain a list of the appropriate assays for an application. For more information, see “Create a plate template” on page 97. 58 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Create or import a plate 4 Create a plate from a template The software includes factory-provided plate templates that you can use as a starting point to create a plate (you can also create your own plate templates). In addition to pre-defined plate parameters, a plate template can also contain a list of the appropriate assays, file name conventions, and results groups for an application. 1. In the Dashboard, click Create Plate From Template to display the Open Plate Template from Library dialog box. 2. In the Define Plate Properties screen, enter the plate name and select the number of wells on the plate. • Find templates by selecting an attribute, entering the text to search for, then clicking Go. (Click Clear to clear the field and enter different search criteria). • Select a template, then click Open. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 59 Chapter 4 Set up and run 4 Create or import a plate IMPORTANT! Enter only alpha-numeric characters in the software. Special characters may not be correctly displayed in some software screens, may cause problems with plate, file, folder, user account, and/or library item names, and may interfere with starting a run and/or importing and exporting library items. IMPORTANT! If you copy/paste sample or plate information into the Assign Plate Contents screen or into a plate import file, copy from a plain text editor such as Notepad. Do not copy from a word processing program such as Microsoft™ Word™, which may include invisible, non-ASCII characters. Non-ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting. • 96—Select if you are using a 96-well standard reaction plate or 8-strip standard tubes with a retainer. • 96-Fast—Select if you are using a 96-well Fast reaction plate or 8-strip fast tubes with a retainer. • 384—Select if you are using a 384-well reaction plate (24-capillary instruments only). 3. (Optional) Enter Owner, Barcode, and Description for the plate. For more information on these parameters, see “Plates library” on page 166. 4. (Optional) In the bottom section of the screen, specify auto-analysis settings for the plate. Refer to the instructions provided with the secondary analysis software. 5. Click Save. 6. Click Assign Plate Contents, then go to “Assign plate contents” on page 61. Import a plate 1. Do either of the following: • Create a plate on another computer where the software is installed, then export (see “Import and export a plate” on page 97). • Create a plate import file (see “Create a plate import file” on page 96. IMPORTANT! If you copy/paste sample or plate information into the Assign Plate Contents screen or into a plate import file, copy from a plain text editor such as Notepad. Do not copy from a word processing program such as Microsoft™ Word™, which may include invisible, non-ASCII characters. Non-ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting. 2. Access the Assign Plate Contents screen: Click the Workflow tab in the Dashboard, then select Assign Plate Contents in the navigation pane. 60 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Assign plate contents 4 3. Click Import, then select the plate import file. 4. Click Assign Plate Contents. Assign plate contents You assign the following information to the wells in a plate before you can run the plate: • Sample names and sample types (required)—Identifies the well positions of each sample for data collection and processing. • Assay (required)—Specifies the parameters that control data collection and primary analysis (basecalling or sizing). All named wells on a plate must have an assigned assay. For more information on assays, see “Assays library” on page 169. • Filename convention (optional)—Specifies file naming. For more information, see “File name convention overview” on page 173. • Results group (optional)—Specifies sample data file storage. For more information on assays, see “Results Group overview” on page 178. Access the Assign Plate Contents screen 1. Access the Assign Plate Contents screen from: • The Define Plate Properties screen by clicking Assign Plate Contents (described above). • The navigation pane by selecting Assign Plate Contents. • The Dashboard by clicking the Workflow tab, then selecting Assign Plate Contents in the navigation pane. 2. Click Show In Wells to specify the attributes to display in wells. Figure 5 shows the Plate View of the Assign Plate Contents screen. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 61 Chapter 4 Set up and run 4 Assign plate contents 1 2 3 4 5 Figure 5 Assign plate Contents 1 Show well attributes 2 Name samples 3 Assign assays, file name conventions, and results groups 4 Assign sample types and user-defined fields 5 Link plate for run 62 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Assign plate contents 4 Name samples and assign sample types in the plate view Note: For other ways to name samples, see “Name samples in the plate view” on page 91 and “Use the table view” on page 93. 1. Click a well, type a sample name directly into the well, then press Enter. 2. Click-drag to select multiple wells. 3. Right-click and select Fill or Fill Series to populate the selected fields. Note: To use Fill Series, type a number as the last character of the named well. The number will increment for each well in the series. Note: You can copy and paste sample names instead of using fill commands. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 63 Chapter 4 Set up and run 4 Assign plate contents 4. At the bottom-right of the Assign Plate Contents screen, expand the Customize Sample Info pane. 5. In the Plate View, click-drag to select the wells of interest. 6. Specify the Sample Type for the selected wells, then press Enter. 7. (Optional) Specify User Defined Fields and Comments. User Defined Fields contain additional attributes you can assign to a plate and are displayed only in Table View. IMPORTANT! Enter only alpha-numeric characters in the software. Special characters may not be correctly displayed in some software screens, may cause problems with plate, file, folder, user account, and/or library item names, and may interfere with starting a run and/or importing and exporting library items. IMPORTANT! If you copy/paste sample or plate information into the Assign Plate Contents screen or into a plate import file, copy from a plain text editor such as Notepad. Do not copy from a word processing program such as Microsoft™ Word™, which may include invisible, non-ASCII characters. Non-ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting. 64 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Assign plate contents 4 8. For sequencing assays, specify Amplicon and Specimen. 9. Repeat steps step 6 through step 8 to assign the Sample Type for all named wells. 10. Go to “Assign assay, file name convention, and results group in the plate view” on page 65. Assign assay, file name convention, and results group in the plate view Note: If a file name convention or results group you created is not listed for the plate, go to “Add assays, file name conventions, and results groups to a plate” on page 95. 1. Select the wells for which to specify an assay. 2. Select the checkbox next to the assay name to assign the assay to the selected wells. 3. Repeat for file name conventions and results group. 4. Select Save Plate. 5. Go to “Print the plate layout” on page 66. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 65 Chapter 4 Set up and run 4 Assign plate contents How file location in file name conventions and results groups work If you do not specify a file name convention, data files are named in this format: <sample name>_ <well>. If you do not specify a results group, files are stored in the location specified in the file name convention or in Preferences4User4Run (see “User preferences” on page 44). If you specify both a file name convention and a results group, files are stored in the location specified in the results group. Print the plate layout 1. In the Assign Plates for Run screen, click View Plate Grid Report. 2. Select Print Preview or Print as needed. 3. To save the report electronically (PDF), print the report and select a PDF printer. 4. Close the report. 5. Go to “Prepare and assemble sample plates” on page 67. 66 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Prepare and assemble sample plates 4 Prepare and assemble sample plates IMPORTANT! Do not use warped or damaged plates. Capillary-to-plate mapping The capillary-to-plate mapping for the default injection order is shown below. The numbers in the wells represent capillary numbers. If you change the injection order in the injection list, injection order in the injection list differs from the following examples. 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 A 1 1 1 1 1 1 1 1 1 1 1 1 A 1 2 3 1 2 3 1 2 3 1 2 3 B 2 2 2 2 2 2 2 2 2 2 2 2 B 4 5 6 4 5 6 4 5 6 4 5 6 C 3 3 3 3 3 3 3 3 3 3 3 3 7 8 9 7 8 9 7 8 9 7 8 9 C D 4 4 4 4 4 4 4 4 4 4 4 4 10 11 D 10 11 12 10 11 12 10 11 12 12 E 5 5 5 5 5 5 5 5 5 5 5 5 13 14 E 13 14 15 13 14 15 13 14 15 15 F 6 6 6 6 6 6 6 6 6 6 6 6 16 17 F 16 17 18 16 17 18 16 17 18 18 G 7 7 7 7 7 7 7 7 7 7 7 7 G 19 20 21 19 20 21 19 20 21 19 20 21 H 8 8 8 8 8 8 8 8 8 8 8 8 H 22 23 24 22 23 24 22 23 24 22 23 24 1 2 1 2 3 4 Figure 6 3500 96-well plate capillary-to-plate Figure 7 3500xL 96-well plate capillary-to-plate mapping mapping (8 capillary) (24 capillary) Injection 1 (wells A1–H1) Injection 1 (wells A1-H3) Injection 2 (wells A2–H2) Injection 2 (wells A4-H6) This pattern repeats for a total of 8 injections Injection 3 (wells A7-H9) Injection 4 (wells A10-H12) 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 67 Chapter 4 Set up and run 4 Prepare and assemble sample plates 1 2 3 4 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 2 3 1 2 3 12 4 4 5 5 6 6 4 4 5 5 6 6 4 4 5 5 6 6 4 4 5 5 6 6 12 1 2 3 1 2 3 4 5 6 4 4 5 5 6 6 4 4 5 5 6 6 4 4 5 5 6 6 4 4 5 5 6 6 4 5 6 12 7 7 8 8 9 9 7 7 8 8 9 9 7 7 8 8 9 9 7 7 8 8 9 9 12 4 5 6 4 5 6 7 8 9 7 8 9 12 7 7 8 8 9 9 7 7 8 8 9 9 7 7 8 8 9 9 7 7 8 8 9 9 12 7 8 9 7 8 9 10 10 11 11 12 12 10 10 11 11 12 12 10 10 11 11 12 12 10 10 11 11 12 12 10 11 12 10 11 12 12 12 10 10 11 11 12 12 10 10 11 11 12 12 10 10 11 11 12 12 10 10 11 11 12 12 12 10 11 12 10 11 12 13 14 15 13 13 14 14 15 15 13 13 14 14 15 15 13 13 14 14 15 15 13 13 14 14 15 15 13 14 15 12 12 13 13 14 14 15 15 13 13 14 14 15 15 13 13 14 14 15 15 13 13 14 14 15 15 12 13 14 15 13 14 15 16 17 18 16 16 17 17 18 18 16 16 17 17 18 18 16 16 17 17 18 18 16 16 17 17 18 18 16 17 18 12 12 12 16 17 18 16 17 18 16 16 17 17 18 18 16 16 17 17 18 18 16 16 17 17 18 18 16 16 17 17 18 18 19 20 21 19 20 21 12 19 19 20 20 21 21 19 19 20 20 21 21 19 19 20 20 21 21 19 19 20 20 21 21 12 12 19 20 21 19 20 21 22 23 24 19 19 20 20 21 21 19 19 20 20 21 21 19 19 20 20 21 21 19 19 20 20 21 21 12 22 23 24 12 22 22 23 23 24 24 22 22 23 23 24 24 22 22 23 23 24 24 22 22 23 23 24 24 12 22 23 24 22 23 24 22 22 23 23 24 24 22 22 23 23 24 24 22 22 23 23 24 24 22 22 23 23 24 24 Figure 8 3500xL 384-well plate capillary-to-plate mapping (24 capillary only) Injection 1 (wells A1, A3, A5; wells C1, C3, C5; wells E1, E3, E5; wells G1, G3, G5; wells I1, I3, I5; wells K1, K3, K5; wells M1, M3, M5; wells O1, O3, O5) Injection 2 (wells B1, B3, B5; wells D1, D3, D5; wells F1, F3, F5; wells H1, H3, H5; wells J1, J3, J5; wells L1, L3, L5; wells N1, N3, N5; wells P1, P3, P5) Injection 3 (wells A2, A4, A6; wells C2, C4, C6; wells E2, E4, E6; wells G2, G4, G6; wells I2, I4, I6; wells K2, K4, K6; wells M2, M4, M6; wells O2, O4, O6) Injection 4 (wells B2, B4, B6; wells D2, D4, D6; wells F2, F4, F6; wells H2, H4, H6; wells J2, J4, J6; wells L2, L4, L6; wells N2, N4, N6; wells P2, P4, P6) This pattern repeats for a total of 16 injections Allelic ladder run requirements We recommend that you inject one allelic ladder for each set of 24 samples in HID runs: • 8-capillary instruments—One allelic ladder per 3 injections • 24-capillary instruments—One allelic ladder per 1 injection Allelic ladders that are injected under the same conditions are recommended to accurately genotype samples in the secondary analysis software (GeneMapper™ ID-X Software v1.3 or later). IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed that can, in turn, cause sizing variation. We recommend the frequency of allelic ladder injections described above to account for normal variation in fragment migration speed. However, during internal HID validation studies, verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment. Results group for one allelic ladder per run folder For a 24-capillary instrument, create a results group that specifies an injection folder, then select this results group for all injections on the plate. For an 8-capillary instrument, create one results group for each set of three injections on the plate (each results group specifies a results group name folder). For more information, see “Results Group example 2: store re-injections in separate folders” on page 185. 68 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Prepare and assemble sample plates 4 Prepare sample plates 1. Pipet samples into the plate according to the plate layout (see “Print the plate layout” on page 66). 2. Briefly centrifuge the plate. 3. Verify that each sample is positioned correctly in the bottom of its well. IMPORTANT! If the contents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each sample is positioned correctly in the bottom of its well. 4. Store the plate on ice and protected from light until you prepare the plate assembly and load the plate in the instrument. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 69 Chapter 4 Set up and run 4 Prepare and assemble sample plates Prepare the plate assembly Prepare the plate assembly on a clean, level surface. Wear gloves when handling septa. Do not heat plates that are sealed with septa. 96-well plate assembly IMPORTANT! Use the correct plate base for the plate in use. Using the wrong plate base may affect performance. See Appendix D, “Catalog numbers” for plate assembly specifications and catalog numbers. 1 2 3 1 Plate retainer 2 Plate with septa 3 Plate base 1. Align the holes in the septa with the wells of the plate, then press down firmly on the septa until the septa lies flat on the plate. 2. Place the plate into the plate base. 3. Snap the plate retainer (cover) onto the plate, septa, and plate base. 4. Verify that the holes of the plate retainer and the septa are aligned. If holes are not aligned, take it apart, then re-assemble. IMPORTANT! The array tips will be damaged if the plate retainer and septa holes do not align correctly. 5. If the contents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each sample is positioned correctly in the bottom of its well. 70 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Prepare and assemble sample plates 4 8-strip tube standard or fast assembly IMPORTANT! Use the correct plate base for 8-tube standard or fast strips. Using the wrong plate base may affect performance. See Appendix D, “Catalog numbers” for plate assembly specifications and catalog numbers. 1 1 2 2 3 3 4 4 5 5 6 Figure 10 8-strip fast tube assembly Figure 9 8-strip standard tube assembly 1 Plate retainer 1 Plate retainer 2 Septa 2 Septa 3 8-strip tubes 3 Retainer 4 96-well tray 4 8-strip tubes 5 Plate base 5 96-well tray 6 Plate base 1. Place the tubes in the 96-well tray. IMPORTANT! The array tips will be damaged if the plate retainer and septa strip holes do not align correctly. 2. (8-strip standard tube only) Place the retainer on the tubes. 3. Align the holes in the septa strip with the retainer (8-strip standard tube) or the tubes (8-strip fast tube), then firmly press down. 4. Place the tray-tube-retainer assembly into the plate base. 5. Snap the plate retainer (cover) onto the plate, septa, and plate base. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 71 Chapter 4 Set up and run 4 Prepare and assemble sample plates 6. Verify that the holes of the plate retainer and the septa strip are aligned. If holes are not aligned, re-assemble and then assemble the plate assembly. 7. If the reagents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each sample is positioned correctly in the bottom of its well. 384-well plate assembly IMPORTANT! Use the correct plate base for 384-well plates. Using the wrong plate base may affect performance. See Appendix D, “Catalog numbers” for plate assembly specifications and catalog numbers. 1. Place the sample plate into the plate base. 2. Place the septum on the plate and press down to seat. 3. If the reagents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each sample is positioned correctly in the bottom of its well. Load the plate in the instrument 1. Click the Tray button on the front panel to move the autosampler to the front position, then open the instrument door. 2. Place the plate in the autosampler with the labels facing you (or the instrument door) and the notched corner of the plate in the notched corner of the autosampler. 3. Close the instrument door to initialize the instrument. Link the plate 1. In the Assign Plates for Run screen, click Link Plate for Run. Note: By default, plate A position is selected. 72 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Quick Start a run 4 2. Go to “Load plates for run and create the injection list” on page 74. Quick Start a run Load the plate in the instrument before proceeding (see “Load the plate in the instrument” on page 72). You can start a run in the Dashboard by selecting a plate with plate contents already assigned. 1. In the Dashboard, click Quick Start Run to display the Select Plate from Library dialog box. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 73 Chapter 4 Set up and run 4 Load plates for run and create the injection list 2. Select a plate, then click Load Plate. 3. Click Start Run from the Load Plates for Run screen. Note: If an install check for the run application type (Sequencing or Fragment) has not been performed, a message is displayed and the run does not start. Load plates for run and create the injection list Load the plate in the instrument (see “Load the plate in the instrument” on page 72) and link the plate (“Link the plate” on page 72) before proceeding. 1. Access the Load Plates for Run screen (Figure 11) from: • The Assign Plate Contents screen by clicking Link Plate for Run. • The navigation pane by selecting Load Plates for Run in the navigation pane. • The Dashboard by clicking the Workflow tab, then selecting Load Plates for Run in the navigation pane. 2. Review the consumables information and the calibration information and ensure the status is acceptable for a run. 3. Enter a Run Name or use the default run name: <Start Instrument Run Date/Time Stamp> YYYY- MM-DD-hh-mm-ss-SSS (milliseconds), for example, “Run 2014-06-10-17-33-46-522” where the run start date is February 5 2009 and the run start time is 15:03:42:096. Note: An instrument run begins when you click Start Run (on the Load Plates for Run screen) and ends when the last injection on the last plate has completed. For example, if you link two plates, then start the run, both plates and any duplicate injections or re-injections are part of the same instrument run. An injection is an instance of 8 or 24 samples (depending on instrument configuration) processed simultaneously under the same conditions. 74 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Load plates for run and create the injection list 4 When you access the Load Plates for Run screen by clicking Load Plates for Run on the Assign Plate Contents screen, the plate is automatically linked (indicated by the active Unlink button). Figure 11 Load Plates for Run 4. If needed, click Unlink, then follow the steps in “Link a plate (if a plate is not linked)” on page 76. 5. As needed, click Switch Plates ( ) to assign the plate to the other position in the autosampler. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 75 Chapter 4 Set up and run 4 Load plates for run and create the injection list 6. Click either of the following: • Create Injection List—Displays the Preview Run screen where you can modify the injection list before starting the run. Go to “Review, modify, or export the injection list in Preview Run” on page 78. • Start Run—Displays the Monitor Run screen. Go to “Monitor the run” on page 80. Note: If an install check for the run application type (Sequencing or Fragment) has not been performed, a message is displayed and the run does not start. Link a plate (if a plate is not linked) If you access the Load Plates for Run screen from the navigation pane, a plate may not be linked (indicated by the active Link Plate button). To link a plate: 76 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Load plates for run and create the injection list 4 1. Click Link Plate to display the Select Plate from Library dialog box. 2. Select a plate, then click Link Plate. 3. Do either of the following: • Click Create Injection List, then go to “Review, modify, or export the injection list in Preview Run” on page 78 • Click Start Run, then go to “Monitor the run” on page 80 Note: If an install check for the run application type (Sequencing or Fragment) has not been performed, a message is displayed and the run does not start. Link a plate from the Recent Plates or Recent Runs tab Instead of clicking Link Plate to select a plate, you can click-drag a plate from the Recent Plates tab (pending plates) or the Recent Runs tab (processed plates). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 77 Chapter 4 Set up and run 4 Review, modify, or export the injection list in Preview Run Review, modify, or export the injection list in Preview Run The Preview Run screen allows you to modify the injection list before you start the run. 1. Access the Preview Run screen (Figure 12) from: • The Load Plates for Run screen by clicking Create Injection List. • The navigation pane by selecting Preview Run. • The Dashboard by clicking the Workflow tab, then selecting Preview Run in the navigation pane. Figure 12 Preview Run screen 78 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Start the run from Preview Run 4 2. Click the icon above the plate to specify the attributes to display in the plate view. 3. Click the Plate tabs to display Plate A or Plate B. The Preview Run screen contains an injection list and a plate view. The injection list is linked to the plate view. Click an injection to select the associated wells in the plate view. IMPORTANT! If the injection list is blank, make sure that you clicked Create Injection List on the Load Plates for Run screen. 4. To modify the injection list at any time before a run or during a run, select an injection, then click Move Up, Move Down, and Delete as needed. 5. To specify a duplicate injection (a replicate injection that uses the same instrument protocol as the original injection), select an injection, then click . Sample data files for each duplicate injection can be saved in a separate folder in the results group folder if specified in the results group. 6. To export the injection list, click Export. Start the run from Preview Run When the injection list is configured, click Start Run. The Monitor Run screen is automatically displayed. IMPORTANT! You must re-inject samples before the run completes unless the Pause after last injection preference is set. Note: If an install check for the run application type (Sequencing or Fragment) has not been performed, a message is displayed and the run does not start. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 79 Chapter 4 Set up and run 4 Export the injection list from Preview Run Export the injection list from Preview Run The injection list can be exported in a CSV, XLS, or TXT export file. The export file lists samples in the order in which they are displayed on the screen. 1. Select Preview Run in the left pane. 2. Click Export. Monitor the run The Monitor Run screen (Figure 13) is automatically displayed when you click Start Run in the Load Plates for Run screen or the Preview Run screen. The current injection is highlighted in green in the plate view. The injection list is linked to the plate view. Click an injection to select the associated wells in the plate view. A selected injection is highlighted in green in the plate view. Figure 13 Monitor Run screen 1. Click the Table Settings button, then specify the columns to show or hide in the injection list. 80 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Monitor the run 4 2. (Optional) Specify the attributes and/or display sample details: • Click the icon above the plate to specify the attributes to display in the plate view. In addition to the attributes available in Preview Run, a Flag attribute is available. If you select the Flag attribute, green marks are displayed for wells with Average QV values that are within range, yellow marks are displayed for wells with Average QV values that are in the suspect range, and red marks are displayed for wells with Average QV values that are out of range. • Place the mouse pointer over a well to display sample details. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 81 Chapter 4 Set up and run 4 Pause a run and load a new plate (flexible plate loading) Pause a run and load a new plate (flexible plate loading) The software allows you to load an additional plate in the instrument at any time during a run, and move injections from the new plate to the top of the injection list. 1. In the Workflow tab, click Monitor Run in the left pane. 2. In the Monitor Run screen, click Pause Run. 3. Click OK to accept the message that indicates that the instrument will pause after completing the current injection. 4. When the run pauses, click Load Plate for Run in the left pane. 5. If both plate positions are filled in the Load Plate for Run screen, click Unlink for one of the positions, then remove the plate. 6. Install the new plate, click Link Plate, then select the plate you want to load. 7. At the bottom of the Load Plate for Run screen, click Create Injection List. 8. Click Monitor Run in the left pane. 9. As needed, change the injection order in the Monitor Run screen. You can move injections from the newly loaded plate up in the injection list to inject from the newly loaded plate before the first plate has finished running. 10. Click Resume Run. 82 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Check sequence or sample quality and re-inject samples 4 Check sequence or sample quality and re-inject samples When an injection is complete, it is flagged with in the Injection and Analysis columns. If the software detects a problem with offscale data or low quality samples, the injection is also flagged with . Note: If the Injection, Analysis, or Flag columns are not displayed, you can click the Table Settings button, then show them in the injection list. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 83 Chapter 4 Set up and run 4 Check sequence or sample quality and re-inject samples Check sequence or sample quality 1. Expand the Flag pane at the bottom right of the screen. The flag table displays a quick preview of sample quality and identifies samples that may need investigation. The flag table is linked to the plate view. 84 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Check sequence or sample quality and re-inject samples 4 2. Click a flag to select the associated well in the plate view: Note: If no samples are listed in this pane, no flags were found and the samples have passed quality checks. • All samples passed • At least one sample is in the suspect range and requires review • At least one sample is offscale or is in the fail range 3. To filter the flag table, select a flag type. To sort the table, double-click column headers. The flags you may see in the flag table are: Flag/Symbols Description Offscale (red) At least one data point in the analysis range has saturated the CCD camera. (green or red) Note: In the View Results screen, an offscale sample is flagged with . QV: Average Quality Value (yellow) or (red) The Average Quality Value (based on CRL, (sequencing) Trace Score, and QV20+ results) is in the Suspect or Fail range. For information, see “Basecalling protocol—QV settings” on page 205. (green, yellow, red) SQ: Sizing Quality (fragment) (yellow) or (red) The Sizing Quality is in the Suspect or Fail range. For information, see “Sizecalling protocol—QC settings” on (green, yellow, red) page 211. IMPORTANT! Normalization is not applied to samples with (red) Sizing Quality. 4. Click a row in the flag table, then click the Sample tab in Instrument Run Views to display the associated data in the Sample view. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 85 Chapter 4 Set up and run 4 Check sequence or sample quality and re-inject samples Re‑inject samples from the Monitor Run screen A re-injection physically re-injects all samples in the capillary array. You can specify whether to collect data for all or only selected samples in the array. By default, you can specify a re-injection before the run completes. To allow re-injections after a run is complete, set the Pause After Last Injection preference (see “User preferences” on page 44). 1. Select the injections or wells to re-inject: Note: Re-inject is dimmed if you select an injection that contains more than one results group, or if you select flags in the flags table that correspond to samples with different results groups. To enable Re-inject, select samples that specify the same results group. To collect data for all wells in an 1. Select the injection in the injection list. injection 2. Click Re-inject. To collect data for only specific 1. Select the injection. wells 2. Select in the array view the capillary that corresponds to the Note: You can also re-inject well or sample of interest (see “Array view” on page 99). specific samples in Review 3. Click Re-inject. Results. To collect data for only samples 1. Select the samples in the flag table (see “Check sequence that contain flags or sample quality” on page 84). 2. Click Re-inject. Note: If you are running an HID plate, see “Re-inject HID allelic ladder samples” on page 88. 2. In the Re-injection dialog box, select options, then click OK. Note: Sample data files for each re-injection can be saved in a separate folder in the results group folder if specified in the results group. 86 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Check sequence or sample quality and re-inject samples 4 If you select a protocol other than the original If you select a protocol other than the original, the software: • Creates a copy of the assay specified for the re-injected well (Original_Assay-1). • Adds the new or modified instrument protocol to Original_Assay-1. • Assigns Original_Assay-1 to the re-injected well only. • Saves the plate (the software does not save the copy of the assay to the library). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 87 Chapter 4 Set up and run 4 Check sequence or sample quality and re-inject samples How re-injections are displayed in the plate view If the Injection Number attribute is selected for display in the plate view, the number of the original injection and the re-injection are shown. 1 2 1 Sample 1 selected for re-injection 2 Re-injection number listed for all samples in the re-injection Note: If you select only specific wells for the re-injection (which physically re-injects all samples for the capillary array but collects data only for the selected wells), the re-injection number is displayed for all samples in the re-injection, not just the samples selected for data collection. Re-inject HID allelic ladder samples If you select to re-inject a sample that includes an allelic ladder in its results group, but the allelic ladder is not part of the injection, the software prompts you to select one or more allelic ladder samples to re-inject. For example: • You are running an 8-capillary instrument, and you have specified one results group for each set of three injections (for more information, see “Results Group example 3: store one allelic ladder per run folder (8‑capillary instruments)” on page 188) • The allelic ladder sample is in Injection 1. • You select for re-injection a sample that is in injection 2. 88 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run Check sequence or sample quality and re-inject samples 4 • The software prompts you to select one or more allelic ladder samples to re-inject. The allelic ladders available to select are from the same plate and within the same results group as the original injection. If the results group does not contain an allelic ladder sample, the software does not prompt you to select one for re-injection. Complete the following actions in the Add Allelic Ladder to Re-injection dialog box: 1. Select one or more allelic ladder samples. IMPORTANT! The software does not display the well location of allelic ladder samples in this dialog box. To identify allelic ladder samples for re-injection, include the well position in the allelic ladder sample name when you assign plate contents. 2. Select whether to collect data for the remaining samples in the allelic ladder re-injection. 3. Select whether to apply a modified instrument protocol to the allelic ladder re-injections, or whether to use the original instrument protocol for the allelic ladder re-injection(s). You will select the modified protocol in the next screen. IMPORTANT! Allelic ladders that are injected under the same conditions are recommended to accurately genotype samples in the secondary analysis software (GeneMapper™ ID-X Software v1.3 or later). 4. Click OK. 5. Specify the remaining re-injection settings as described in “Re‑inject samples from the Monitor Run screen” on page 86. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 89 Chapter 4 Set up and run 4 Review completed injections in Review Results Review completed injections in Review Results You can review results for any completed injections. Select the injection, then click Review Results. The samples for the injection are loaded in the Samples Table in Review Results. For more information, see Chapter 5, “Review sequencing results”. Pause, resume, or stop a run Pause and resume As needed, click: • Pause—Pauses the run after the current injection completes (the symbol is not displayed in the injection list because the injection continues to completion). • Resume—Resumes the run. Abort or terminate As needed, click: • Abort—Immediately aborts the current injection and pauses the instrument run. You can resume the run or terminate the injection list. Do not click Delete to stop an injection. IMPORTANT! You can stop the current injection only when the front panel indicator is blinking green. If you click Abort when the front panel indicator is solid green, the physical injection is already completed (although the software is still processing the information) and a message is displayed indicating that there is no injection in process. • Terminate injection list—Stops the instrument run. Terminate is active only after you click Pause or Abort. 90 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run More features in Assign Plate Contents 4 More features in Assign Plate Contents Name samples in the plate view To name samples in the Plate View: To name one • Click a well, then type a sample name directly into the field, then press Enter. sample or • Copy and paste a name from another well. To set the direction for the cursor when you press Enter: Click to set the Enter key to move the cursor vertically to the next row. Click to set the Enter key to move the cursor horizontally to the next column. To name • Click a named well. multiple • Click-drag multiple wells. samples • Right-click and select Fill or Fill Series to populate the selected fields. Note: To use Fill Series, type a number as the last character of the named well). You can also copy and paste sample names. To name all • Select all wells. wells at one • Select assays, file name conventions, and results group for the plate. time • Enter name and select sample type (in the Customize Sample Info pane) for the whole plate. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 91 Chapter 4 Set up and run 4 More features in Assign Plate Contents Customize the plate view 1. Click Show In Wells to specify the attributes to display in wells. 2. Click Select Wells to select wells with a specific attribute. 3. Click Zoom In, Zoom Out, and Fit as needed. 92 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run More features in Assign Plate Contents 4 View the capillary-to-plate map Click Array Selection to select wells by injection. Click again to turn off array selection. Use the table view 1. Click Table View. 2. Click the Sample Name field, then type a name. 3. 3. Click next to each field, then select a setting. 4. Right-click a column header, then select Fill or Fill Series to populate the selected fields (to use Fill Series, type a number as the last character of the named well). Note: You can double-click column headers to sort columns. Multi-column sorting is supported (see “Sort by one or multiple columns” on page 94 below). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 93 Chapter 4 Set up and run 4 More features in Assign Plate Contents Sort by one or multiple columns Double-click column headers to sort. To sort by multiple columns: • Double-click a column header to sort the column. • Alt+Shift-click another column header to sort another column. • Alt+Shift-click a third column header to sort a third column. Numbers in the column headers reflect sort order. Customize a table Click the Table Settings button, then specify the columns to show or hide. Click: • Apply—To use the settings for this session only. • Save to Preferences—To save for future use by all users. Preferences are saved for the logged-in user. • Restore Defaults—To restore factory-default settings. 94 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run More features in Assign Plate Contents 4 Add assays, file name conventions, and results groups to a plate To add an assay, file name convention, or results group from the library, click Add from Library at the bottom of the Assign Plate Contents screen. Create a plate import template The software allows you to import plate information from files that you create in another application. To create a template for importing plate information, set up a plate in the software and then export it to create a file that contains the correct header and column information for importing: 1. In the Dashboard, click Create Plate from Template. 2. In the Open Plate Template from Library dialog box: a. Select a filter to display the plate template type of interest. b. Select a plate template, then click Open. 3. Enter a name for the plate, then specify the capillary length and polymer type for the plate. 4. Click Assign Plate Contents. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 95 Chapter 4 Set up and run 4 More features in Assign Plate Contents 5. In the Assign Plate Contents screen, click Export. Note: Before you click Export, you can assign other plate elements to the plate import template as described in “Assign plate contents” on page 61. 6. Select a file type for the plate import template. 7. Enter a name and location for the plate record template. 8. Click Save. The figure below shows the format of the exported plate. Create a plate import file 1. Open a plate import template (see “Create a plate import template” on page 95). 2. Save the plate import template under a new name. 3. Enter sample names (required). 96 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run More features in Assign Plate Contents 4 4. (Optional) Enter information for the remaining columns. Note: If you specify assay, results group, or file name convention names, the names you enter must exactly match the names of existing items in the library. IMPORTANT! If you copy/paste sample or plate information into the Assign Plate Contents screen or into a plate import file, copy from a plain text editor such as Notepad. Do not copy from a word processing program such as Microsoft™ Word™, which may include invisible, non-ASCII characters. Non-ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting. 5. Save the plate import file. Edit a plate You can edit a plate from: • Library—Select a plate, then click Edit. • Dashboard—Click Edit Existing Plate. • Define Plate Properties screen—Select Open Plate4Edit Existing Plate. • Assign Plate Contents screen—Select Open Plate4Edit Existing Plate. Import and export a plate You can import and export plates from: • Plates library—Plates in .xml format for use on another 3500 instrument. See “Import and export a library entry” on page 165. • Define plate properties— Plates in TXT, CSV, and XLS format—Files you create that contain plate information in a specific format. • Assign Plate Contents— Plates in TXT, CSV, and XLS format—Files you create that contain plate information in a specific format. Create a plate template A plate template contains default settings that you can edit when you create a plate from the template. 1. Create a plate (see “Create a new plate” on page 167). 2. (Optional) Add sample names and sample types (see “Name samples and assign sample types in the plate view” on page 63). 3. (Optional) Add the assays, file name conventions, and results groups appropriate for this plate template’s application (see “Add assays, file name conventions, and results groups to a plate” on page 95). Adding assays, file name conventions, and results groups to the plate template automatically displays these items in the Assign Plate Contents screen when you open the plate template. You do not have to add these items from the library for each plate you create. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 97 Chapter 4 Set up and run 4 More features in Monitor Run 4. (Optional) Click Show In Wells to specify the attributes to display in wells in the template. 5. Select Save Plate4Save As Template. The software displays the template icon below the plate layout. Specify the default plate type for the Open Plate dialog box Specify the default plate type for the Open Plate dialog box in Preferences. Save electronic version of reports When you print any report, you can select a PDF printer to save the report to PDF. More features in Monitor Run Review the Instrument Run views Select an injection, then click an instrument run view tab. As needed: • Click to zoom in and out. • Click to detach a view and display it in a separate window that you can move around on the screen. To locate a detached view, click the 3500 task bar icon. 98 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 4 Set up and run More features in Monitor Run 4 Array view The Array view shows the color data (based on the dominant fluorescence color) for each capillary as a function of instrument scan number (time). Adjust the brightness and color by using the slider bars above the view. Sample view The Sample view shows the relative dye concentrations as a function of instrument scan number (time) for the selected capillary. You can select and deselect the dye colors to display. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 99 Chapter 4 Set up and run 4 More features in Monitor Run EPT view The EPT view (ElectroPhoresis Telemetry) shows instrument data conditions (laser power, temperatures, electrophoresis voltage) as a function of time. In the legend to the right of the EPT view, you can select and deselect the traces to display in the view. You can view the EPT plot for completed or terminated runs. 100 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 5 Review sequencing results ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 ■ Access the View Sequencing Results screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 ■ Review results for the currently running sequencing plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 ■ Review previously run sequencing samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 ■ Review sequence quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 ■ Review traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 ■ Understand Quality Values (QVs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 ■ Re‑inject samples from Review sequencing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 ■ View, print, and save (PDF) trace quality reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 ■ Export sequencing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 ■ Modify sequence data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Workflow Review sequencing results Review sequence quality (page 104) Re‑inject samples from Review sequencing results (page 109) View, print, and save (PDF) trace quality reports (page 110) Export sequencing results (page 111) 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 101 Chapter 5 Review sequencing results 5 Access the View Sequencing Results screen Access the View Sequencing Results screen Access the View Sequencing Results screen from: • The Monitor Run screen by clicking Review Results. • The navigation pane by selecting View Sequencing Results. • The Dashboard by clicking View Run Results. Review results for the currently running sequencing plate To view results for completed injections in the current run while a run is in progress: 1. Navigate to View Sequencing Results4Trace Quality View. 2. Select one or more samples, then click Open Trace to display the data in the Trace pane. Note: The basecaller version listed in the basecalling protocol is limited to a 3‑digit number. The version listed in sequencing results is a 4-digit number. The fourth digit is an internal number used by the software. 102 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 5 Review sequencing results Review previously run sequencing samples 5 Review previously run sequencing samples If you access the View Sequencing Results screen when no run is in progress and no plate is linked, no samples are listed. (If the plate from the most recent run is linked, the results from that plate are displayed.) To view results for samples other than those from the most recent run, click Import, then select the samples to review. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 103 Chapter 5 Review sequencing results 5 Review sequence quality Review sequence quality 1. Display Metric Analysis results to review sample basecalling and trimming results. 2. Click the Table Settings button, then specify the columns to show or hide. 3. Double‐click column headers to sort columns. Multi‐column sorting is supported (see “Sort by one or multiple columns” on page 94). 104 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 5 Review sequencing results Review sequence quality 5 4. Review the results: Result Description Trace The average basecall Quality Value (QV) of basecalls in the clear range sequence of a Score trace. The clear range is the region of the sequence that remains after excluding the low-quality or error-prone sequence at the 5′ and 3′ ends. The clear range is calculated by the KB Basecaller using QVs. CRL The longest uninterrupted segment of basecalls with an average Quality Value (QV) ≥20. In addition to evaluating the QV of a basecall, the software considers the QV of adjacent basecalls within a 21-bp moving window to determine a contiguous read length based on quality values: the software starts from the 5' end and calculates the average QV across a moving window size of 21, sliding 1 bp at a time, to the 3' end. The resulting longest contiguous segment is determined as the CRL. QV20+ The total number of bases in the entire trace with Quality Value ≥20. Trace Pass/fail/check determined by the settings in the Basecalling protocol QV Settings tab. Score Quality CRL Quality QV20 Quality PUP Score A measure of noise or pull-up that is determined by taking the mean of the ratios of signal strength calculated for each basecalled peak: primary peak/secondary peak under the primary peak. A higher value indicates less baseline or secondary noise. A lower value indicates an elevated baseline or secondary noise. Example 1: Main called base signal strength is 1,000 RFU and the largest secondary peak beneath it is 10 RFU; PUP=100 Example 2: Main called base signal strength is 1,000 RFU and the largest secondary peak beneath it is 100 RFU; PUP=10 5. Review warnings: a. Scroll to the right of the Metric Analysis table to display the Warning column. b. Display the Analysis Status legend. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 105 Chapter 5 Review sequencing results 5 Review traces c. Review warnings: Result Description Success Basecalling and trimming successful. Success with warning Basecalling successful, trimming not successful. Warning messages are listed in the Warning/Error Message column (default position is the last column in the table). Fail Basecalling and trimming failed, no results generated. Error Basecalling and trimming failed due to internal software error, no results generated. Unclassified No analysis performed. 6. (Optional) Click Minimize and Restore to collapse and expand the samples table. Review traces 1. Select the samples of interest in the samples table, then click Open Trace. 2. Select items from the trace toolbar to manipulate the trace as needed. Place the mouse pointer over a button for the description of the button. 3. (Optional) Modify trace display: • Use the Tile Viewer options to display up to four traces at a time. • Set trace colors in Preferences (see “Overview” on page 41). 106 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 5 Review sequencing results Review traces 5 4. Set the category of base for the Tab key. Place mouse pointer on QV barbar to display QV value 5. Review traces: press Tab to review bases from left to right in a trace. Press Shift+Tab to move right to left. Bases QV bar Place mouse pointer Mixed base in trace to display QV 6. Click the tabs at the bottom of the trace pane for different views of the data. Display thumbnails Viewing options Peak Place mouse pointer Move slider to (analyzed data) in trace to zoom 1. Click the View Thumbnails button to display results as thumbnails. scale vertically 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 107 Chapter 5 Review sequencing results 5 Understand Quality Values (QVs) 2. Sort as needed. 3. To compare signal across all samples on a plate, select Uniform Y Scaling. 4. Click the View Tables button to close the thumbnail pane. Understand Quality Values (QVs) Quality value ranges We recommend the following ranges for QVs (set in Preferences, see “Overview” on page 41): • Pure bases: Low QV < 15, Medium QV = 15 to 19, High QV = 20+ (default). • Mixed bases: Low QV < 5, Medium QV = 5 to 10, High QV > 10 (investigate to determine the best range for your application). Pure base versus mixed base QVs Pure bases and mixed bases have the same probability of error for the associated basecall (10–q/10). Note the following: • High-quality pure bases typically have QVs of 20 or higher. • The distribution of quality values for mixed bases differs dramatically from that of pure bases. • Mixed bases have a maximum QV of 20. • Review all mixed base calls. 108 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 5 Review sequencing results Re‑inject samples from Review sequencing results 5 Quality values (QV) and probability of error (Pe) QV Pe QV Pe 1 79.0% 30 0.10% 5 32.0% 35 0.032% 10 10.0% 40 0.010% 15 3.2% 45 0.0032% 20 1.0% 50 0.0010% 25 0.32% 60 0.00010% Re‑inject samples from Review sequencing results Before the run completes, select a sample with suspect or failing flags, then click Re-inject. For information on making a re-injection before a run completes, see “Re‑inject samples from the Monitor Run screen” on page 86. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 109 Chapter 5 Review sequencing results 5 View, print, and save (PDF) trace quality reports View, print, and save (PDF) trace quality reports View Trace Reports Note: You can set defaults for the reports in Preferences (see “Set preferences (optional)” on page 41). 1. Click View Trace Reports, then select a report type to view and print. 2. (Optional) Modify report settings. You can specify additional report settings in Preferences (see “Set preferences (optional)” on page 41). 3. Double-click different elements in the QC report to open the Trace view and display the associated sample. 4. To print the report, click Print, then preview or print. 5. To save the report electronically (PDF), print the report and select a PDF printer. 110 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 5 Review sequencing results Export sequencing results 5 Report options • QC—One-page bar chart that shows trace score statistics and results for each selected sample. • Plate—One-page per plate for all selected samples that shows the well-location thumbnail raw data traces with color-coded headers that reflect Trace Score quality. • Trace Score, CRL, and QV20+—One-page bar chart that shows trace score, CRL, or QV20+ statistics and results for each selected sample. • CRL Distribution—One-page bar chart that shows CRL statistics and CRL results distribution for all selected samples. • Signal Strength—One-page graph that shows the average sequencing dye signal strength for all selected samples. Export sequencing results 1. Filter the table of interest. 2. Select an export option: Results, Reports, or Traces. 3. Select the export options and the location for the export file, then click OK. The file(s) are exported to the specified location with the following naming conventions: • Results—export_ReportName.txt • Reports—ReportName.* (* is the format you selected: .txt, .xls, .pdf, or .html) • Traces—FileName.* (* is the format you selected: .annotation.txt, .phd.1, .scf, .fsta, .qual, or .seq) Modify sequence data To edit, modify, or further analyze sequence data, import the sample data files into a secondary analysis software application such as SeqScape™ Software 3 (or later), MicroSEQ™ ID Analysis Software v3.0 (or later), Variant Reporter™ Software 2 (or later), and Sequence Analysis (SeqA) Software 6 (or later). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 111 6 Review fragment/HID analysis results ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 ■ Access the View Fragment/HID Results screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 ■ Export the injection list from Samples view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 ■ Review results for the currently running fragment/HID analysis plate . . . . . . . . . . . . . . . . . . . . . . . 114 ■ Review previously run fragment analysis/HID samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 ■ Review sample quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 ■ Review normalized data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 ■ Review plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 ■ Review sizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 ■ Re-inject samples from Review fragment results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 ■ View, print, and save (PDF) sample quality reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 ■ Export sizing results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 ■ Modify fragment analysis or HID data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Workflow Review analysis results Review sample quality (page 115) Re-inject samples from Review fragment results (page 122) View, print, and save (PDF) sample quality reports (page 122) Export sizing results (page 123) 112 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 6 Review fragment/HID analysis results Access the View Fragment/HID Results screen 6 Access the View Fragment/HID Results screen Access the View Fragment Results screen from: • The Monitor Run screen by clicking Review Results. • The navigation pane by clicking View Fragment Results. • The Dashboard by clicking View Run Results. Export the injection list from Samples view The injection list can be exported in a CSV, XLS, or TXT export file. The export file lists samples in the order in which they are displayed on the screen. 1. Select View Fragment/HID results in the left pane. 2. Create a table setting that includes Injection Start Date column. 3. Sort the table by sample file name, then by Injection Start Date column (which also includes the time of the injection). 4. Click Export. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 113 Chapter 6 Review fragment/HID analysis results 6 Review results for the currently running fragment/HID analysis plate Review results for the currently running fragment/HID analysis plate If you access the View Fragment Results screen while an instrument run is in progress, the samples table lists results for completed injections in the current run. Select one or more samples in the samples table to display their data in the plot view and sizing table view. 114 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 6 Review fragment/HID analysis results Review previously run fragment analysis/HID samples 6 Review previously run fragment analysis/HID samples If you access the Results screen when no run is in progress and no plate is linked, no samples are listed. (If the plate from the most recent run is linked, the results from that plate are displayed.) To view results for samples other than those from the most recent run, click , then select the samples to review. By default, the Fragment Samples view is selected. If you are importing HID files, click HID Samples. Review sample quality 1. In the samples view, click the Table Settings button, then specify the columns to show or hide. 2. Double-click Offscale, Pull-Up (fragment), Broad Peak (HID), and SQ columns to sort suspect and failing flags to the top of the table. Multi-column sorting is supported (see “Sort by one or multiple columns” on page 94). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 115 Chapter 6 Review fragment/HID analysis results 6 Review sample quality Flag/ Symbols Description Normalization Limit • —Sample was collected with a normalization size standard, SS Norm Factor is within range. —Sample was collected with a normalization size standard, sample SS Norm Factor is not within range. No Data—Normalization is enabled, but Sizing Quality is . NO—Sample was not collected with a normalization size standard. N/A—Sample was not collected on a 3500 instrument. For more information, see “Review normalized data” on page 117. Note: If the Sizing Quality is , normalization is not applied, even if the SS Norm Factor is within the normalization range. SS Norm Factor The size standard normalization factor that is applied to all peaks. See “Size standard normalization feature” on page 331. This factor is saved in the data file and applied during GeneMapper™ ID‑X Software analysis if normalization is enabled in the GeneMapper™ ID‑X Software Analysis Method►Peak Detector tab. Avg Normalization The averaged peak height of the size standard peaks that were used to calculate the size PH standard normalization factor. Offscale At least one data point in the analysis range has saturated the CCD camera. Note: In the Monitor Run screen, an offscale sample is flagged with . Spectral Pull-Up At least one peak contains a pull-up peak. (fragment analysis A pull-up peak is identified when the peak height of the minor peak is £X% of and within ±Y only) data point of the major peak, where X and Y are values you specify. Broad Peak (HID At least one peak exceeds the Broad Peak threshold. analysis only) Broad peaks affect Sizing Quality. See Chapter 8, “Manage library resources”. Note: The value displayed when you place the mouse pointer over a Broad Peak flag is an internal value and does not reflect the peak width. SQ: Sizing Quality The Sizing Quality is in the Fail or Suspect range. Place the mouse pointer over a flag to display the Sizing Quality value for the sample. 3. Click a flag in the samples table, or select samples in the samples table to display the associated data in the plot view and sizing table view. 4. (Optional) Modify the sample view: • Right-click the Size Standard field to view the size standard for a sample. • Click Minimize and Restore to collapse and expand the samples table. 116 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 6 Review fragment/HID analysis results Review normalized data 6 Review normalized data Normalization corrects for instrument, capillary, and injection variability. When specified in the primary analysis protocol, the software calculates a size standard normalization factor for each sample. The size standard normalization factor is used as a multiplier to adjust the peak height of the sample peaks relative to the size standard peaks. A sample is normalized if it is collected with a normalization size standard (specified in the primary analysis protocol [sizecalling or QC] in the assay). Note: If the Sizing Quality is , normalization is not applied, even if the size standard normalization factor is within the normalization limits set in the instrument protocol. Ensure that you use the normalization size standard appropriate for your application. How normalization is applied To normalize, the software: 1. Determines if the data was collected on a 3500/3500xL Genetic Analyzer. 2. Determines if the sample was collected with a normalization size standard definition file (normalization is enabled). 3. If normalization is enabled, the software calculates a size standard normalization factor for the sample using multiple size standard fragments. The size standard normalization factor is calculated by dividing the Normalization Target from the instrument protocol by the observed average peak height of the size standard fragments in the samples. 4. Compares the size standard normalization factor to the thresholds (set in the instrument protocol). 5. If the calculated size standard normalization factor is within the normalization factor range, multiplies the peak heights of the sample by the calculated size standard normalization factor. 6. If the calculated size standard normalization factor is outside the normalization factor range, multiplies the peak heights of the sample by the maximum or minimum normalization factor threshold setting. For example, if the normalization factor range is 0.3 to 3.0 and the calculated size standard normalization factor is 5, the software applies a size standard normalization factor of 3.0. 7. Displays normalization information in the Samples view. Normalization factor in secondary analysis If normalization is applied in the 3500 Series Data Collection Software v3.3, the calculated size standard normalization factor is stored with the raw data and is applied to the raw data in the GeneMapper™ Software 5 and the GeneMapper™ ID‑X Software v1.2 or later secondary analysis software. You can turn normalization off and on in the analysis method used in the GeneMapper™ Software 5 and GeneMapper™ ID‑X Software v1.2 or later secondary analysis software. If normalization is not applied in the 3500 Series Data Collection Software v3.3 (either a normalization size standard was not used, or sizing failed ), normalization cannot be applied in the secondary analysis software. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 117 Chapter 6 Review fragment/HID analysis results 6 Review plots Review plots 1. Select the samples of interest in the samples table. 2. Select items from the plot toolbar to manipulate the plot as needed. Place the mouse pointer over a button for the description of the button. IMPORTANT! If you first view a 4-dye sample, then view a 5-dye sample, you must manually select the fifth dye. It is not automatically selected when you switch to a 5-dye sample. 3. Click to apply scaling settings to plots: Enter the range for Y axis and X axis, then click the Zoom buttons. IMPORTANT! You must open Plot Settings each time you access the View Results screen, then click Zoom. Scaling settings are not automatically applied when you access this screen, or when you click Apply. To apply scaling settings to all samples in the samples table, select all of the samples in the samples table to display them in the plot view, specify the scaling settings, click Zoom, then click Page Up and Page Down in the plot view to move through the samples. If the button is dimmed, the Plot Settings dialog is open. Click the 3500 task bar icon, then select Plot Settings. 4. Display multiple plots as needed: In the Plot Settings Display tab, select Checkerboard. 5. Click a peak to label it (to label all peaks, see “Label peaks” on page 119). Zoom on data 1. Place the pointer above the top of the plot or to the left of the plot at the start of the area you want to zoom, then click to turn the pointer to . 2. With the still above the plot or to the left of the plot, click-drag to the end of the area you want to zoom. Do not drag the inside the plot area. Doing so changes back to a pointer and does not zoom as expected. You can also click zoom and fit buttons to zoom . Change plot settings Click (Plot Settings) in the plot view toolbar. For information on plot settings, click in the plot settings tabs. If the button is dimmed, the Plot Settings dialog is open. Click the 3500 task bar icon, then select Plot Settings. 118 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 6 Review fragment/HID analysis results Review plots 6 Overlay samples 1. Select samples in the samples view to display the plots. 2. Click Overlay All. When Combine Dyes is selected, the plot view displays one plot with all samples and all dyes. When Separate Dyes is selected, the plot view displays one plot per dye. Each dye plot contains all samples. Label peaks 1. Select samples in the samples view to display the plots. 2. Click (Plot Settings) in the plot view toolbar, then select the Labels tab. 3. Label peaks: If you have ... Then ... Already specified 1. Select Show Peak Labels. default labeling 2. Click Label Peaks. preferences 3. Click Apply. IMPORTANT! You must open Plot Settings each time you access the View Results screen, then click Label Peaks. Labeling settings are not automatically applied when you access View Results, or when you click Apply. Not specified 1. In Labels to Show, select the needed labels. default labeling 2. In Labeling Options: preferences • Select Show Peak Labels. • To label all peaks with the selected labels, click Label Peaks (make sure All is selected). • To label selected peaks, select the category from the Label Peaks list (Height, Area, Size), specify the range to label for the selected category (for example, if you select Height, specify the height range of the peaks to label), then click Label Peaks. • Select Retain Labels. 3. Click Save to Preferences to save these settings for future use. You can change preferences at any time. 4. Click Apply. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 119 Chapter 6 Review fragment/HID analysis results 6 Review plots View thumbnails Click the View Thumbnails button to display the traces for the samples selected in the samples view and the dyes selected in the plot view. Rename samples To rename sample files: 1. In the Sample Name column, select the samples to rename, or click the Sample Name column header to select the entire column. 2. Click Rename. 3. In the Search field, enter the sample name to change. 4. In the Rename field, enter the new name. 5. Click Search, then click Rename. 120 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 6 Review fragment/HID analysis results Review sizing 6 Sort by one or multiple columns Double-click column headers to sort. To sort by multiple columns: • Double-click a column header to sort the column. • Alt+Shift-click another column header to sort another column. • Alt+Shift-click a third column header to sort a third column. Numbers in the column headers reflect sort order. Review sizing The Sizing Table View displays: • For fragment samples—All dyes • For HID samples—Size standard dye only (orange or red) Set up the sizing table 1. Select samples in the samples table to display the plots. 2. In the sizing table, click the Table Settings button, then specify the columns to show or hide. 3. Filter the table as needed. 4. Double-click column headers to sort columns. Multicolumn sorting is supported (see “Sort by one or multiple columns” on page 94). 5. Selecting rows in the sizing table, then click Label Selected Peaks. Examine the size standard plot 1. In the plot view toolbar, deselect all dye colors except the size standard dye color (red or orange). 2. In the sizing table, select the size standard peaks of interest. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 121 Chapter 6 Review fragment/HID analysis results 6 Re-inject samples from Review fragment results 3. Click Label Selected Peaks to label the size standard peaks in the plot view. Note: If labels are not displayed, click (Plot Settings) in the plot view toolbar, then select Show Labels in the Labels tab. Click Save to Preferences to retain this setting. 4. Ensure that all size standard peaks are present and correctly labeled. Overlay the sizing curve 1. Click (Plot Settings) in the plot view toolbar. 2. Select Overlay Sizing Curve in the Display tab. Re-inject samples from Review fragment results Before the run completes, select a sample with suspect or failing flags, then click Re-inject. For information on making a re-injection before a run completes, see “Re‑inject samples from the Monitor Run screen” on page 86. View, print, and save (PDF) sample quality reports 1. Select the samples of interest in the samples table. 2. Click Reports, then select a report type to view and print. Reports are displayed in the sizing table view at the bottom of the screen. Reports are displayed in the sizing table view at the bottom of the screen. 3. (Optional) Modify report settings. 4. To print the report, click Print, then preview or print. 122 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 6 Review fragment/HID analysis results Export sizing results 6 Report options • Sizing—One page per selected sample that shows the quality ranges set in the sizecalling or QC protocol, the quality values for the sample, and the electropherogram for the sample. Plot zooming is not retained in the report. • Overlay—One page for all selected samples that shows the size standard dyes overlaid with the size standard curves. • Plate—One page per plate for all selected samples that shows the well-location thumbnail traces with color-coded headers that reflect sizing quality. Plot zooming is not retained in the report. Export sizing results 1. Set up the sizing table (see “Set up the sizing table” on page 121). All rows and columns displayed in the sizing table are exported. 2. Click Export Results. Modify fragment analysis or HID data To edit, modify, or further analyze fragment analysis or HID data, import the sample data files into a secondary analysis software application such as: • Fragment analysis—GeneMapper™ Software 5 (or later) • HID—GeneMapper™ ID‑X Software v1.2 (or later) 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 123 7 Run calibrations and install checks Section 7.1 Run spatial and spectral calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 ■ Run a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 ■ Run a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Section 7.2 Run an install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 ■ Run a Sequencing install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 ■ Run a fragment/HID install check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 124 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spatial calibration 7 Section 7.1 Run spatial and spectral calibrations Run a spatial calibration Spatial calibration overview The software uses images collected during the spatial calibration to establish a relationship between the signal emitted by each capillary and the position where that signal falls on, and is detected by, the CCD camera. When to perform a spatial calibration Perform a spatial calibration after you: • Remove or replace the capillary array. • Replace the capillary array when it expires. Note: When the instrument reads the information from a newly installed capillary array, you are required to run a spatial calibration and a spectral calibration before you can run plates. • Open the detector door or move the detection cell. • Move the instrument. Perform a spatial calibration IMPORTANT! Do not open the instrument door during a spatial calibration run. Doing so will stop the run and require you to restart the software. 1. In the Dashboard, preheat the oven if you will be selecting the Fill option for the calibration (fill the array with polymer). 2. Click the Maintenance menu, then select Calibration4Spatial. Note: The screen does not display results unless you have previously performed a spatial calibration. 3. Select No Fill, or select Fill to fill the array with polymer before starting the calibration. 4. Select Perform QC Checks. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 125 Chapter 7 Run calibrations and install checks 7 Run a spatial calibration 5. Click Start Calibration. During the calibration, the software performs quality checks and calculates the following values: Attribute Calculation Threshold Average peak height (sum of all peak heights) • 8-cap: 6,400 RFU divided by • 24-cap: 3,000 RFU (number of peaks) Individual peak height Peak height 1,000 RFU Uniformity (peak height similarity) (standard deviation) 0.2 divided by (average peak height) Capillary spacing max. spacing - min. spacing 2 pixels The display updates as the run progresses. • A signal optimization factor is calculated for each capillary using a fitted curve method. The fitted curve method minimizes background and reduces noise. The adjusted spatial intensity, not the spatial intensity displayed for the capillary, is used to calculate the signal optimization factor. The signal optimization factors are applied during data collection to minimize optical variation effects and increase signal uniformity between capillaries. • If any of the QC check calculations do not meet a threshold condition, a Spatial QC Check error message is displayed. 126 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spatial calibration 7 Evaluate the spatial calibration profile When the run is complete: 1. Evaluate the spatial calibration profile to ensure that you see: • One sharp peak for each capillary. Small shoulders are acceptable. • One marker (+) at the apex of every peak. No off-apex markers. • An even peak profile (all peaks about the same height). 2. If the results meet the criteria above, click Accept Results. If the results do not meet the criteria above, the Accept button is dimmed. Go to “Spatial calibration troubleshooting” on page 298. IMPORTANT! Do not log off or close the software before clicking Accept Results. Spatial calibration results are not saved until you click Accept Results. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 127 Chapter 7 Run calibrations and install checks 7 Run a spatial calibration Example spatial profiles 8-capillary spatial 24-capillary spatial Export spatial calibration results 1. Click Export Result. 2. Enter an export file name. 3. Select the export file type, then click Save. 128 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 The export file contains the following results: • Capillary Number • Position (pixels) • Spacing • Intensity View and print a calibration report Note: Spatial and spectral calibration reports include the date on which a capillary array is installed for the first time on the instrument. Install standard reports use the most recent install date if a capillary array was removed and re-installed on the instrument. 1. Click View Report. 2. (Optional) In the Report screen, click toolbar options to manipulate the report. Place the mouse pointer over an item for a description of the item. 3. To print the report, click Print. Save historical reports (PDF) for record keeping IMPORTANT! Save a report electronically for record keeping. The software does not save historical results. Only the most recent results are maintained in the software. 1. Click View Report. 2. Click Print. 3. In the Printer dialog box, select a PDF printer. 4. Specify a name and location for the report. Run a spectral calibration Spectral calibration overview A spectral calibration creates a de-convolution matrix that compensates for dye overlap (reduces raw data from the instrument) in the dye data stored in each sample file. IMPORTANT! To calibrate a custom dye set using AnyDye, first create the dye set, then select the name of the custom dye set from the Dye Set list. The AnyDye selection in the Dye Set list contains default settings. It does not correspond to custom dye sets created with the AnyDye dye set template. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 129 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration When to perform a spectral calibration Perform a spectral calibration when you: • Use a dye set that you have not previously calibrated • Replace the capillary array for maintenance purposes • Replace the capillary array when it expires (the expiration date is indicated on the packaging and the RFID tag) Note: When the instrument reads the information from a newly installed capillary array, you are required to run a spatial calibration and a spectral calibration before you can run plates. • See a decrease in spectral separation (pull-up/pull-down in peaks) in the raw or analyzed data Note: For sequencing applications, you can skip this process, and run the Sequencing install check. If you select Keep Spectral Calibration Data in the Install Check, the software runs a spectral calibration for the dye set during a sequencing check and allows you to save the spectral calibration data. For information, see “Run an install check” on page 144. Estimated run time Note: The run times listed below do not include the time for the oven to preheat to 60°C. Application Standard Polymer type Run time (min) Sequencing Sequencing standard POP-6™polymer £135 Sequencing Sequencing standard POP-7™ polymer £40 Fragment analysis Matrix standard Any polymer £75 Prepare for spectral calibration Before you begin If you have not already done so, perform a spatial calibration (see page 125). Prepare the instrument 1. In the Dashboard, check consumables status (see “Check consumables status” on page 37). Ensure that: • Consumables are not expired • Adequate injections remain for consumables 2. Ensure that buffer levels are at the fill lines (see “Check buffer fill levels” on page 39). 3. Set the oven temperature, then click Start Pre-heat. Pre-heat the oven and detection cell while you prepare for a run (detection cell temperature is set by the software). Pre-heating helps mitigate subtle first-run migration rate effects. The pre-heat function automatically turns off after 2 hours. 130 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 We recommend that you pre-heat the oven for at least 30 minutes before you start a run if the instrument is cold. Temperatures are displayed in red as they warm to the set-points. When temperatures are at the set-point they are displayed in green. Temperatures may fluctuate slightly when they reach the set-point as they stabilize. 4. Check the pump assembly for bubbles and run the Remove Bubble wizard if needed (see “Remove bubbles from the polymer pump” on page 268). Prepare the spectral calibration standard Prepare the matrix or sequencing standard appropriate for your application as described in the product insert. See Appendix D, “Catalog numbers” for catalog numbers. If peaks are offscale for G5, F, and E5 dye sets, dilute the matrix standard and repeat the calibration. Prepare the standard plate IMPORTANT! Do not use warped or damaged plates. 1. Load the standards in any injection position in the plate. The example below shows injection position 1, but you can specify the starting well for an injection position. IMPORTANT! You do not create a plate in the software for the install check. 8-capillary A1 through H1 96-well plate 24-capillary A1 through H1, A2 through H2, and A3 through H3 96-well plate 24-capillary Columns 1, 3, and 5 in rows A, C, E, G, I, K, M, O 384-well plate Note: 384-well plates are not supported on 8‑capillary instruments. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 131 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration 2. Briefly centrifuge the plate that contains the standards. 3. Verify that each standard is positioned correctly in the bottom of its well. IMPORTANT! If the contents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each standard is positioned correctly in the bottom of its well. 4. Store the plate on ice until you prepare the plate assembly and load the plate in the instrument. 5. Prepare the plate assembly as described in “Prepare the plate assembly” on page 70. Load the plate in the instrument 1. Click the Tray button on the front panel to move the autosampler to the front position, then open the instrument door. 2. Place the plate in the autosampler with the labels facing you (or the instrument door) and the notched corner of the plate in the notched corner of the autosampler. 3. Close the instrument door to initialize the instrument. 132 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 Perform a spectral calibration IMPORTANT! Do not change E‑Signature settings during a spectral calibration. IMPORTANT! If you change polymer type, spectral calibrations for the original polymer type are not retained. 1. Access the Spectral Calibration screen. Note: The screen does not display results until you perform a spectral calibration. To view previous calibration data, click History View. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 133 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration 2. Select the number of wells, standard, and dye set. 3. Select the plate position for the plate loaded in the instrument. Note: You do not create a plate in the software for the calibration. 4. Specify the starting well for the injection position in which you loaded the standard in the plate. 5. For Chemistry Standard and Dye Set, select: Note: For the BigDye™ Direct DNA PCR Amplification/ Clean-up/Cycle Sequencing kit, use dye set Z. Chemistry standard Dye set Application Sequencing standard v1.1 E Sequencing BigDye™ Terminator v1.1 matrix standard E Sequencing Sequencing standard v3.1 Z Sequencing BigDye™ Terminator v3.1 matrix standard Z Sequencing DS-32 Matrix Standard Kit (Dye Set F) F Fragment analysis DS-02 Matrix Standard Kit (Dye Set E5) E5 Fragment analysis DS-30 Matrix Standard Kit (Dye Set D) D Fragment analysis DS-33 Matrix Standard (Dye Set G5) G5 Fragment analysis DS‑36 Matrix Standard Kit (Dye set J6, 6‑dye) J6 Fragment analysis DS-37 Matrix Standard Kit (Dye set J6‑T, 6‑dye) J6-T Fragment analysis Custom AnyDye Fragment analysis IMPORTANT! The E‑signature function creates a record when a spectral calibration is performed, but does not record the dye set calibrated. To include the dye set calibrated in the E‑signature record, enter the dye set in the E-Sig Comments field. 6. (Optional) Select Allow Borrowing. Selecting this option instructs the software to automatically replace information from failed capillaries with information from an adjacent passing capillary with the highest Quality value. For more information, see “What you see during a spectral calibration” on page 138. 134 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 7. Click Start Run. The following occurs: • A Progress Information message with a dimmed Cancel button is displayed. The message is displayed for several minutes while the instrument checks system components. You cannot cancel the calibration during this initialization period. When the initialization is complete, the Progress Information message automatically closes. The Abort button is displayed at the top of the screen. • If you used the default setting "Perform run 2 and 3 if run 1 fails", the instrument sets up three injections (see “What you see during a spectral calibration” on page 138 for information on the number of injections performed). • The Capillary Run Data display updates after each injection is complete. • The status bar updates during Run 1. IMPORTANT! The status bar does not update during Run 2 or Run 3. • Passing and failing capillaries are shown in green and red respectively. Borrowed capillaries are shown in yellow with an arrow indicating the adjacent capillary from which results were borrowed. To display the result for each capillary (spectral data, Quality Value, and Condition Number) below the run results table, click a capillary in the table. Note: The results displayed when you click a borrowed capillary are the passing results borrowed from the adjacent capillary. To determine the reason that a capillary fails, view the spectral calibration report. See “View and print a calibration report” on page 129. For all spectral calibration injections (even capillaries that are green in the Overall row), evaluate the data as described in the next section. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 135 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration Spectral Quality Values and Condition Numbers Spectral Quality Value A spectral Quality Value reflects the confidence that the individual dye emission signals can be separated from the overall measured fluorescence signal. It is a measure of the consistency between the final matrix and the data from which it was computed. A Quality Value of 1.0 indicates high consistency, providing an ideal matrix with no detected pull-up/pull-down peaks. In rare cases, a high Quality Value can be computed for a poor matrix. This can happen if the matrix standard contains artifacts, leading to the creation of one or more extra peaks. The extra peaks cause the true dye peak to be missed by the algorithm, and can lead to a higher Quality Value than would be computed with the correct peak. Therefore, it is important to visually inspect the spectral calibration profile for each capillary. Condition Number A Condition Number indicates the amount of overlap between the dye peaks in the fluorescence emission spectra of the dyes in the dye set. If there is no overlap in a dye set, the Condition Number is 1.0 (ideal conditions), the lowest possible value. The condition number increases with increasing peak overlap. The ranges that the software uses to determine if a capillary passes or fails are: Dye set Quality Value minimum Condition Number maximum AnyDye 0.8 (default) 20.0 (default) D 0.95 8.5 E 0.95 5.5 E5 0.95 6.0 F 0.95 8.5 G5 0.95 13.5 J6 or J6-T 0.95 8.0 Z 0.95 5.5 136 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 Evaluate the spectral calibration data IMPORTANT! Do not accept a spectral calibration until you examine the data for all capillaries. When a spectral calibration completes successfully, the Overall row displays green, red, or yellow results. For each capillary: 1. Click a capillary to display the spectral and raw data for a capillary. 2. Check that the data meet the following criteria: Attribute Acceptance criteria Example Order of the peaks in the spectral 4-dye: blue-green-yellow-red profile (intensity vs pixel) from left to right 5-dye: blue-green-yellow-red-orange 6-dye: blue-green-yellow-red-purple- orange Order of the peaks in the raw data Sequencing (matrix standard only): profile from left to right 4-dye: red-yellow-blue-green Order of the peaks in the raw data Fragment analysis: profile from left to right • 4-dye: red-yellow-green-blue • 5-dye: orange-red-yellow-green- blue Extraneous peaks in the raw data None E5: profile (intensity vs scan) Note: The E5 profile may include extraneous peaks outside the matrix peak region, which can be ignored. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 137 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration (continued) Attribute Acceptance criteria Example Peak morphology in the spectral • No gross overlaps, dips, or other profile (intensity vs pixel) irregularities • Peaks are separate and distinct • Peak apexes are separate and distinct (the tails will overlap) Note: The peak morphology of G5 (shown to the right, top), F, and J6 (shown to the right, bottom) may not be as rounded and symmetrical as the peak morphology for other dye sets (shown above) due to the effect of variable binning (a feature that reduces signal variation between dyes of different fluorescent efficiencies). 3. As needed, zoom on the spectral profile traces to determine if the data meet the criteria (see “Zoom on data” on page 118). 4. If the data for all capillaries meet the criteria above, click Accept Results. 5. If any capillary data does not meeting the criteria above, click Reject Results, then go to “Spectral calibration troubleshooting” on page 299. Zoom on data 1. Place the pointer above the top of the plot or to the left of the plot at the start of the area you want to zoom, then click to turn the pointer to . 2. With the still above the plot or to the left of the plot, click-drag to the end of the area you want to zoom. Do not drag the inside the plot area. Doing so changes back to a pointer and does not zoom as expected. You can also click zoom and fit buttons to zoom . What you see during a spectral calibration A spectral calibration can run up to three injections. The number of injections performed depends on: • The number of capillaries that pass or fail during an injection • Whether you select the Allow Borrowing option Note: The first time you perform a spectral calibration (for each dye set) after installing a new capillary array, you may notice pull-down peaks (or mirror image peaks). While the run is in progress, these pull-down peaks will eventually correct themselves. Once the run completes the electropherogram, the pull-down peaks disappear. 138 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 Capillary information sharing A spectral calibration can share capillary information: • Between injections – If a capillary in an injection does not meet the spectral Quality Value and Condition Number limits shown on page 105, the software automatically uses the information from that capillary in a different injection. • Within an injection – If a capillary in an injection does not meet the spectral Quality Value and Condition Number limits shown on page 105 and the Allow Borrowing option is selected, the software can also use the information from a capillary to the left or the right of that capillary, if the values are higher than those for that capillary in a different injection. Spectral calibration with Borrowing disabled When Borrowing is disabled, all capillaries must pass (meet the spectral Quality Value and Condition Number limits) for the calibration to pass. Injection 1 • The software evaluates the Quality Value and Condition Number of all capillaries. • If all capillaries pass, the calibration is complete, and injections 2 and 3 are not performed. • If any capillaries fail, injection 2 is performed. Injection 2 • The software evaluates the Quality Value for each capillary across injections 1 and 2 and uses the information from the capillary with the highest Quality Value. • If all capillaries now pass, the calibration is complete and injection 3 is not performed. • If the same capillary fails in both injection 1 and 2, injection 3 is performed. Injection 3 • The software evaluates the Quality Value for each capillary across injections 1, 2, and 3 and the information from the capillary with the highest Quality Value. • If all capillaries now pass, the calibration passes. • If the same capillary fails in injection 1, 2, or 3, the calibration fails. Spectral calibration with Borrowing enabled When Borrowing is enabled, all capillaries have to pass (meet the spectral Quality Value and Condition Number limits) within the borrowing limits: • 8-capillary instruments – One adjacent-capillary borrowing event allowed • 24-capillary instruments – Up to three adjacent-capillary borrowing events allowed (the number of allowed borrowing events can be decreased in Preferences). The software identifies a borrowed capillary with an arrow pointing from the capillary from which the data is borrowed. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 139 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration Injection 1 • The software evaluates the Quality Value and Condition Number of all capillaries. • If all capillaries pass, the calibration is complete, and injections 2 and 3 are not performed. • If any capillaries fail, the software borrows from an adjacent capillary. • If, after borrowing, >1 or > 3 capillaries fail, injection 2 is performed. Injection 2 • The software evaluates the quality values between adjacent capillaries in injection 2 and for each capillary across injections 1 and 2 and uses the information with the highest Quality Value for each capillary. • If all capillaries pass, the calibration is complete and injection 3 is not performed. • If, after borrowing, >1 or > 3 capillaries from injection 1 or 2 do not pass, injection 3 is performed. Injection 3 • The software evaluates the quality values between adjacent capillaries in injection 3 and for each capillary across injections 1, 2, and 3, then uses the information with the highest Quality Value for each capillary. • If all capillaries now pass, the calibration passes. • If after borrowing, >1 or > 3 capillaries from injection 1, 2, or 3 do not pass, the calibration fails. 140 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 Example spectral calibration data Dye Set E created from Sequencing Standard Dye Set Z created from Sequencing Standard 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 141 Chapter 7 Run calibrations and install checks 7 Run a spectral calibration Dye Set G5 created from Matrix Standard Set DS-33 Dye Set J6 created from Matrix Standard Set DS-36 Export spectral calibration results To export spectral calibration results: 1. Click Export Spectral Calibration Results. 2. Specify an export file name and location, then click Save. The export file contains the following results: • Capillary Number • Condition Number • Scan Number 142 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a spectral calibration 7 • Borrowed From Capillary • Quality Value • Peak Height • Reason For Failure • Run From Injection View and print a calibration report Note: Spatial and spectral calibration reports include the date on which a capillary array is installed for the first time on the instrument. Install standard reports use the most recent install date if a capillary array was removed and re-installed on the instrument. 1. Click View Report. 2. (Optional) In the Report screen, click toolbar options to manipulate the report. Place the mouse pointer over an item for a description of the item. 3. To print the report, click Print. Save historical reports (PDF) for record keeping IMPORTANT! Save a report electronically for record keeping. The software does not save historical results. Only the most recent results are maintained in the software. 1. Click View Report. 2. Click Print. 3. In the Printer dialog box, select a PDF printer. 4. Specify a name and location for the report. View the spectral calibration history Only the most recent spectral calibration for each dye set is maintained in the software. Select History View, then select a dye set to view the associated calibration history. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 143 Chapter 7 Run calibrations and install checks 7 Run a Sequencing install check Section 7.2 Run an install check Run a Sequencing install check When to perform a sequencing install check If an install check is not performed when your instrument is installed, you must perform an install check before you can run plates. We recommend that you run an install check monthly to verify that the instrument meets specifications. The sequencing install check has an option to include and save the spectral calibration. If you select this option and you accept the sequencing install standard results, you do not need to run a separate spectral calibration (described in “Run a Sequencing install check” on page 144) for the dye set. Estimated run time Note: The run times listed below do not include the time for the oven to preheat to 60°C. • General sequencing (BDTv3.1): ~1 hour • MicroSeq ID: 2 hours • BDTv1.1POP6: ~2.5 hours Prepare for the sequencing install check Before you begin If you will not save the spectral calibration from the sequencing install run, perform a spectral calibration (see “Perform a spectral calibration” on page 133). Prepare the instrument 1. In the Dashboard, check consumables status (see “Check consumables status” on page 37). Ensure that: • Consumables are not expired • Adequate injections remain for consumables 2. Ensure that buffer levels are at the fill lines (see “Check buffer fill levels” on page 39). 3. Set the oven temperature, then click Start Pre-heat. Pre-heat the oven and detection cell while you prepare for a run (detection cell temperature is set by the software). Pre-heating helps mitigate subtle first-run migration rate effects. The pre-heat function automatically turns off after 2 hours. We recommend that you pre-heat the oven for at least 30 minutes before you start a run if the instrument is cold. 144 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a Sequencing install check 7 Temperatures are displayed in red as they warm to the set-points. When temperatures are at the set-point they are displayed in green. Temperatures may fluctuate slightly when they reach the set-point as they stabilize. 4. Check the pump assembly for bubbles and run the Remove Bubble wizard if needed (see “Remove bubbles from the polymer pump” on page 268). Prepare the sequencing install check standard Prepare the BigDye™ Terminator v1.1 or v3.1 Sequencing Standard as described in the product insert. See Appendix D, “Catalog numbers” for catalog numbers. Note: If you are using the BigDye™ Direct DNA PCR Amplification/Clean-up/Cycle Sequencing kit and you will use the sequencing install data for spectral calibration, use the v3.1 Sequencing Standard. Prepare the standard plate IMPORTANT! Do not use warped or damaged plates. 1. Load the standards in any injection position in the plate. The example below shows injection position 1, but you can specify the starting well for an injection position. IMPORTANT! You do not create a plate in the software for the install check. 8-capillary A1 through H1 96-well plate 24-capillary A1 through H1, A2 through H2, and A3 through H3 96-well plate 24-capillary Columns 1, 3, and 5 in rows A, C, E, G, I, K, M, O 384-well plate Note: 384-well plates are not supported on 8‑capillary instruments. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 145 Chapter 7 Run calibrations and install checks 7 Run a Sequencing install check 2. Briefly centrifuge the plate that contains the standards. 3. Verify that each standard is positioned correctly in the bottom of its well. IMPORTANT! If the contents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each standard is positioned correctly in the bottom of its well. 4. Store the plate on ice until you prepare the plate assembly and load the plate in the instrument. 5. Prepare the plate assembly as described in “Prepare the plate assembly” on page 70. Load the plate in the instrument 1. Click the Tray button on the front panel to move the autosampler to the front position, then open the instrument door. 2. Place the plate in the autosampler with the labels facing you (or the instrument door) and the notched corner of the plate in the notched corner of the autosampler. 3. Close the instrument door to initialize the instrument. Perform a sequencing install check 1. Access the Sequencing Install Standard screen. 146 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a Sequencing install check 7 2. Select the chemistry type. Note: BDTv3.1 with POP-6™ polymer is not available for the install check (it can be used for application runs). If your application uses BDTv3.1 with POP-6™ polymer, select BDTv.1 for the sequencing install check, then perform a separate spectral calibration using the Z dye set. Do not select Keep Spectral Calibration Data. 3. Select the number of wells and plate position in the instrument. Note: You do not create a plate in the software for the install check. 4. Specify the starting well for the injection position in which you loaded the standard in the plate. Note: If you navigate away from the Install Standard screen after you start the install check, the starting well may be reset to A01. This is a display issue only; the starting well you specify is used for the install check. 5. (Optional) If you have not already run a spectral calibration, select Keep Spectral Calibration Data to save the sequencing install standard run (if it passes) as a spectral calibration. Note: The spectral calibration record will only be saved if the Keep Spectral Calibration Data option is checked on the screen. If you uncheck the option, create a separate spectral calibration from the Maintenance menu. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 147 Chapter 7 Run calibrations and install checks 7 Run a Sequencing install check 6. Click Start Run. IMPORTANT! Do not accept a sequencing installation standard run until you examine the data. 148 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a Sequencing install check 7 What you see during a sequencing install check The instrument performs one run, then evaluates: • Spectral data, if you specified to keep spectral data • Sequence data The Capillary Run Data display (Figure 14) is updated after the run is complete: • The spectral calibration status is displayed in the first row of the run results table. Passing and failing capillaries in the install check run are shown in green and red respectively for the CRL (contiguous read length) criteria. Borrowed capillaries (spectral only) are shown in yellow with an arrow indicating the adjacent capillary from which results were borrowed. The spectral result for each capillary is displayed below the run results table. • The sequencing install standard status is displayed in the third row of the run results table (CRL Pass/Fail). • The Quality Value and Condition Number for each capillary is displayed below the table. Note: The values shown in this figure are examples only. Figure 14 Capillary Run Data Pass/fail criteria for the optional spectral calibration The software evaluates the Quality Value and Condition Number for each capillary (for more information, see “Spectral Quality Values and Condition Numbers” on page 136). Borrowing is automatically enabled. 1 borrowing event is allowed for 8-capillary instruments. Up to 3 borrowing events are allowed for 24-capillary instruments. For more information, see “Spectral calibration with Borrowing enabled” on page 139. The number of borrowing events can be decreased (see “System preferences” on page 42). Thresholds used by the software for pass/fail are listed in the following table: Dye set Quality Value minimum Condition Number maximum E 0.95 5.5 Z 0.95 5.5 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 149 Chapter 7 Run calibrations and install checks 7 Run a Sequencing install check Pass/fail criteria for the sequencing install check The software calculates the Contiguous Read Length for each capillary. Capillaries that are below the threshold fail. The remaining results that the software displays are for information only. Result Description Contiguous Read The longest uninterrupted segment of basecalls with an average Quality Value Length (CRL) (QV) ≥20. In addition to evaluating the QV of a basecall, the software considers the QV of adjacent basecalls within a 21-bp moving window to determine a contiguous read length based on quality values: the software starts from the 5' end and calculates the average QV across a moving window size of 21, sliding 1 bp at a time, to the 3' end. The resulting longest contiguous segment is determined as the CRL. CRL Pass/Fail • BDTv1.1—Capillaries with a CRL <600 bp fail. • BDTv3.1 (General Sequencing)—Capillaries with a CRL <500 bp fail. • MicroSEQ™ ID – Capillaries with a CRL <600 bp fail. For information only— The alignment of the base-called sample sequence with the known reference of the sequencing install standard is used to calculate the following results. CRL Basepair Accuracy CRL Basepair Accuracy is determined by basepair comparison between the base-called sample sequence and the known reference sequence of the sequencing install standard within the contiguous read length region calculated (as described in the CRL definition above). Read Length The length of read (in bases) at which base calling accuracy is ³98.5%. The Read Length is measured only within the following regions: • BDTv1.1: 20–619 bp (maximum value is 600) • BDTv3.1 (General Sequencing): 40–539 bp (maximum value is 500) • MicroSEQ™ ID: 20–619 bp (maximum value is 600) The Read Length value is derived from basecall accuracy, and not from quality values. Basepair Accuracy Basepair Accuracy calculates the percent of accurate basecalls in the known (Read Length Accuracy) reference sequence within the Read Length of the sequencing install standard. CRL Median and SD Median and standard deviation determined for all capillaries. 150 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a Sequencing install check 7 Evaluate sequencing install standard data When a sequencing install standard run completes successfully, the CRL Pass/Fail row displays green or red results. For each capillary: 1. Click a capillary to display the spectral and raw data profiles for a capillary. 2. Check that the data meet the following criteria: Attribute Acceptance Criteria Example Order of the peaks in the spectral 4-dye: blue-green-yellow-red profile (intensity vs pixel) from left to right Extraneous peaks in the raw data None E5: profile (intensity vs scan) Note: The E5 profile may include extraneous peaks outside the matrix peak region, which can be ignored. Extraneous peaks in the raw data None profile (intensity vs scan) Peak morphology in the spectral • No gross overlaps, dips, or other profile (intensity vs pixel) irregularities • Peaks separate and distinct • Peak apexes are separate and distinct (the tails will overlap) 3. (Optional) Review the CRL Basepair Accuracy to determine discrepancies from the reference sequence. If you observe large discrepancies (for example, 5–10 contiguous miscalled bases in the middle of a sequence), review the data. If you see a raw data peak larger than the adjacent peaks with baseline pull-up in all 4-dye color channels, it may indicate the presence of a bubble. Check the pump, run the Remove Bubbles wizard (see “Remove bubbles from the polymer pump” on page 268), then repeat the run as needed. 4. If the data for the required number of capillaries meets the criteria above (at least 7 capillaries for 8-capillary instruments, at least 22 capillaries for 24‑capillary instruments), click Accept Results. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 151 Chapter 7 Run calibrations and install checks 7 Run a Sequencing install check 5. If the data for the required number of capillaries do not meet the criteria above (at least 7 capillaries for 8-capillary instruments, at least 22 capillaries for 24‑capillary instruments): a. (Optional) If you want to generate a report for the failed calibration, click View Report before you click Reject Results. To save the report electronically, select a PDF printer. b. Click Reject Results. For troubleshooting information, see “Sequencing install standard troubleshooting” on page 302. IMPORTANT! If you reject results, the spectral calibration is not saved. Example sequencing install check results View previously run install standards Select History View, then select an install standard to view the associated install check information. 152 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a fragment/HID install check 7 View and print an install check report Note: Ensure that all dyes are selected before viewing the report. The report may contain incomplete data if all dyes are not selected. Note the following: • Install check reports include the most recent install date if a capillary array was removed, then re-installed on the instrument. Spatial and spectral calibration reports include the date on which a capillary array is installed on the instrument for the first time. • The sorting in the Install Standard screen is not applied to the report. • You can generate a report for a failed install check run before you click Reject Results. 1. Click View Report. Note: The Date Performed field reflects the date that the install check was accepted, not the date it was run. 2. (Optional) In the Report screen, click toolbar options to manipulate the report. Place the mouse pointer over an item for a description of the item. 3. To print the report, click Print. 4. To save the report electronically (PDF), print the report and select a PDF printer. Save historical reports (PDF) for record keeping IMPORTANT! Save a report electronically for record keeping. The software does not save historical results. Only the most recent results are maintained in the software. 1. Click View Report. 2. Click Print. 3. In the Printer dialog box, select a PDF printer. 4. Specify a name and location for the report. Run a fragment/HID install check When to perform a fragment/HID install check If an install check is not performed when your instrument is installed, you must perform an install check before you can run plates. We recommend that you run an install check monthly to verify that the instrument meets specifications. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 153 Chapter 7 Run calibrations and install checks 7 Run a fragment/HID install check Estimated run time Note: The run times listed below do not include the time for the oven to preheat to 60°C. • Fragment install check (POP-7™ Polymer, 50‑cm capillary array)—≤ 90 minutes • HID install check (POP-4™ Polymer, 36‑cm capillary array)—≤ 30 minutes Prepare for the fragment/HID install check Before you begin If you will not save the spectral calibration from the sequencing install run, perform a spectral calibration (see “Perform a spectral calibration” on page 133). Prepare the instrument 1. In the Dashboard, check consumables status (see “Check consumables status” on page 37). Ensure that: • Consumables are not expired • Adequate injections remain for consumables 2. Ensure that buffer levels are at the fill lines (see “Check buffer fill levels” on page 39). 3. Set the oven temperature, then click Start Pre-heat. Pre-heat the oven and detection cell while you prepare for a run (detection cell temperature is set by the software). Pre-heating helps mitigate subtle first-run migration rate effects. The pre-heat function automatically turns off after 2 hours. We recommend that you pre-heat the oven for at least 30 minutes before you start a run if the instrument is cold. Temperatures are displayed in red as they warm to the set-points. When temperatures are at the set-point they are displayed in green. Temperatures may fluctuate slightly when they reach the set-point as they stabilize. 4. Check the pump assembly for bubbles and run the Remove Bubble wizard if needed (see “Remove bubbles from the polymer pump” on page 268). Prepare the fragment/HID install check standard Prepare the standard as described in the product insert. See Appendix D, “Catalog numbers” for catalog numbers. • Fragment analysis: DS‑33 GeneScan™ Installation Standards with GeneScan™ 600 LIZ™ Size Standard v2.0 • HID: AmpFℓSTR™ Identifiler™ Allelic Ladder (For HID install check) 154 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a fragment/HID install check 7 Prepare the standard plate IMPORTANT! Do not use warped or damaged plates. 1. Load the standards in any injection position in the plate. The example below shows injection position 1, but you can specify the starting well for an injection position. IMPORTANT! You do not create a plate in the software for the install check. 8-capillary A1 through H1 96-well plate 24-capillary A1 through H1, A2 through H2, and A3 through H3 96-well plate 24-capillary Columns 1, 3, and 5 in rows A, C, E, G, I, K, M, O 384-well plate Note: 384-well plates are not supported on 8‑capillary instruments. 2. Briefly centrifuge the plate that contains the standards. 3. Verify that each standard is positioned correctly in the bottom of its well. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 155 Chapter 7 Run calibrations and install checks 7 Run a fragment/HID install check IMPORTANT! If the contents of any well contain bubbles or are not located at the bottom of the well, briefly centrifuge the plate, remove the plate from the centrifuge, and verify that each standard is positioned correctly in the bottom of its well. 4. Store the plate on ice until you prepare the plate assembly and load the plate in the instrument. 5. Prepare the plate assembly as described in “Prepare the plate assembly” on page 70. Load the plate in the instrument 1. Click the Tray button on the front panel to move the autosampler to the front position, then open the instrument door. 2. Place the plate in the autosampler with the labels facing you (or the instrument door) and the notched corner of the plate in the notched corner of the autosampler. 3. Close the instrument door to initialize the instrument. Perform the fragment/HID install check 1. Access the Fragment Install Standard or HID Install Standard screen. 2. Select the plate type (number of wells). 3. Select the plate position in the instrument. Note: You do not create a plate in the software for the install check. 156 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a fragment/HID install check 7 4. Specify the starting well for the injection position in which you loaded the standard in the plate. Note: If you navigate away from the Install Standard screen after you start the install check, the starting well may be reset to A01. This is a display issue only; the starting well you specify is used for the install check. 5. Click Start Run. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 157 Chapter 7 Run calibrations and install checks 7 Run a fragment/HID install check What you see during a fragment install check The instrument performs one run and indicates the number of observed allele and size standard peaks. The Capillary Run Data display is updated after the run is complete. The number of observed size standard and allele peaks is shown. Results for each allele are shown at the bottom of the screen in the Run Information table. Number of peaks per capillary Plot and allele size/height for selected capillary Allele results for all capillaries Pass/fail criteria for the fragment/HID install check The software evaluates peaks in the data for each capillary. To be identified as a possible allele, peaks must be within the following ranges (nominal allele size, or reference bin size, is hard-coded): Fragment analysis HID analysis All markers between ±0.4 bp and ±0.5 • All markers except TH01: ±0.7 bp of nominal size for the allele bp of nominal size for the allele • TH01: – Seven markers are ±0.7 bp of nominal size for the allele – Three markers are ±0.5 bp of nominal size for the allele For all peaks that are within the nominal size range, the software calculates the Average Peak Height and the Sizing Precision. Peaks that meet the following thresholds pass. 158 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a fragment/HID install check 7 Result Description Threshold Min Peak Height Minimum of peak heights for observed allele peaks of the • Fragment: >175 RFU included capillaries. • HID: >400 RFU Sizing Precision Standard deviation of the observed allele fragment sizes <0.15 for expected alleles Pass/Fail Alleles with a sizing precision and minimum peak height that do not meet thresholds fail. For information only Nominal Size Expected allele fragment peak size (bp). Mean Average fragment size for the observed allele peaks. Peak Height % >Min Percentage of observed allele peaks with a peak height above the minimum threshold. Sizing Accuracy Difference between the expected allele size and the mean allele size. Evaluate fragment install standard data 1. Examine the number of size standard and allele peaks found for each capillary. 2. If all capillaries pass, click Accept Results. If any capillaries fail, the Accept Results button is disabled. Evaluate the raw data for failed capillaries. You can deselect 1 failed capillary for 8‑capillary instruments or 2 failed capillaries for 24‑capillary instruments to recalculate results and then click Accept. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 159 Chapter 7 Run calibrations and install checks 7 Run a fragment/HID install check Example fragment install standard results Example HID install standard results View previously run install standards Select History View, then select an install standard to view the associated install check information. 160 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 7 Run calibrations and install checks Run a fragment/HID install check 7 View and print an install check report Note: Ensure that all dyes are selected before viewing the report. The report may contain incomplete data if all dyes are not selected. Note the following: • Install check reports include the most recent install date if a capillary array was removed, then re-installed on the instrument. Spatial and spectral calibration reports include the date on which a capillary array is installed on the instrument for the first time. • The sorting in the Install Standard screen is not applied to the report. • You can generate a report for a failed install check run before you click Reject Results. 1. Click View Report. Note: The Date Performed field reflects the date that the install check was accepted, not the date it was run. 2. (Optional) In the Report screen, click toolbar options to manipulate the report. Place the mouse pointer over an item for a description of the item. 3. To print the report, click Print. 4. To save the report electronically (PDF), print the report and select a PDF printer. Save historical reports (PDF) for record keeping IMPORTANT! Save a report electronically for record keeping. The software does not save historical results. Only the most recent results are maintained in the software. 1. Click View Report. 2. Click Print. 3. In the Printer dialog box, select a PDF printer. 4. Specify a name and location for the report. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 161 8 Manage library resources ■ Overview of libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 ■ General library procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 ■ Plates library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 ■ Assays library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 ■ File Name Conventions library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 ■ Results Group library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 ■ Instrument protocol library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 ■ Dye sets library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 ■ Size standards library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 ■ Basecalling protocols library (primary analysis—sequencing) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 ■ Sizecalling protocols library (primary analysis—fragment) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 ■ QC protocols library (primary analysis—HID) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Overview of libraries The Library workflow contains the following libraries: • Items that you select when you set up a run: – Plates—Contains factory-provided plate templates that you can use to create plates for each run. – Assays—Contains factory-provided assay templates that you cannot modify. You can also create new assays. – Optional Filename Conventions—Contains factory-provided file name conventions that you cannot modify. You can also create new file name conventions. – Optional Results Groups—Contains factory-provided results groups that you cannot modify. You can also create new results groups. • Items that you select when you create an assay: – Instrument protocols – Primary analysis protocols—Basecalling (sequencing), sizecalling (fragment analysis), QC (HID analysis) • Items you select when you create instrument sizecalling and QC protocols: – Dye sets – Size standards 162 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources General library procedures 8 Factory-provided template and locked items The software libraries include factory-provided items that are optimized for different applications (for example, instrument protocols with specific run modules and primary analysis protocols with specific settings). You can use the factory-provided items directly. If the factory-provided items do not suit your needs, you can do one of the following: • Duplicate and modify a factory-provided item, and save the item with a new name. • Create a new item. Entries in the library may be flagged with the following symbols: • Factory-provided. Cannot be edited or deleted. • Template. • Locked. If the SAE module is enabled on your system, a locked item can be unlocked and modified by the user who created it, the administrator, or another user with unlock permissions. For information, see Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)” . General library procedures Access libraries Click the Library tab to access the Library workflow. You can click Main Workflow, or select Dashboard or any other menu item at any time to advance from the Library workflow. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 163 Chapter 8 Manage library resources 8 General library procedures Create a new entry from a factory-provided template or locked entry IMPORTANT! Auditing of an item depends on whether it is created directly from the library or from within another item (for example, you can create an assay directly from the library, or within a plate in the Assign Plate Contents screen). For more information on auditing, see “Review the object audit history” on page 232. 1. Select the factory-provided entry in the library. 2. Click Duplicate. 3. Enter a name for the item. 4. Select the item, then click Edit. 5. Modify parameters as needed (see the appropriate section for information). 6. Click Save. Delete a library entry IMPORTANT! Auditing of an item depends on whether it is deleted directly from the library or from within another item (for example, you can delete an assay directly from the library, or within a plate in the Assign Plate Contents screen). For more information on auditing, see “Review the object audit history” on page 232. Note: You cannot delete or factory-provided items. Select an item, then click Delete. Deleting a library entry does not affect existing items that contain the entry. (When you select an item to include in a higher-level item, a copy of that item is included in the higher-level item. For example, when you select an instrument protocol to include in an assay, a copy of the instrument protocol is included in the assay. If you delete the instrument protocol, the copy of the instrument protocol in the assay remains intact.) For information on how deleted items are tracked in auditing, see “Audit action” on page 233. 164 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources General library procedures 8 Edit a library entry IMPORTANT! Auditing of an item depends on whether it is edited directly from the library or from within another item (for example, you can edit an assay directly from the library, or within a plate in the Assign Plate Contents screen). For more information on auditing, see “Review the object audit history” on page 232. Note: To edit a plate template, select the template from the main workflow. Go to Define Plate Properties4Open Plate4Edit Existing Template. 1. Select an item, then click Edit. 2. Modify parameters as needed. 3. Click Save. Import and export a library entry To import or export .xml files for use with other instruments that use the same version of the software: • Import—Click Import, then select the .xml file to import. If any items in the import file exist in the library, the software displays a message and gives you the option to replace or skip the item. • Export—Select one or more entries, then click Export, then specify a location for the export file. To select multiple entries, Shift-click to select contiguous entries, Ctrl-click to select non- contiguous entries. IMPORTANT! You must save a plate before you export it. View audit and e‑signature histories for library entries Note: An administrator can also view audit and e-signature histories in the SAE module. For information, see Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)”. To view the audit or e-signature history for a library entry: 1. Select the item in the library. 2. Click View Audit History or View E-Signature History (active only if the selected item is enabled for e-sig). Note: Factory-provided items do not list creation date in the audit history. If you duplicate a factory-provided item, the new item contains an audit history that starts with the duplication date listed as the creation date. 3. For more information, see “Display audit histories” on page 231. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 165 Chapter 8 Manage library resources 8 Plates library Sort and search library entries Sort by one or multiple columns Double-click column headers to sort. To sort by multiple columns: • Double-click a column header to sort the column. • Alt+Shift-click another column header to sort another column. • Alt+Shift-click a third column header to sort a third column. Numbers in the column headers reflect sort order. Search In each library, you can select a category to search, then enter the text to search for. The list of categories corresponds to the column headers in each library. Click Go to search. Click Clear to remove the search criteria. Customize a table Click the Table Settings button, then specify the columns to show or hide. Click: • Apply—To use the settings for this session only. • Save to Preferences—To save for future use by all users. Preferences are saved for the logged-in user. • Restore Defaults—To restore factory-default settings. Plates library The Plates library contains all plates that have been saved in the software (plates that have been run and plates that have not yet been run). 166 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Plates library 8 Plate overview Plate definition A plate associates sample attributes (sample information and analysis information) with a well position. A plate defines how samples are analyzed during capillary electrophoresis and how sample files are named and stored after analysis. When you create a plate, you specify: • Plate type (sequencing, fragment, mixed, or HID) • Number of wells, capillary length, and polymer type When you set up a plate for a run, you add assays, optional file name conventions, and optional results groups to wells in the plate. If you add these items from the library, a copy of the items is added to the plate, and can be modified independently from the original items stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Plate templates The Plates library includes templates that specify the appropriate application type, polymer, and capillary length. You can use these template to create new plates. To create your own templates, see “Create a plate template” on page 97. Plate template names reflect the run module associated with the plate (a plate specifies an assay, an assay specifies an instrument protocol, and an instrument protocol specifies a run module which contains data collection settings). Appendix B, “Run modules and dye sets” lists the run time and size or base range collected for each run module. Create a new plate If you are running a stand-alone version of the software (a version that is not installed on the instrument computer), you can create plates, then export them for use on the instrument computer. 1. Access the Plates library. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 167 Chapter 8 Manage library resources 8 Plates library The list of items in the library may be filtered based on the library filtering user preference. Click Disable Filters to show all items in the list. 2. Click Create. The software switches to the Workflow tab. 3. To create a new plate, specify settings (“Define plate properties” on page 168). To create a new plate based on an existing plate, click New Plate, then select an option. Select a plate, click Open, then specify settings. 4. Select a Save option. Define plate properties Setting Description Plate Details Name Plate name. Names must be unique. Number of Wells • 96 well —For standard 96-well plates standard reaction plates and 8‑strip standard tubes with retainers. • 96 Fast tube—For Fast 96-well plates and Fast 8-strip tubes with retainers. • 384 well—For 384-well plates (24-capillary instruments only) Plate Type Sequencing, Fragment, or Mixed (Sequencing and Fragment). Capillary Length and Capillary length and polymer type with which the plate will be used. Polymer Owner, Barcode, Optional text entries. Description (optional) You can use these entries to search for plates in the Plates library and in run logs (Tools4View Run Logs). Autoanalysis Settings to communicate with secondary analysis software. For information, see the instructions provided with the secondary analysis software. 168 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Assays library 8 Assays library Assay overview An assay contains the instrument protocol (dye set and run module) and primary analysis protocol needed to collect data and basecall or sizecall a sample. Assays, File Name Conventions, and Results Groups may already be listed in the plate template when you create a plate from a template. An assay contains: • One or more instrument protocols appropriate for the sample type/dye set for which the assay will be used • A primary analysis protocol that depends on your application: – Sequencing—Basecalling protocol – Fragment—Sizecalling protocol – HID—QC protocol You must assign an assay to all named sample wells on a plate before you can link a plate and run it. When you create an assay, you add one or more instrument protocols and a primary analysis protocol. If you add these items from the library, a copy of the items is added to the assay, and can be modified independently from the original items stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Create a new assay 1. Access the Assays library. The list of items in the library may be filtered based on the library filtering user preference. Click Disable Filters to show all items in the list. 2. Select an assay. 3. Click Edit. Note: You can also create an assay from the Assign Plate Contents screen. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 169 Chapter 8 Manage library resources 8 Assays library The fields in the Edit Assay dialog box (Figure 15) are described in “Assay settings” on page 171. 170 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Assays library 8 Figure 15 Create new assay Assay settings Setting Description Assay Name Name of the assay. Locked Prevents the item from being edited. Color Color code for the assay when it is displayed in the Assign Plate Contents screen (if Assay Color is selected for Show In Wells). Application Type Sequencing or Fragment. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 171 Chapter 8 Manage library resources 8 Assays library (continued) Setting Description Do you wish to When you select Yes, allows you to select or create additional instrument protocols assign multiple instrument for the assay. The software creates one injection for each instrument protocol protocols to this assay? specified in an assay. Instrument Protocol Instrument protocol for data collection. For information, see “Instrument protocol settings” on page 190. Basecalling Protocol Protocol for basecalling, trimming, and quality determination. (sequencing) For information, see “Basecalling protocol—Analysis settings” on page 203. Sizecalling Protocol Protocol for primary analysis (peak detection and sizing) and quality determination. (fragment analysis) For information, see “Sizecalling protocol—Analysis settings” on page 208 . QC Protocol (HID) Protocol for primary analysis (peak detection and sizing) and quality determination. For information, see “QC protocols library (primary analysis—HID)” on page 211. 172 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources File Name Conventions library 8 File Name Conventions library File name convention overview A File Name Convention (FNC) specifies the naming convention for sample data files. It is an optional component in a plate. If you do not specify a file name convention, data files are named in this format: <sample name>_<well> The file extension is determined by the application you run: • Sequencing—AB1 (you can also set Preferences to export additional file formats. See “Set preferences (optional)” on page 41.) • Fragment analysis—FSA • HID—HID Note: The file location specified in a file name convention is used only if a results group is not specified for a well. When you set up a plate for a run, you can optionally add file name conventions to the plate. If you add this item from the library, a copy of the item is added to the plate, and can be modified independently from the original item stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Create a new file name convention If factory-provided file name conventions do not suit your needs, you can create new file name conventions: 1. Access the File Name Conventions library. 2. Click Create. Note: You can also create a file name convention from the Assign Plate Contents screen. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 173 Chapter 8 Manage library resources 8 File Name Conventions library 3. In the Create New File Name Conventions dialog box (Figure 16), select attributes and delimiters (“File name convention settings” on page 176). IMPORTANT! Enter only alpha-numeric characters in the software. Special characters may not be correctly displayed in some software screens, may cause problems with plate, file, folder, user account, and/or library item names, and may interfere with starting a run and/or importing and exporting library items. As you select attributes, the software displays a preview of the file name. 174 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources File Name Conventions library 8 4. To add delimiters between items in the Selected Attributes list: a. Ctrl-click or Shift-click to select two or more attributes. b. Select a delimiter. c. Select the Add between attributes check box. d. Click Add. 5. Save the file name convention: • If you are creating the file name convention from the Library, click Save. • If you are creating the file name convention from the Assign Plate Contents screen, click Apply to Plate or Save to Library. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 175 Chapter 8 Manage library resources 8 File Name Conventions library Figure 16 Create new File Name convention File name convention settings Setting Description Name Name of the file name convention. Names must be unique. Locked When enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. 176 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources File Name Conventions library 8 (continued) Setting Description Color Color code for the file name convention when it is displayed in the Assign Plate Contents screen (if File Name Convention Color is selected for Show In Wells). Preview of Interactively displays the attributes you select. name Available • Amplicon Name (from Customize Sample • Polymer Type attributes Info in sequencing assays) • Run name • Analysis Protocol Name (primary analysis • Sample Type protocol) • Specimen Name (from Customize Sample Info • Assay Name in sequencing assays) • Capillary Number • Time of Run (run start time) • Custom Text fields (£3) • Unique Time Stamp Integer - (numeric string in • Date of Run milliseconds that does not correspond to the • Injection Number current time) • Instrument Name • User-defined Fields (up to 5; specified in “Assign plate contents” on page 61) • Instrument Protocol • User Name (available only when security is • Owner Name (plate owner) enabled in the SAE module) • Plate Name • Well Position IMPORTANT! The maximum allowed length of a file name, including the path, is 240 characters. The software warns you if your selections will possibly exceed the maximum, but allows you to save the file name convention. However, you will see a pre‑check validation error when you start a run if the file name will exceed 240 characters. Delimiters Symbols you can include in the file name: Dash (-), Dot (.), Underscore (_), Plus (+), Dollar ($). Custom text Text to display for the custom text attribute fields. File location The file location in the file name convention is used only if no results group is specified for a well. The Results Group file location overrides the File Name Convention file location. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 177 Chapter 8 Manage library resources 8 Results Group library Results Group library Results Group overview A Results Group is used to name, sort, and customize the folders in which sample data files are stored. It is an optional component in a plate. Note: The file location specified in a results group overrides the file location in the file name convention specified for a well. When you set up a plate for a run, you can optionally add results groups to wells in the plate. If you add this item from the library, a copy of the item is added to the plate, and can be modified independently from the original item stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Allelic ladder location (HID analysis) To accurately genotype samples, the GeneMapper™ ID-X Software requires at least one allelic ladder sample per run folder. (Multiple allelic ladder samples in a single run folder can also be used for analysis.) We recommend that you run one allelic ladder for a set of 24 samples: • 8-capillary instruments—One allelic ladder per 3 injections • 24-capillary instruments—One allelic ladder per 1 injection Note: Run HID validation studies to determine the required number of allelic ladders for your application. See “Results Group example 3: store one allelic ladder per run folder (8‑capillary instruments)” on page 188 for a results group example that places three injections in each run folder for 8-capillary instruments. 178 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Results Group library 8 Create a new Results Group If factory-provided results groups do not suit your needs, you can create new results groups: 1. Access the Results Group library. 2. Click Create. Note: You can also create a results group from the Assign Plate Contents screen. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 179 Chapter 8 Manage library resources 8 Results Group library 3. In the Create Results Group dialog box (Figure 17 on page ), select attributes and delimiters (“Results group settings” on page 182). As you select attributes, the software displays a preview of the results group name. 4. To add delimiters between items in the Selected Attributes list: a. Ctrl-click or Shift-click to select two or more attributes. b. Select a delimiter. c. Select the Add between attributes check box. d. Click Add. 5. Save the results group: • If you are creating the results group from the Library, click Save. • If you are creating the results group from the Assign Plate Contents screen, click Apply to Plate or Save to Library. The Results Group file location overrides the File Name Convention file location. 180 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Results Group library 8 Figure 17 Create New Results Group 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 181 Chapter 8 Manage library resources 8 Results Group library Results group settings Setting Description Name Name of the results group. Names must be unique. The Results Group Name is a required attribute, you cannot remove this attribute from the Selected Attribute list. Locked When enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. Color Color code for the results group when it is displayed in the Assign Plate Contents screen (if Results Group Color is selected for Show In Wells). Preview of Interactively displays the attributes you select. name Available • Results Group Name (required) • PA Protocol Name (primary attributes • Assay Name analysis=basecalling protocol) • Injection Number • Plate Name • IP Name (instrument protocol) • Prefix • Logged-in User Name (available only • Start Instrument Run Date/time Stamp when security is enabled in the SAE • Suffix module) Delimiters Symbols you can include in the results group name: Dash (-), Dot (.), Underscore (_), Plus (+), Dollar ($). Prefix/suffix Text to display for the prefix or suffix text attribute fields. text Select re- • Store reinjection sample files in a separate reinjection folder (same level as injection folders). injection folder • Store reinjection sample files with original sample files (same level). option 182 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Results Group library 8 (continued) Setting Description Select folder Location: option • Default file location (specified in Preferences4User4Run Setup) • Custom location Sub-folder options: • Include an instrument run name folder (run name can be user-defined in the Load Plates for Run screen) • Include a results group name folder • Include an injection folder Results group example 1: store files by plate name Two default, factory-provided, results groups are provided that store sample data files by plate name: • Figure 18 shows the factory-provided PN_Injfolder_RG results group and the folders created when it is used. This results group creates a folder for each injection. • Figure 19 shows the factory-provided PN_RG results group and the folders created when it is used. This results group does not create a folder for each injection. All samples for a plate are stored in the same folder. If you include two plates in a run, a separate folder is created for each plate. Figure 18 PN_Injfolder_RG results group 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 183 Chapter 8 Manage library resources 8 Results Group library Figure 19 PN_RG results group 184 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Results Group library 8 Results Group example 2: store re-injections in separate folders Figure 20 shows an example results group that specifies a sample file storage location of: C:\Example\instrument run (IR) folder\result group name folder[results group name+start instrument run date/time stamp+logged in user name]\injection name or re-injection name folder. The numbers in the figure relate the elements in the results group with the elements in the file hierarchy created by a run that uses this results group (see Figure 23). Figure 20 Results group example Figure 21 shows the injection list for a run that specifies duplicate and re-injections. The numbers in the figure relate the elements in the injection list with the elements in the file hierarchy created by this run (see Figure 23). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 185 Chapter 8 Manage library resources 8 Results Group library Figure 21 Injection list example Figure 22 shows an example file name convention that specifies a sample name syntax of: sample name.(primary) analysis protocol name.unique time stamp integer The numbers in the figure relate the elements in the file name convention with the files created by a run that uses file name convention (see Figure 23). Figure 22 File name convention example Figure 23 shows the folders and files generated by the results group, file name convention, run name, and injections shown in Figure 20, Figure 21, and Figure 22. 186 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Results Group library 8 Figure 23 Folder hierarchy and file naming example 1 File location from results group 2 Instrument Run Name folder from results group 3 Results group Name folder from results group 4 Injection folder from results group • Duplicate injections ( ) have the same base folder name as the original injection. In the example above, the following injections are duplicates: 1 and 2, 4 and 5. • Re-injections ( ) have the same base folder name as the original injection, and are indicated with _n where n is the number of re-injections. In the example above, Injection 6 is a re-injection of Injection 5. 5 Run name (default or user-defined) from injection list 6 Results group name syntax from results group 7 File name syntax from file name convention 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 187 Chapter 8 Manage library resources 8 Results Group library Results Group example 3: store one allelic ladder per run folder (8‑capillary instruments) We recommend that you run one allelic ladder for each set of 24 samples (see “Allelic ladder location (HID analysis)” on page 178). To store one allelic ladder per run folder on an 8-capillary instrument, create one results group for each set of three injections on the plate. Each results group specifies a results group name folder. Because you assign one results group to a set of three injections, all 24 sample data files, including the allelic ladder, are stored in the same results group folder. The example below shows one results group; for a full 96-well plate, create three more with the same settings, but different names, for example, Injection 4 through 6, Injection 7 through 9, and Injection 10 through 12. 188 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Instrument protocol library 8 Instrument protocol library Instrument protocol overview An instrument protocol contains the parameters that control the instrument during data acquisition. An instrument protocol is a required element of an assay for all applications. When you create an assay, you add one or more instrument protocols to the assay. If you add these items from the library, a copy of the items is added to the assay, and can be modified independently from the original items stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Create a new instrument protocol If factory-provided instrument protocols do not suit your needs, you can create new instrument protocols: 1. Access the Instrument Protocols library. 2. Click Create. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 189 Chapter 8 Manage library resources 8 Instrument protocol library 3. In the Create New Instrument Protocol dialog box (Figure 24), select an application type: Sequencing, Fragment, or HID. The run module selection list is filtered based on the application you select. Figure 24 Create New Instrument Protocol Note: Normalization parameters circled in red are displayed for fragment analysis and HID applications only. 4. Specify settings (“Instrument protocol settings” on page 190). 5. Save the assay: • If you are creating the assay from the Library, click Save. • If you are creating the assay from the Assign Plate Contents screen, click Apply to Plate or Save to Library. Instrument protocol settings Setting Description Application Type • Sequencing • Fragment analysis • HID 190 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Instrument protocol library 8 (continued) Setting Description Capillary Length, Polymer, Capillary length, polymer type, and dye set with which the protocol will be used Dye set Run module Factory-provided modules that specify instrument control parameters. For more information, see Appendix B, “Run modules and dye sets”. Protocol name Name of the protocol. Names must be unique. Locked When SAE is enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. Useful when your system includes the SAE module (described in Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)”. Description Optional text entry. Oven temperature (°C) Temperature setting for main oven throughout run. Run voltage (kVolts) Final sample electrophoresis separation run voltage. Prerun voltage (kVolts) Pre run voltage setting before sample injection. Injection voltage (kVolts) Injection voltage setting for sample injection. Run time (sec) Length of time data is collected after voltage is ramped up to the run voltage and the run starts. PreRun time (sec) Prerun voltage time. Injection time (sec) Sample injection time. Data delay (sec) Time from the start of separation to the start of sample data collection. Advanced options - Do not change unless advised otherwise by Thermo Fisher Scientific support personnel Voltage tolerance (kVolts) Maximum allowed voltage variation. Voltage # of Steps (nk) Number of voltage ramp steps to reach Run Voltage. Voltage step interval (sec) Dwell time at each voltage ramp step. First read out time (ms) The interval of time for a data point to be produced. First ReadOut time should be equal to Second ReadOut time. Second read out time (ms) The interval of time for a data point to be produced. Second ReadOut time should be equal to First ReadOut time. Fragment and HID protocols only: Normalization parameters - Leave at default settings (for information on how these parameters are used, see “Review normalized data” on page 117). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 191 Chapter 8 Manage library resources 8 Dye sets library (continued) Setting Description Normalization Target The expected average RFU for the subset of peaks in the GS600 LIZ™ v2 size standard used for normalization. The default value for each run module has been experimentally determined based on the average peak height of selected peaks in the GS600 size standard with a specific injection time. IMPORTANT! If you change the injection time in an instrument protocol, adjust the Normalization Target proportionately. For example, for an instrument protocol with an injection time of 10 seconds and a Normalization Target of 2000: if you change the injection time to 15 seconds (50% increase), change the Normalization Target to 3000 (50% increase). Normalization Factor The passing range for Normalization Factor (default range is 0.3 to 3.0). Thresholds IMPORTANT! Increasing the factor threshold above 3.0 may cause amplification of noise. If the calculated Normalization Factor is outside the Normalization Factor range, the software multiplies the peak heights of the sample by the low or high Normalization Factor threshold setting (for example, if the Normalization Factor range is 0.3 to 3.0 and the calculated Normalization Factor is 5, the software applies a Normalization Factor of 3.0). Normalization Factor Average peak height of the subset of peaks in the GS600 LIZ™ v2 size standard used for normalization divided by the Normalization Target. Samples are flagged with in results if Normalization Factor is within threshold range, or with if it is out of threshold range. Dye sets library Dye set overview A dye set defines the following for an instrument protocol: • Dye color(s) • Order of the dye peaks in the standard • Spectral analysis parameters When you create an instrument protocol, you add a dye set to the protocol. If you add this item from the library, a copy of the item is added to the assay, and can be modified independently from the original item stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. 192 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Dye sets library 8 Create a new dye set If factory-provided dye sets do not suit your needs, you can create new dye sets: 1. Access the Dye Sets library. 2. Click Create. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 193 Chapter 8 Manage library resources 8 Dye sets library 3. In the Create New Dye Set dialog box (Figure 25), specify settings (“Dye set settings” on page 195). Figure 25 Create New Dye Set 4. Click Save. 194 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Dye sets library 8 Dye set settings Setting Description Dye Set Name Name of the dye set. Names must be unique. Locked When enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. Useful when your system includes the SAE module (described in Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)”). Chemistry The standard for which you are creating the dye set: Sequencing Standard or Matrix standard Dye Set Template Factory-provided template upon which to base the dye set. The Any Dye template can be used for applications that do not use all of the dye colors contained in the matrix standard kits used for spectral calibration. For information, see “Create a new dye set using the AnyDye template” on page 195. Arrange Dyes Displays the dyes and the peak order for the dye set template selected. Editable only for AnyDye template: • Dye Selection—Specifies the dyes to use for calibration • Reduced Selection—Specifies the dyes used in the samples. For example, if you use the 5 dye kit and have samples with only blue peaks, you can "reduce" or deconvolute with blue and orange (size standard) dyes only. Parameters Specifies the Quality Value, Condition Number, Scan, and Sensitivity requirements for the dye set. Notes Optional text entry. Create a new dye set using the AnyDye template If factory-provided dye sets do not suit your needs, you can create new dye sets: 1. Access the Dye Sets library. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 195 Chapter 8 Manage library resources 8 Dye sets library 2. Click Create. Figure 26 Create New Dye Set using the AnyDye template 3. Enter a dye set name. 4. Select a chemistry and the AnyDye dye set template. 5. Select the dye colors that are present in your dye set by selecting or deselecting the checkboxes in the Dye Selection row. An internal ID number is assigned to each selected dye color based on the emission wavelength, from the shortest to the longest wavelength. IMPORTANT! The internal ID numbers are not displayed in the software. Figure 27 illustrates that the internal ID numbers change based on the dyes that are selected (from left to right). The internal ID numbers are shown in parentheses in the examples below for illustrative purposes. 196 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Dye sets library 8 Figure 27 Internal ID examples—IDs that correspond to dye colors are determined by the dyes that are selected (the internal ID numbers shown in the example are not shown in the software) 1 Example 1—With all dyes selected, the internal ID numbers that are assigned to dye colors are: Blue (1), Green (2), Yellow (3), Red (4), Purple (5), Orange (6). 2 Example 2—With the Purple dye deselected, the internal ID numbers that are assigned to dye colors are: Blue (1), Green (2), Yellow (3), Red (4), Orange (5)—the internal ID of the Orange dye changes to 5. 3 Example 3—With the Blue, Yellow, and Purple dyes deselected, the internal ID numbers that are assigned to dye colors are: Green (1), Red (2), Orange (3)—the internal ID of Green changes to 1, Red changes to 2, and Orange changes to 3. 6. Specify the migration order of the dyes in the Calibration Peak Order row. Because the software does not allow you to set the migration order by color, you must enter the internal ID numbers associated with the dyes as determined in the previous step. IMPORTANT! The Calibration Peak Order fields do not correspond to the dye colors displayed above the Calibration Peak Order fields. Figure 28 through Figure 30 show examples of how to set migration order for the selected dyes. The internal ID numbers are shown in parentheses in the examples below for illustrative purposes. Figure 28 Example 1—For migration order of Orange, Red, Yellow, Blue, Green, Purple, specify Calibration Peak Order of 6 (Orange), 4 (Red), 3 (Yellow), 1 (Blue), 2 (Green), 5 (Purple). Figure 29 Example 2—For migration order of Orange, Red, Yellow, Blue, Green, specify Calibration Peak Order of 5 (Orange), 4 (Red), 3 (Yellow), 1 (Blue), 2 (Green). 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 197 Chapter 8 Manage library resources 8 Size standards library Figure 30 Example 3—For migration order of Orange, Red, Green, specify Calibration Peak Order of 3 (Orange), 2 (Red), 1 (Green). Note: The Reduced Selection row contains checkmarks to indicate that the dye will be used for calibration. If a dye is not selected in the Dye Selection row, it is unselected in the Reduced Selection row and is not used for calibration. 7. (Optional) Expand the Parameters section, then specify remaining settings. 8. Click Save. Size standards library Size standard overview A size standard defines the sizes of known fragments. It is used to generate a standard curve. The standard curve is used to determine the sizing of unknown samples. When you create a sizecalling (fragment) or QC (HID) protocol, you add a size standard to the protocol. If you add this item from the library, a copy of the item is added to the protocol, and can be modified independently from the original items stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Normalization size standards provided The library contains factory-provided normalized size standards that you can use to normalize fragment analysis and HID data: • Fragment analysis: – GS600LIZ+Normalization – GS600(60-600)LIZ+Normalization—For applications that have primer peaks that obscure the 20 and 40-mer peaks of the GS600 size standard. • HID: – GS600(80-400)LIZ+Normalization 198 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Size standards library 8 Normalization corrects for instrument, capillary, and injection variability. For each sample, the software calculates a normalization factor based on a threshold setting. The normalization factor is used as a multiplier to adjust the peak height of the sample peaks relative to the GS600 LIZ™ v2.0 size standard peaks. IMPORTANT! Normalization is not applied to samples with failing sizing quality. Select a size standard definition file appropriate for your application that accurately sizes samples. For example, if your application includes small fragments that may be obscured by primer peaks, or large fragments that may not be present due to slower migration rates, specify a size standard definition file that eliminates these fragments from sizing. For more information, see “Review normalized data” on page 117. Create a new size standard If factory-provided size standards do not suit your needs, you can create new size standards: 1. Access the Size Standards library. 2. Click Create. 3. In the Create New Size Standard dialog box (Figure 31), enter a size standard name. 4. (Optional): • Select the Locked check box. When enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. Useful when your system includes the SAE module (for more information, see Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)”). • Enter a description. 5. Select a dye color. 6. Enter sizes in the list on the left. Separate sizes with a comma, space, or return. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 199 Chapter 8 Manage library resources 8 Size standards library 7. Click Add Sizes. 8. Click Save. Figure 31 Create New Size Standard Modify a factory-provided normalization size standard 1. Select a factory-provided normalization size standard (indicated in the name with “+Normalization.”). 2. Click Duplicate. 3. Edit the copy of the normalized size standard. The size standard peaks used to normalize the data are displayed in gray and are not editable. 4. Click Save. 200 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Basecalling protocols library (primary analysis—sequencing) 8 Basecalling protocols library (primary analysis— sequencing) Basecalling protocol overview A basecalling protocol is the required primary analysis protocol for sequencing applications. A basecalling protocol defines the settings used by the sequencing basecallers to assign base calls to each detected peak and assign a quality value: • Analysis settings • Ranges for the sequencing quality flags displayed in View Results When you create a sequencing assay, you add a basecalling protocol to the assay. If you add this item from the library, a copy of the item is added to the assay, and can be modified independently from the original item stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Create a new basecalling protocol If factory-provided basecalling protocols do not suit your needs, you can create new basecalling protocols: 1. Access the Basecalling Protocols library. 2. Click Create. 3. In the Analysis Settings tab of the Create New Basecalling Protocol dialog box (Figure 32), specify settings “Basecalling protocol—Analysis settings” on page 203. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 201 Chapter 8 Manage library resources 8 Basecalling protocols library (primary analysis—sequencing) 4. Click QV Settings. In the QV Settings tab of the Create New Basecalling Protocol dialog box (Figure 33), specify settings (see “Basecalling protocol—QV settings” on page 205). QV settings are quality value ranges used in the following screens: • Monitor Run screen—The state of the QV flag: – If all three values are in the pass range, the QV flag in Monitor Run is set to (green). – If any values are in the suspect range, the QV flag in Monitor Run is set to (yellow). – If any values are in the fail range, the QV flag in Monitor Run is set to (red). • View Sequencing Results4Metric Analysis Results table—The pass/check/fail status for Trace Score Quality, CRL Quality, and QV20+ Quality results. 5. Click Save. Figure 32 Create New Basecalling Protocol—Analysis Settings 202 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Basecalling protocols library (primary analysis—sequencing) 8 Figure 33 Create New Basecalling Protocol—QV Settings Basecalling protocol—Analysis settings Setting Description Name Name of the protocol. Names must be unique. Locked When SAE is enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. Useful when your system includes the SAE module (described in Chapter 9, “Use Security, Audit, and E-Sig functions (SAE Module)”. Description Optional text entry. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 203 Chapter 8 Manage library resources 8 Basecalling protocols library (primary analysis—sequencing) (continued) Setting Description Basecaller Basecalling algorithm used to identify bases. Note: The basecaller version listed in the basecalling protocol is a 3-digit number. The version listed in sequencing results is a 4-digit number. The fourth digit is an internal number used by the software. Mobility file Compensates for mobility differences between dyes used to label the DNA. Quality • Basecall Assignment (ambiguous bases): Threshold – Do not assign Ns to basecalls – Assign Ns to basecalls with QV<15—Bases with a QV less than the threshold display N instead of the base letter • Ending base—Last base on which to perform basecalling: • At PCR Stop • After X number of Bases • After X number of Ns in X number of Bases • After X number of Ns Note: If you have PCR products with sequences that end while data is still being collected, select the At PCR Stop check box. Mixed bases When enabled, determines the secondary peak height ratio where the secondary peak is threshold considered a potential mixed base. Reaching the threshold is a necessary but not sufficient condition for the basecalling algorithm to call a mixed base. 204 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Basecalling protocols library (primary analysis—sequencing) 8 (continued) Setting Description Analyzed Data Determines scaling of the processed traces. This parameter does not affect the accuracy of the Scaling basecalling. • True Profile—The processed traces are scaled uniformly so that the average height of peaks in the region of strongest signal is about equal to a fixed value. The profile of the processed traces will be very similar to that of the raw traces. • Flat Profile—The processed traces are scaled semi-locally so that the average height of peaks in any region is about equal to a fixed value. The profile of the processed traces will be flat on an intermediate scale (> about 40 bases). Clear range • Use clear range minimum and maximum—Specifies the first and last base in the range to methods consider, or trims the specified number of bases from the 3¢ end. • Use quality values—Sets a window with a specified number of allowed low-quality bases by removing bases until there are < X number of bases per Z number of bases with QV < Y. • Use identification of N cells—Sets a window with a specified number of allowed ambiguous base calls (Ns) by removing bases until there are < X number of Ns per Y number of bases. Basecalling protocol—QV settings Setting Description Contiguous The longest uninterrupted segment of basecalls with an average Quality Value (QV) ≥20. Read Length In addition to evaluating the QV of a basecall, the software considers the QV of adjacent basecalls within a 21-bp moving window to determine a contiguous read length based on quality values: the software starts from the 5' end and calculates the average QV across a moving window size of 21, sliding 1 bp at a time, to the 3' end. The resulting longest contiguous segment is determined as the CRL. Trace Score The average basecall Quality Value (QV) of basecalls in the clear range sequence of a trace. The clear range is the region of the sequence that remains after excluding the low-quality or error-prone sequence at the 5′ and 3′ ends. The clear range is calculated by the KB Basecaller using QVs. QV20+ The total number of bases in the entire trace with Quality Value ≥20. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 205 Chapter 8 Manage library resources 8 Sizecalling protocols library (primary analysis—fragment) Sizecalling protocols library (primary analysis—fragment) Sizecalling protocol overview A sizecalling protocol is the required primary analysis protocol for fragment applications. A sizecalling protocol defines peak detection, sizing, and quality values. When you create a fragment assay, you add a sizecalling protocol to the assay. If you add this item from the library, a copy of the item is added to the assay, and can be modified independently from the original item stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. Create a new sizecalling protocol 1. Access the Sizecalling Protocols library. 2. Click Create. 3. In the Analysis Settings tab of the Create New Sizecalling Protocol dialog box (Figure 34), specify settings (see “Sizecalling protocol—Analysis settings” on page 208). 4. Click QC Settings. In the QC Settings tab of the Create New Sizecalling Protocol dialog box (Figure 35), specify settings (“Sizecalling protocol—QC settings” on page 211). 206 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Sizecalling protocols library (primary analysis—fragment) 8 5. Click Save. IMPORTANT! Normalization is not applied to samples with Size Quality flags. Specify analysis settings that accurately detect and size the size standard, and QC settings with appropriate pass fail ranges. The software does not support re-analyzing data with new settings. Figure 34 Create New Sizecalling Protocol—Analysis Settings 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 207 Chapter 8 Manage library resources 8 Sizecalling protocols library (primary analysis—fragment) Figure 35 Create New Sizecalling Protocol—QC Settings Sizecalling protocol—Analysis settings Setting Description Protocol Name of the protocol. Names must be unique. Name Description Optional text entry. Size standard Size standard definition in the software that corresponds to the dye set used in the chemistry. To apply normalization, select a normalization size standard (see “Normalization size standards provided” on page 198). 208 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources Sizecalling protocols library (primary analysis—fragment) 8 (continued) Setting Description Analysis The range (in data points) to analyze: Range • Full Range to analyze the entire scan region as collected by the genetic analysis instrument, including the primer peak. • Partial Range to analyze only data points within a specified range. Enter Start Point in data points after the primer peak and before the first required size standard peak. Enter a Stop Point after the last required size standard fragment. Start and Stop points may vary from instrument to instrument and platform to platform. Display raw data to determine the appropriate analysis range. Data points outside the specified analysis range are ignored. Note: Ensure the Analysis Range contains all size standard fragments included in the Sizing Range specified below. Sizing Range The size range (in base pairs) appropriate for the kit you are using: • All Sizes for the software to analyze fragments of all sizes in the Analysis Range. • Partial Sizes for the software to analyze only fragments within a specified range. Enter a Start Size and a Stop Size appropriate for the size standard used. Size Calling • Local Southern—(default) Determines the fragment sizes using the reciprocal relationship Method between fragment length and electrophoretic mobility. • 3rd Order Least Squares—Uses regression analysis to build a best-fit size calling curve. • 2nd Order Least Squares—Uses regression analysis to build a best-fit size calling curve. • Cubic Spline Interpolation—Forces the sizing curve through all the known points of the selected size standard. • Global Southern Method—Compensates for standard fragments with anomalous electrophoretic mobility (similar to least squares methods). Primer Peak If the primer peaks in your application obscure peaks of interest, select Present. Selecting Present instructs the algorithm to ignore primer peaks. Primer peaks are still displayed in the trace. Note: If this setting does not allow detection of the 20- and 40-mer peaks for samples that use the GS600 LIZ™ size standard, running samples with the GS600(60‑600)LIZ+Normalization may allow detection of the peaks. Peak Specify the threshold (RFU) for peak detection for each dye color. Peaks below the threshold are Amplitude not detected. Thresholds For example, if you use the default values of 175, peaks with heights equal to or greater than 175 are detected. Peaks with heights below 175 are still displayed in the electropherogram plots but are not detected or labeled. Note: Use the same peak amplitude thresholds in secondary analysis software. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 209 Chapter 8 Manage library resources 8 Sizecalling protocols library (primary analysis—fragment) (continued) Setting Description Smoothing Select an option to smooth the outline of peaks and reduce the number of false peaks detected: • None (default) to apply no smoothing. Best if the data display sharp, narrow peaks of interest. • Light to provide the best results for typical data. Light smoothing slightly reduces peak height. • Heavy for data with very sharp, narrow peaks of interest. Heavy smoothing can significantly reduce peak height. Baseline Specify a window to adjust the baseline signals of all detected dye colors to the same level for an Window improved comparison of relative signal intensity. Note the following: • A small baseline window relative to the width of a cluster, or grouping of peaks spatially close to each other, can result in shorter peak heights. • Larger baseline windows relative to the peaks being detected can create an elevated baseline, resulting in peaks that are elevated or not resolved to the baseline. Min. Peak Half Specify the minimum full peak width at half maximum Peak Height required for peak detection. Width The range is 2 to 99 data points. Polynomial Polynomial Degree cannot be greater than Peak Window Size. Degree Adjust to affect the sensitivity of peak detection. You can adjust this parameter to detect a single base pair difference while minimizing the detection of shoulder effects and/or noise. The peak detector calculates the first derivative of a polynomial curve fitted to the data within a window that is centered on each data point in the analysis range. Using curves with larger polynomial degree values allows the curve to more closely approximate the signal and, therefore, captures more of the peak structure in the electropherogram. Peak Window Enter a window width in data points for peak detection sensitivity. If more than one peak apex is Size within the window, all are labeled as a single peak. Note the following: • The maximum value is the number of data points between peaks. • The Peak Window Size setting is limited to odd numbers. To increase peak detection sensitivity: Increase polynomial degree, decrease peak window size. To decrease peak detection sensitivity: Decrease polynomial degree, increase peak window size. Slope • Peak Start—The peak starts when the first derivative (slope of the tangent) in the beginning Thresholds of the peak signal before the inflection point becomes equal to or exceeds the “Peak Start” Peak Start value. This threshold is set to 0 by default, which means that the peak will normally start at and End the leftmost point where the slope of the tangent is closest to 0° (horizontal line). A value other than 0 moves the peak start point toward its center. The value entered must be non-negative. • Peak End—The peak ends when the first derivative (slope of the tangent) in the end of the peak signal after the inflection point becomes equal to or exceeds the “Peak End” value. This value is set to 0 by default, which means that the peak will normally end at the rightmost point where the slope of the tangent is closest to 0° (horizontal line). A value other than 0 moves the peak end point toward its center. The value entered in this field must be non-positive. 210 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources QC protocols library (primary analysis—HID) 8 Sizecalling protocol—QC settings Setting Description Size Quality The Pass Range and the Fail Range for the SQ flag displayed in View Fragment Results. Results that are within the Pass range are flagged as (Pass). Results that are within the Fail range are flagged as (Fail). Results that are between the Pass and Fail ranges are flagged (Check). For example, with a Pass Range of 0.75 to 1.0 and a Fail Range of 0.0 to 0.25, any result ³0.75 is , any result <0.25 is , and any result between ³0.25 to <0.75 is . How Size Quality is determined The Size Quality algorithm evaluates the similarity between the fragment pattern for the size standard dye specified in the size standard definition and the actual distribution of size standard peaks in the sample, calculates an interim SQ (a value between 0 and 1). Assume Defines the expected linear range. Useful in large fragment size standards where non-linearity might Linearity be expected. Pull-Up The pull-up ratio and tolerance for pull-up peak identification. A pull-up peak is identified when the peak height of the minor peak is: • £X% (pull-up ratio) of the major peak and • Within ±Y data point (pull-up scan) of the major peak When at least one peak is identified as a pull-up peak, the (Check) flag is displayed for the Spectral Pull-Up quality flag in View Fragment Results. QC protocols library (primary analysis—HID) QC protocol overview A QC protocol is the required primary analysis protocol for HID applications. A QC protocol defines peak detection, sizing, and quality values. When you create an HID assay, you add a QC protocol to the assay. If you add this item from the library, a copy of the item is added to the assay, and can be modified independently from the original item stored in the library. For information on how changes are tracked if auditing is enabled, see “Audit action” on page 233. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 211 Chapter 8 Manage library resources 8 QC protocols library (primary analysis—HID) Create a new QC protocol If factory-provided QC protocols do not suit your needs, you can create new QC protocols: 1. Access the QC Protocols library. 2. Click Create. 3. In the Analysis Settings tab of the Create New QC Protocol dialog box (Figure 36), specify settings (“QC protocol—Analysis settings” on page 214). 4. Click QC Settings. In the QC Settings tab of the Create New QC Protocol dialog box (Figure 37), specify settings (“QC protocol—QC settings” on page 217). 212 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources QC protocols library (primary analysis—HID) 8 5. Click Save. IMPORTANT! The default values in the QC protocol templates (other than peak amplitude threshold values) have been optimized for each kit. You must optimize and validate peak amplitude threshold values during internal HID validation. If you modify other settings, ensure that the size standard is accurately detected and sized with the new settings. IMPORTANT! Normalization is not applied to samples with Size Quality flags. The software does not support re-analyzing data with new settings. Figure 36 Create New QC Protocol—Analysis Settings 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 213 Chapter 8 Manage library resources 8 QC protocols library (primary analysis—HID) Figure 37 Create New QC Protocol—QC Settings QC protocol—Analysis settings Setting Description Protocol Name Name of the protocol. Names must be unique. Description Optional text entry. Size standard Size standard definition in the software that corresponds to the dye set used in the chemistry. To apply normalization, select a normalization size standard (see “Normalization size standards provided” on page 198). Analysis Range Select Full to collect data points for the entire scan region, including the primer peak. You can specify a limited analysis range in the GeneMapper™ ID‑X Software. Note: If you select Partial, ensure that the Analysis Range contains all size standard fragments included in the Sizing Range specified below. 214 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources QC protocols library (primary analysis—HID) 8 (continued) Setting Description Sizing Range Select Partial, then specify 80 to 400 to limit the fragment sizes evaluated for the size standard. If you specify sizes outside this range, the Sizing Quality may fail. Size Calling Method Select the method to determine the molecular length of unknown fragments appropriate for the AmpFℓSTR™ kit you use: • Local Southern—(default) Determines the fragment sizes using the reciprocal relationship between fragment length and electrophoretic mobility. The unknown fragment is surrounded by two known-sized fragments above and one below, then two below and one above. The results are averaged and the size of the allele is determined. • 3rd Order Least Squares—Uses regression analysis to build a best-fit size calling curve. • 2nd Order Least Squares—Uses regression analysis to build a best-fit size calling curve. • Cubic Spline Interpolation—Forces the sizing curve through all the known points of the selected size standard. • Global Southern Method—Compensates for standard fragments with anomalous electrophoretic mobility (similar to least squares methods). IMPORTANT! If you modify peak detection settings, ensure that the size standard is accurately detected and sized with the new settings. Normalization is not applied to samples with Size Quality flags. The software does not support re-analyzing data with new settings. For more information on peak detection parameters, see the GeneMapper™ ID‑X Software Reference Guide. Use Smoothing Select an option to smooth the outline of peaks and reduce the number of false peaks detected: • None to apply no smoothing. Best if the data display sharp, narrow peaks of interest. • Light (default) to provide the best results for typical data. Light smoothing slightly reduces peak height. • Heavy for data with very sharp, narrow peaks of interest. Heavy smoothing can significantly reduce peak height. Use Baselining Specify a window to adjust the baseline signals of all detected dye colors to the same level for an improved comparison of relative signal intensity. Note the following: • A small baseline window relative to the width of a cluster, or grouping of peaks spatially close to each other, can result in shorter peak heights. • Larger baseline windows relative to the peaks being detected can create an elevated baseline, resulting in peaks that are elevated or not resolved to the baseline. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 215 Chapter 8 Manage library resources 8 QC protocols library (primary analysis—HID) (continued) Setting Description Peak Amplitude IMPORTANT! Optimize these thresholds during internal HID validation. Threshold Specify the threshold (RFU) for peak detection for each dye color. Peaks below the threshold are not detected. For example, if you use the default values of 175, peaks with heights equal to or greater than 175 are detected. Peaks with heights below 175 are still displayed in the electropherogram plots but are not detected or labeled. Note: Use the same peak amplitude thresholds in secondary analysis software. Minimum Peak Half Specify the smallest half peak width at full height for peak detection. The range is 2 to 99 Width data points. Polynomial Degree Adjust to affect the sensitivity of peak detection. You can adjust this parameter to detect a single base pair difference while minimizing the detection of shoulder effects and/or noise. The peak detector calculates the first derivative of a polynomial curve fitted to the data within a window that is centered on each data point in the analysis range. Using curves with larger polynomial degree values allows the curve to more closely approximate the signal and, therefore, captures more of the peak structure in the electropherogram. Peak Window Size Enter a window width in data points for peak detection sensitivity. If more than one peak apex is within the window, all are labeled as a single peak. Note the following: • The maximum value is the number of data points between peaks. • The Peak Window Size setting is limited to odd numbers. To increase peak detection sensitivity: Increase polynomial degree, decrease peak window size. To decrease peak detection sensitivity: Decrease polynomial degree, increase peak window size. Slope Thresholds Not recommended for use with AmpFℓSTR™ kit data. Peak Start • Peak Start—The peak starts when the first derivative (slope of the tangent) in the Slope Thresholds beginning of the peak signal before the inflection point becomes equal to or exceeds Peak End the “Peak Start” value. This threshold is set to 0 by default, which means that the peak will normally start at the leftmost point where the slope of the tangent is closest to 0° (horizontal line). A value other than 0 moves the peak start point toward its center. The value entered must be non-negative. • Peak End—The peak ends when the first derivative (slope of the tangent) in the end of the peak signal after the inflection point becomes equal to or exceeds the “Peak End” value. This value is set to 0 by default, which means that the peak will normally end at the rightmost point where the slope of the tangent is closest to 0° (horizontal line). A value other than 0 moves the peak end point toward its center. The value entered in this field must be non-positive. 216 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 8 Manage library resources QC protocols library (primary analysis—HID) 8 QC protocol—QC settings Setting Description Size Enter the Pass Range and the Fail Range for the SQ flag displayed in View HID Results. Quality Results that are within the Pass range are flagged as (Pass). Results that are within the Fail range are flagged as (Fail). Results that are between the Pass and Fail ranges are flagged (Check). For example, with a Pass Range of 0.75 to 1.0 and a Fail Range of 0.0 to 0.25, any result ³0.75 is , any result <0.25 is , and any result between ³0.25 to <0.75 is . How Size Quality is determined The Size Quality algorithm evaluates the similarity between the fragment pattern for the size standard dye specified in the size standard definition and the actual distribution of size standard peaks in the sample, calculates an interim SQ (a value between 0 and 1). Weighting The Broad Peak (BD) threshold specified in the QC Protocol - QC Settings tab affects the SQ. To determine the final SQ value, the software: • Evaluates size standard peak widths in the sample in the dye color specified in the size standard definition. • If the width of any size standard peak in the sizing range exceeds the broad peak threshold, applies a 0.5 weighting factor: • Interim SQ × (1 – 0.5) Note: The GeneMapper™ ID‑X Software allows you to set broad peak weighting. For more information, see the GeneMapper™ ID‑X Software Reference Guide. Broad Enter the maximum peak width (in base pairs). Peak When a peak width is greater than the threshold, the (Check) flag is displayed for the BD (Broad Peak) quality flag in View HID Results. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 217 9 Use Security, Audit, and E-Sig functions (SAE Module) ■ Administrators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 ■ Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Administrators Administrators overview of system security auditing and electronic signature This option is available if your system includes a license for the Security, Audit, and E-Signature module. The Security, Audit, E-Signature module (SAE module) provides the following functionality: • System security—Controls user access to the software. A default Administrator user account is provided, and additional user accounts and permissions can be user-defined. • Auditing—Tracks changes made to library items, actions performed by users, and changes to the SAE settings. The software automatically audits some actions silently. You can select other items for auditing and specify the audit mode. Provides reports for audited library items, SAE changes, and actions. • Electronic signature (e-sig)—Determines if users are permitted, prompted, or required to provide a user name and password when performing certain functions. Can be configured so that a predefined list of functions can be performed only if the data used for the functions is signed (for example, you can run a plate only if the calibration data for the system has been signed). Example applications You can configure the SAE module in a variety of ways: • Require multiple e-sigs. • Require specific users or users with specific permissions to e-sign. • Allow only certain users to approve reviewed samples. 218 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Configure the security system Access the Security screen The Security screen allows you to control restrictions and security policies for all user accounts, and set up notifications when certain security events occur. Access the Security screen. Figure 38 Security screen 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 219 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Set account setup and security policies Security policies apply to all user accounts. 1. Under Account Setup, specify user name limits. IMPORTANT! The software allows spaces in user names (Define name spacing). Use spaces in user names with caution. For information, see “Spaces in user names” on page 221. 2. Specify the allowed characters in user names: spaces and alpha, numeric, upper/ lower case, and special characters (@, commas, periods, semicolons, dashes, underscores, and tildes). 3. Specify password limits and whether users can paste copied text into the password field. 4. Specify the required characters in passwords: spaces and alpha, numeric, upper/ lower case, and special characters (any non-space, non-alpha, or non-numeric characters). 5. Specify password reuse. You cannot disable the password reuse restriction. Note: Do not disable the Account Suspension feature. 6. Click Setup Messaging Notification to specify when and how to notify the administrator of certain security events. For information, see “Set up messaging notifications” on page 221. 7. Click Save Settings. The new settings are applied to the logged-in user the next time the user logs in. 220 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Spaces in user names If you allow spaces in user names, be aware of the following issues: • Leading and trailing spaces in user names are difficult to detect on the screen or in printed reports. • The number of consecutive spaces in a user name is difficult to determine on the screen or in printed reports. Spaces in user names may cause confusion when searching for an audit or e-sig record associated with a user name. To find a record associated with a user name, you must specify the user name exactly, including leading, consecutive, and trailing spaces. Set up messaging notifications 1. In the Security screen (Figure 38), click Setup Messaging Notifications to display the Setup Notifications dialog box. 2. Select the events for notification: • Number (#) of failed authentications over specified time interval—A user attempts to log in with an incorrect password. The message indicates the number of failed authentications. • Session timeout for a user—No activity occurred in a user account for the specified period of inactivity. • Account suspension for failed authentication—The user exceeds maximum number of allowed failed authentications (login attempts with an incorrect password). • Notification for SAE activation —Not supported. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 221 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators 3. Select the notification method: • Pop-up dialog—The software immediately displays a pop-up message to the current user if an event is triggered by the current user. The message instructs the user to inform a system administrator of the event. • Message when Admin logs in—If an event triggers notification, the next time any user with an Administrator role logs in, the software displays a list those events, indicating the time each event occurred and the user who triggered the event. The Administrator has the option of acknowledging the event, which removes it from the notification list. 4. Click OK. Manage user accounts Create or edit a user account The software includes a default Administrator user account with permissions (defined by the account user role) to perform all functions in the software. Create a user account 1. Access the Users screen. 222 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 2. Click Create to display the New User dialog box. 3. Enter User Name, Password, First Name, MI (middle initial – optional) and Last Name. Click a field to display the field limits, which are specified in Security settings. Note: First Name, MI (middle initial), and Last Name are used to create User Full Name, which is displayed as the name of the logged-in user. Note: You cannot change the User Name after you save the user account. 4. Select Pre-expired to require the user account to specify a new password at first log in. The Password Expires On date is specified in Security settings. 5. Select the user role (described in “Create or edit a user role” on page 225) and the electronic signature state (determines if a user account has permission to electronically sign objects). Leave the status set to Active. Note: The Dx User function is not supported. 6. (Optional) Enter email (for information only), phone, and comments. 7. Click Save. If the Save button is dimmed, it indicates an invalid entry in a field. Click a field to display the limits for the field, then enter a valid entry. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 223 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators The Users screen displays the following information for each user account: • User • Last Modified On • Full Name • Created Date • Dx User (not supported in research use • Password Change Date (by either user or only mode.) administrator) • Role • Email (for records only) • Status • Phone • Password Expired (true=yes, false=no) • Comments Edit a user account 1. In the Users screen, select a user account, then click Edit. Note: If you select multiple users, only Status and Role will be changed. 2. Edit settings as needed. You cannot edit the user name of an existing user. 3. Click Save. Activate a suspended user account 1. Select the user. 2. Click Edit. 3. Change the status from Suspended to Active. Delete (inactivate) a user account You cannot delete a user because user records are required for auditing. To disable a user account, inactivate it. 1. Select the user. 2. Click Edit. 3. Change the status from Active to Inactive. 4. Click Save. 224 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Determine the name of the logged-in user To display the full name of the logged-in user: Place the mouse pointer on the Logout menu. The full name of the logged-in user is also displayed in the Load Plates for Run screen and the Monitor Run screen. Create or edit a user role User roles determine the permissions associated with a user account. Three default user roles are included in the software. You can modify two of them, and can create your own roles with customized settings as needed: • Administrator (cannot be edited or deleted) • Scientist • Technician To determine the permissions for these roles or to edit these roles, select the role, then click Edit. Create a user role 1. Access the Roles screen. 2. Click Create. 3. Enter a role name and (optional) comment. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 225 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators 4. Select permissions (described below). To select all permissions in a category, select the checkbox next to the category. 5. Click Save Role. User role permissions Category Permissions Setup Create plate/plate template Run • Start plate run • Edit default instrument run name • Manage injection list • Duplicate injection • Re-inject Primary Analysis • Edit sample (names) • Export sequencing results • Assay • Create • File name convention • Edit • Results group • Delete • Instrument protocol • Import • PA protocol • Export • QC protocol • Size standard • Dye sets Plates and templates • Edit • Delete • Import • Export Locking/Unlocking • Assays • File name convention • Results group • Instrument protocols • PA protocols • Size standards • Dye sets 226 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 (continued) Category Permissions Preferences • Edit system preferences • Export system preferences • Import system preferences • Edit user preferences • Import user preferences • Export user preferences (all) Calibrations • Perform spatial calibration • Perform spectral calibration Install check Run install standard check Archiving • Archive • Purge • Restore SAE configuration Log in to timed-out user sessions Edit a user role 1. In the Roles screen, select a user role, then click Edit. 2. Edit settings as needed. You cannot edit the Administrator user role. 3. Click Save. View and print a user report 1. Select the User or Roles tab. Click View Report. 2. In the Report screen, click toolbar options to manipulate the report as needed. Place the mouse pointer over an item for a description of the item. 3. To print the report, click Print. Close the report. Save electronic copies (PDF) of the report To save the report electronically (PDF), print the report and select a PDF printer. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 227 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Manage auditing Access the Audit Settings screen The Audit Settings screen controls the events that are audited, and the reasons available to users when audit mode is set to Prompt or Required. Access the Audit Settings screen. 228 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Figure 39 Audit Settings 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 229 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Select objects to audit 1. Select the objects and/or actions to audit. Objects you can select for auditing (audit records displayed in Actions you can select for auditing Object Audit History): (audit records displayed in Action • Dye set Log): • Size standard • Export assay • Instrument protocol • Export plate record • PA protocol (primary analysis) • Assay • Plate template • File name convention • Results group • Plate • Sample files Note: For a list of items that the system audits silently in addition to the configurable items listed above, see “Generate audit reports” on page 231. 2. Set the Audit Mode for each item you enable for auditing: • Prompt—The event is audited, a reason prompt is displayed, but the user can cancel and continue without entering a reason. • Required—The event is audited, a reason prompt is displayed, and the user must specify a reason. • Silent—The event is audited, no reason prompt is displayed. 3. Click Save Settings. 230 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Create audit reason settings You can create, modify and delete the reasons that are available for selection in the Audit Reason dialog box (displayed when a user performs an audited action). 1. To require users to select a pre-defined reason in the Audit Reason dialog box (displayed when a user performs an audited action), select the Select a reason from the list for your change checkbox. Users are not permitted to enter a reason. 2. As needed, click Create, or select a reason, then click Edit or Delete. Generate audit reports Display audit histories 1. Access the Audit Reports screen. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 231 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Note: To access the Audit Reports screen, the user role for an account must specify the Configure SAE permission. Users without the Configure SAE permission can view object audit histories for individual entries in the libraries by selecting entries, then clicking View Audit History (see “View audit and e‑signature histories for library entries” on page 165). 2. Select a tab to display: • Object Audit History—The most recent audit for all user objects (samples and objects in the Library) that have been audited. • System Configuration History—SAE configuration records, including audit history for each user account. • Action log—System-specified audit events. 3. (Optional) Modify the display: • Sort the table. See “Sort by one or multiple columns” on page 94. • Specify filters (date range, user name, action, object or record type, object or record name, reason), then click Go. Note: The Reason field in System Configuration History is not used. • Select a record, then click Show Object History or Show Audit Details. • In the history dialog box, select a record, then click Show Audit Details. • Click Table Settings, then specify the columns to show or hide. Review the object audit history Audit records The Object Audit History lists the most recent audit for the user objects listed below (samples and objects in the libraries) that have been audited. • Dye set • Plate template • Size standard • File name convention • Instrument protocol • Results group • PA protocol (primary analysis) • Plate • Assay • Sample files 232 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Audit action Possible actions for all objects are: update, create, and delete. Audit records are generated under the following conditions: Action Description Update The auditing of updates depends on whether an object is modified or overwritten: • Modified—A record is created when an object is modified. • Updated—A record is not created when an object is overwritten in the library. Example: You create a plate, then create a results group from within the plate and save it to the library. You then open the plate, edit the results group from within the plate, then save it to the library. A message indicates that the results group already exists and asks if you want to overwrite it. You click Yes. This action is considered a creation of a new results group, not a modification of the existing results group. No Update record is created; a Create record is created. Create A record is created when you: • Create an item in the library. • Create an item from within another item. • Modify an item from within another item, then overwrite the item in the library when you save it (as described in the "Updated" bullet above). Note: An audit record is not created when a sample file is generated. However, an audit record is generated when a sample is renamed. Delete The auditing of deletions depends on the item deleted: • Items in the library—A record is retained until it is deleted from the library. The deletion of the item from the library is not audited. For example, if you delete a size standard from the library, no audit record for the deletion is listed in the Object Audit Detail History. • Items within other items—The deletion of an item from within another item is audited. Display the object history The object history shows the audit history for the object and for all objects contained in the selected object. For example, when you create an assay, a copy of the instrument protocol and the primary analysis protocol (and therefore dye set, and size standard) are included in the assay object. The objects contained within an object have audit histories distinct from the audit history of the objects stored in the Library. To display the history for an object: Select the object, then click Show Object History. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 233 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators System configuration history Table 1 Audit – System configuration history Record Type Action Corresponds to Security settings Update • Enable security • Disable security • Modify security policies: – Session timeout settings Account settings Update • Modify user name settings • Modify password settings • Modify security policies: – Password expiration – Account suspension Audit reason for change Update Modify reason for change Create Create reason for change Delete Delete reason for change Audit settings Update • Enable auditing • Disable auditing Audit type Update Modify audit settings E-Signature function Update • Modify the authorities for a “prompt before” function • Modify the Enable state of either a “check after” or “prompt before” function E-Signature settings Update • Enable e-signature • Disable e-signature E-Signature type Update • Modify e-signature settings • Modify the enable state of an E-Signature Type Role assignment Create • Create a new user account • Assign a different user role to an existing user account Delete Assign a different user role to an existing user account Role permissions Update Modify user role permissions Create Create a user role - Creates one role assignment record for each permission in a role Delete Delete a user role - Creates one role delete record for each permission in the deleted role 234 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Table 1 Audit – System configuration history (continued) Record Type Action Corresponds to User account Update • Edit • Suspend Create Create new user account User role Update Modify user role Create Create user role Delete Delete user role Action log The action log lists system-specified audit events. All items in the action log are audited silently, except for the items noted as configurable. Configurable items may include comments in the action log. Table 2 Audit – Action log Category Action Assay Assay exported successfully Log In • User logged in • Login failed • User logged out Maintenance Wizards • Remove Bubbles Wizard started • Change Polymer Type Wizard started • Change Array Wizard started • Replenish Polymer Wizard started • Fill Polymer Wizard started • Water Wash Wizard started • Instrument Shutdown Wizard started Plate Plate exported successfully Run • Start • Pause • Resume • Stop (Abort injection) • Terminate (injection list) SAE Configuration • Export 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 235 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Table 2 Audit – Action log (continued) Category Action System Audit Records • Archive • Purge • Restore System Action Records • Archive • Purge • Restore User Profile • Export View and print audit reports 1. Display the records of interest. 2. Filter the list to decrease the time required to generate reports. IMPORTANT! You cannot cancel a report after you click a view button. 3. Click View Audit Summary Report or View Audit Detailed Report. 236 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 4. (Optional) In the Report screen, click toolbar options to manipulate the report. Place the mouse pointer over an item for a description of the item. 5. To print the report, click Print. IMPORTANT! Font setting changes are activated after you close, then reopen the report. IMPORTANT! If you change font settings before you generate a report, the report may not be generated. Generate the report again. 6. To save the report electronically (PDF), print the report and select a PDF printer. Archive, purge, and restore audit records The audit archive function makes a copy of audit records. Purge makes a copy of audit records, and then deletes them. You can use the Restore function to restore purged audit records. Archive and purge To selectively archive or purge (delete) system configuration or action audit records: 1. Select records in the appropriate screen. 2. Click Archive Audit Records or Purge Audit Records. 3. If you select Archive: • Specify a location and name for the .asz audit archive file. • (Optional) Click Yes to Purge (delete) the records after archive. Restore To restore system configuration or action audit records, click Restore, then select the .asz file to restore. Export audit records As needed, you can export audit records to a TXT file for additional manipulation and reporting outside the software. 1. Display the records of interest. 2. Select the records to export. 3. Click Export Audit Records. 4. Specify a name and location for the export TXT file. 5. Click Save. Note: If you export audit records for samples that are not in their original location (samples have been deleted or moved), an error message is displayed. Return sample data files to their original location, then export again. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 237 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Manage electronic signature (E-sig) IMPORTANT! Changes to e-signature settings are not activated until you log out of the software, then log back in. Access the E‑Signature Settings screen Access the E-Signature Settings screen. 238 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Select the actions that allow signature IMPORTANT! Do not change e-sig settings during a spectral calibration. By default, no events require electronic signature. To use e-sig, enable events that require an e- signature. 1. Select the checkbox next to an item in the E-Signature Type list to identify events for which to allow electronic signature. This selection activates the E-Sig button for the selected items; it does require an electronic signature for these selections. 2. (Optional) For each item that you select: a. From the top-right of the screen, select a function after which the system will prompt for electronic signature. This selection presents an e-sig prompt to users when they perform a function. Users can sign or can continue without signing. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 239 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators b. From the bottom-right of the screen, select a function (for example, start run) before which the system will check for required e-sigs. This selection presents an e-sig prompt to users when they start a run if the required signatures have not previously been made. Users must sign before they can continue. For “check before” functions, you can also: • Change the number of signatures required. • Set a special authority for a signature: click the Authorities Required field, then select the user account or the user role to require for e-sig of this function. By default, each required signature needs no special authority; any user can sign. • Click Apply. 3. Click Save Settings. 4. Log out of the software, then log back in, to activate the settings. E-signature settings Table 3 E-signature settings: functions to prompt after E-Signature type Function to prompt after Approve Dye Set Save Approve Size Standard Save Approve Spatial Calibration Accept Approve Spectral Calibration Accept Approve Instrument Protocol Save Approve Sizecalling Protocol Save Approve Basecalling Protocol Save Approve QC Protocol Save Approve Assay Save 240 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 Table 3 E-signature settings: functions to prompt after (continued) E-Signature type Function to prompt after Approve Plate Template Save Approve Plate Save Approve Sample Save Approve Sequencing Install Standard Results Accept Approve MicroSeqID Install Standard Results Accept Approve BDTv1.1 POP6 Install Standard Results Accept Approve Fragment Install Standard Results Accept Approve HID Install Standard Results Accept Table 4 E-signature settings: functions to check before Function to check Signatures and authorities required (defaults if E-Signature type before enabled) Approve Spatial Calibration Start Run 1 signature, any authorities (any user, any user role) Approve Spectral Calibration Approve Plate Approve Sequencing Install Standard Results Approve MicroSeq ID Install Standard Results Approve BDTv1.1 POP6 Install Standard Results Approve Fragment Install Standard Results Approve HID Install Standard Results How the software prompts electronic signature before a run If the system is configured to check that data is signed before starting a run and the data for the run is not signed, a message is displayed when the user clicks Start Run. Example The e-sig system is configured to require signatures from two users (one from the user account named Administrator, and the other from any user account with a scientist user role) for a spatial calibration before it can be used in a run. The spatial calibration has not been signed. A user starts a run. The following message is displayed: 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 241 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Before the run can start, the following users must sign: • The Administrator user • Any other user with the Scientist role specified and electronic signature enabled in their user account If a user that does not meet the specified criteria signs, a message is displayed to indicate which users have e-signed. Generate e-sig reports Display e-sig records 1. Access the E-Signature Reports screen. 242 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 2. (Optional) Edit display settings: • Specify filters (date range, user name, object type, object name), then click Go. • Select a record, then click Show Object History. • In the history dialog box, select a record, then click Show E-Signature Details. • Double-click column headers to sort. Multi-column sorting is supported (see “Sort by one or multiple columns” on page 94). • Customize the table (see “Customize a table” on page 94). 3. The records that are displayed (if they are specified in E-Signature settings) are: • Approve Dye Set • Approve Plate • Approve Size Standard • Approve Sample • Approve Spatial Calibration • Approve BDTv1.1POP6 Install Standard Results • Approve Spectral Calibration • Approve Sequencing Install Standard Results • Approve Instrument Protocol • Approve Microseq ID Install Standard Results • Approve Sizecall Protocol • Approve Fragment Install Standard Results • Approve Basecall Protocol • Approve HID Install Standard Results • Approve QC Protocol • Approve Assay • Approve Plate Template View and print e-signature reports 1. Display the records of interest as described in “Display e-sig records” on page 242. Note: Filter the list to decrease the time required to generate reports. 2. Click View E-Sig Summary Report or View E-Sig Detailed Report. 3. (Optional) In the Report screen, click toolbar options to manipulate the report. Place the mouse pointer over an item for a description of the item. 4. To print the report, click Print. 5. To save the report electronically (PDF), print the report and select a PDF printer. 6. Close the report. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 243 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Administrators Export e-sig records As needed, you can export e-sig records to a TXT file for additional manipulation and reporting outside the software. 1. Display the records of interest as described in “Display e-sig records” on page 242. 2. Select the records to export. 3. Click Export E-Sig Records. 4. Specify a name and location for the export TXT file. 5. Click Save. Export and import user accounts security audit and electronic signature settings Export user accounts, security, audit, and electronic signature settings 1. In any screen in the SAE module, click Export in the navigation pane. 244 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Administrators 9 2. Select the items to export: • User Profiles—Contains all settings in the following screens: – Edit User—All user accounts with Active status – User Role—All user roles and associated permissions (in case a user account specifies a user role that does not exist on the system into which you import the profiles) • System Configuration—Contains all settings in the following screens: – Security—Account setup and security policies – Audit—Objects selected for auditing, audit modes, and reasons – E-Signature Settings—Objects selected for E-Signature, functions, number of signatures, and authorities – User Roles—All user roles and associated permissions 3. Click Export. 4. Specify the name and location for the exported .dat file, then click Save. A message is displayed when the export completes. Import user accounts, security, audit, and electronic signature settings 1. In any screen in the SAE module, click Import in the navigation pane. 2. Select the .dat file to import, then click Open. A message is displayed asking if you want to overwrite the current system configuration. Click Yes. If any imported user accounts already exist on the system, you are prompted to overwrite or skip each account. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 245 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Users Users Users overview of System Security Audit Trail and E-Signature The Security, Audit, E-Signature (SAE) module provides the following functionality: • System security—Controls user access to the software. • Auditing—Tracks changes made to library items, actions performed by users, and changes to the SAE settings. • Electronic signature (e-sig)—Requires users to provide a user name and password when performing certain functions. Depending on the way that your administrator configures these features, you may see the following dialog boxes and prompts when you use the software. 246 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Users 9 Security Log in Enter your user name and password to access the software. Your access to functions in the software is based on the permissions associated with your user account. Functions for which you do not have permissions are dimmed. If your system is configured for password expiration, you will periodically be prompted to change your password. If your system is configured to monitor failed log in attempts, you will be locked out of the software if you incorrectly enter your user name or password more than a specified number of times. Permissions If your user account does not have permission to perform a function in the software, the associated menu commands are dimmed. Determine the name of the logged-in user To display the full name of the logged-in user: Place the mouse pointer on the Logout menu. The full name of the logged-in user is also displayed in the Load Plates for Run screen and the Monitor Run screen. Change your password when it expires When your password is about to expire, a message is displayed when you log in. To change your password, select Tools4Change Password. Enter your current password, enter the new password two times, then click OK. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 247 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Users Activate a suspended account If your system is configured to suspend a user account for failed logins, and you enter an incorrect user name and password more than the allowed number of times, your user account is suspended, and the Log In dialog box indicates that your account is inactive. To activate a suspended account, you can either: • Wait until the suspension period ends or • An administrator can change the account status from Suspended to Active Note: While a user is suspended, a different user can click Reset, then log in. Session timeout If your system is configured to timeout and there is no user activity for the specified time, the Log In dialog box indicates that your user session has timed out. You must enter your user name and password to access the software. Note: The administrator or another user with permission to log in to timed-out sessions can click Reset, then log in. 248 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) Users 9 Audit If your system is configured for auditing, you may be prompted to specify a reason when you make certain changes in the software. Based on your system configuration, you can either select a reason or enter a reason for change. Electronic signature If your system is configured for electronic signature, you may be prompted to provide your user name and password when you perform certain actions in the software. If an item is set to require two signatures, the signers are not required to sign at the same time. When the first signer signs, the E-Sig status is set to Partially Signed. When the second signer signs, the E-Sig status is set to Signed. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 249 Chapter 9 Use Security, Audit, and E-Sig functions (SAE Module) 9 Users You may also be permitted to sign objects such as plates, calibrations, or other library items. If e-sig is enabled for items, any of the following may apply: • The E-Signature button is enabled in the library or the calibration. • You are prompted to sign as described in “How the software prompts electronic signature before a run” on page 241. • The Open Plates dialog box or the library displays an “Is signed” column that reflects the e-sig status of an item. 250 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 10 Maintain the instrument ■ Maintenance schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 ■ Use the maintenance calendar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 ■ Review the Notifications Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 ■ Export the consumables log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 ■ Clean the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 ■ Install buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 ■ Replenish, change, flush, and store polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 ■ Change and store a capillary array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 ■ Maintain the pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 ■ Shutdown move and reactivate the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 ■ Maintain the computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 ■ Service Log and Usage Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Maintenance schedule WARNING! This section lists the common tasks required to maintain the instrument in good working condition. Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids. IMPORTANT! Use only the cleaning agents listed in this guide. Use of cleaning agents other than those listed in this guide may damage the instrument. Review calendar reminders 1. Review the calendar reminders list in the Dashboard daily, then perform the scheduled tasks. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 251 10 Chapter 10 Maintain the instrument Maintenance schedule 2. When you complete a task, click to mark it as complete, click to mark it as dismissed. Completed and dismissed tasks are: • Recorded in the Notification Log. See “Review the Notifications Log” on page 256. • Removed from the Calendar Reminder section, and they do not appear again unless they are repeating tasks. Dismissed tasks can be logged in the Notifications Log. Note: It is the end users’ responsibility to comply with maintenance prompts displayed in the software by completing the maintenance tasks at the recommended frequencies as shown in “Maintenance schedule” on page 251. Daily instrument maintenance tasks Clean the assemblies, anode buffer container, and cathode buffer container, and ensure that the outside of the assemblies is dry. IMPORTANT! Use only the cleaning agents listed in this guide. Use of cleaning agents not listed in this manual can impair instrument function. Task Frequency For information, see ... Click Refresh, then check consumables on the Before each run “Check system status in the Dashboard—View the gauges on the Dashboard to Dashboard” on page 36 see the status for anode buffer container, cathode buffer container, and polymer. Visually inspect the level of fluid inside the anode “Prepare the Anode Buffer Container buffer container and the cathode buffer container. The (ABC)” on page 258 fluid must line up with the fill line. “Prepare the Cathode Buffer Container (CBC)” on page 259 Ensure that the CBC septa are properly seated on the “Ensure proper installation of CBC container. septa” on page 40 Ensure that the plate assemblies are properly “Prepare the plate assembly” on assembled. page 70 Align the holes in the plate retainer with the holes in the septa to avoid damaging capillary tips. Ensure that the plate assemblies and the cathode “Load the plate in the instrument” on buffer container are positioned on the plate deck page 72 properly. They should sit securely on the deck. Ensure the array locking lever on the capillary array is Figure 42 secured. Check for bubbles in the pump block and channels. Daily or before “Remove bubbles from the polymer each run pump” on page 268 Use the Remove Bubble wizard to remove bubbles. Check the loading-end header to ensure that the “Install or change the capillary array” capillary tips are not crushed or damaged. on page 266 252 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Maintenance schedule 10 (continued) Task Frequency For information, see ... Ensure that the pump block is in pushed back Daily Figure 42 position. Clean the instrument surfaces of dried residue, spilled “Clean the instrument” on page 257 buffer, or dirt. Check for leaks and dried residue around the buffer- “Check calendar reminders” on pin valve, check valve, and array locking lever. page 36 If leaks persist, contact Thermo Fisher Scientific. Weekly instrument maintenance tasks Task Frequency For information, see ... Check the storage conditions of the used arrays to ensure Weekly “Store a capillary array” on page 267 the array tip is covered in the reservoir. Run the Wash Pump and Channels wizard. “Wash the pump chamber and channels” on page 268 Use a lab wipe to clean the anode buffer container valve Figure 42 pin assembly on the polymer delivery pump. Restart the computer and instrument. “Restart the instrument and the computer” on page 279 Monthly instrument maintenance tasks Task Frequency For information, see ... Run install check. Monthly or as needed “Run an install check” on page 144 Flush the pump trap. “Flush the water trap (pump trap)” on page 269 Empty the oven condensation Figure 42 reservoir. Replace cathode buffer container “Prepare the Cathode Buffer Container septa. (CBC)” on page 259 Clean the autosampler. “Clean the instrument” on page 257 Clean the drip tray. Check disk space. “Monitor disk space” on page 276 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 253 10 Chapter 10 Maintain the instrument Maintenance schedule (continued) Task Frequency For information, see ... If Security, Audit, and E-sig is Monthly or as needed “Archive and purge” on page 237 enabled, archive and purge audit records. Defragment the hard drive. Monthly, or before fragmentation “Defragment the computer hard drive” on reaches 10% page 277 Quarterly maintenance tasks Task Frequency For information, see ... Archive and purge audit records Every three months “Archive and purge” on page 237 Annual planned maintenance tasks Call your Thermo Fisher Scientific representative to schedule annual planned maintenance. As-Needed instrument maintenance tasks Task Frequency For information, see ... Change the tray. As needed “Clean the instrument” on page 257 Remove dried polymer from the capillary tips with a lint-free tissue moistened with deionized water. Archive and purge library objects. Chapter 8, “Manage library resources” Dashboard4Manage4Archive or Dashboard4Manage4Purge. 254 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Use the maintenance calendar 10 Use the maintenance calendar The Maintenance calendar is a monthly or daily view of the routine maintenance tasks scheduled for your instrument. When a task is due to be performed, it is listed in the Calendar Reminders list in the Dashboard (see “Review the Notifications Log” on page 256). To access the maintenance calendar, click the Maintenance tab, then click Schedule. A set of recommended tasks are scheduled in the calendar, flagged with FR (Factory Repeating) in the monthly view and F (Factory) in the daily view. User-specified repeating tasks are flagged with R (Repeating) in the monthly view. Weekly factory repeating tasks in calendar Monthly factory repeating tasks in calendar • Clean the anode buffer cup pin-valve assembly on the • Replace cathode buffer container septa polymer delivery pump • Clean drip tray • Restart instrument and computer • Clean autosampler • Check disk space • Defragment hard drive • Run install check • Flush pump trap You can change the priority of factory tasks, but you cannot remove them from the calendar or alter the frequency at which the notifications for the tasks are displayed. Additionally, we suggest that you add to the maintenance calendar: • The regular maintenance tasks. • A maintenance task to replace a consumable based on its installation date (for example, create a task to replace the polymer for two days before the polymer will expire). Create calendar entries To create a new scheduled task, click Create and follow the prompts. The following figure is an example of scheduled events in the calendar. The Month and Day tabs allow you to view your schedule in different formats. Click Detach to move the calendar window. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 255 10 Chapter 10 Maintain the instrument Review the Notifications Log View the Planned Maintenance Report 1. Click the Maintenance tab, then click Schedule. 2. Click Planned Maintenance Report. 3. Specify the date range, then click OK. 4. Select Print as needed. 5. To save the report electronically (PDF), print the report and select a PDF printer. Review the Notifications Log The Notifications Log is a history of the action taken on calendar reminders messages in the Dashboard (see “Review calendar reminders” on page 251). 1. Access the Notifications Log. 2. View the Notification Log Report and print as needed. Note: Multi-column sorting is supported (see “Sort by one or multiple columns” on page 94). 256 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Export the consumables log 10 Export the consumables log 1. In any screen, select Tools4Export Consumables Log. 2. Select a location and enter a name for the export file. IMPORTANT! In the exported file, the conditioning reagent part description incorrectly lists "Polymer Pouch". However, the lot number that is listed is correct for the conditioning reagent. Clean the instrument IMPORTANT! Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids. IMPORTANT! Use only the cleaning agents listed in this guide. Use of cleaning agents other than those listed in this guide may damage the instrument. 1. Ensure the oven is closed. 2. Press the Tray button on the front of the instrument to move the autosampler to the forward position. 3. Wipe off any liquid on or around the autosampler using a lint-free tissue. 4. Clean off any polymer build-up crystals on the instrument, including the capillary tips, with deionized water and lint-free tissue. 5. Clean the array plug with deionized water and lint-free tissue. 6. Clean out the drip trays with deionized water, or ethanol, and lint-free tissue. Note: The drip tray can be removed. Install buffers IMPORTANT! Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids. IMPORTANT! Use only the parts listed in Appendix D, “Catalog numbers”. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 257 10 Chapter 10 Maintain the instrument Install buffers Prepare the Anode Buffer Container (ABC) 1. Check the expiration date on the label to ensure that the ABC is not expired and will not expire during use. 2. Allow the refrigerated ABC to equilibrate to room temperature before first use. Do not remove the seal until you have completed step 5. 3. Verify that the seal is intact. Do not use if the buffer level is too low or the seal has been compromised. A fill tolerance of ±1 mm is acceptable. 4. Invert the ABC, then tilt it slightly to move most of the buffer to the larger side of the container. The smaller side of the container should contain <1 mL of the buffer. 5. Verify that the buffer is at the fill line. 6. Peel off the seal at the top of the ABC. 7. With the RFID label toward instrument, place the ABC into the anode-end of the instrument, below the pump. Position the anode in the large chamber of the ABC, then push the ABC up and back to install. IMPORTANT! The RFID label must be facing the instrument (away from you) to ensure that the RFID information is read accurately by the instrument. 8. Close the instrument door to re-initialize. 9. In the Dashboard, click Refresh, then check the Quick View section for updated status. 258 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Install buffers 10 Prepare the Cathode Buffer Container (CBC) 1. Check the expiration date on the label to ensure that the CBC is not expired and will not expire during use. 2. Allow refrigerated CBC to equilibrate to ambient temperature. 3. Wipe away condensation on the CBC exterior with a lint-free tissue. Condensation can cause arcing and termination of the run. 4. Check that the seal is intact. Do not use if the buffer level is too low or the seal has been compromised. A fill tolerance of ±0.5 mm is acceptable. 1 1 Fill line 5. Tilt the CBC back and forth gently and carefully to ensure that the buffer is evenly distributed across the top of the baffles. If you do not tilt the CBC back and forth, the buffer sticks to the baffles because of surface tension. 6. Verify that the buffer is at or above the fill line. 7. When ready to install the CBC, place the container on a flat surface (such as a lab bench) and peel off the seal. 8. Wipe off any buffer on top of the CBC with a lint-free tissue. Ensure that the top of the container is dry. Moisture can cause arcing, termination of a run, or damage to the instrument. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 259 10 Chapter 10 Maintain the instrument Install buffers 9. Place the septa (Cat. No. 4410715) on the CBC. a. Align the buffer septum (the part that is symmetrical) over the 24 holes of the CBC. b. Push the septum lightly into the holes to start, then push firmly to seat it. IMPORTANT! Look at the CBC from the side and ensure that there is no gap between the container and the lip of the septum. c. Align the capillary washing septum over the other chamber of the CBC. The capillary washing septum has 48 slitted holes and one rounded starter tab for alignment. Position the rounded starter tab of the septum over the larger alignment hole on the washing chamber, then press down. 260 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Install buffers 10 d. Push the septum lightly into the holes to start, then push firmly to seat it. 3 4 1 2 1 Buffer chamber and septum 2 Washing chamber and septum 3 Starter tab 4 Alignment hole IMPORTANT! Ensure the septa are securely seated to prevent displacement of the septa during operation. Inspect the underside of the CBC lid to ensure that all capillary washing septum blades are fully engaged and unobstructed. IMPORTANT! A correctly inserted capillary washing septum shows a clear, linear slit in all wells. To easily view the slits, illuminate the CBC from the bottom, such as with an LED screen, and observe the CBC from above. If any holes do not show a clear, linear slit, firmly press down on those specific holes to ensure that the septum blades are visually engaged and the slit of light is unobstructed. 1 2 Figure 40 Illuminated CBC with the capillary washing septum correctly in place 1 Starter tab positioned in alignment hole 2 Linear slit visible 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 261 10 Chapter 10 Maintain the instrument Replenish, change, flush, and store polymer 1 2 Figure 41 Illuminated CBC with the capillary washing septum not in place 1 Starter tab positioned in alignment hole 2 Linear slit not visible 10. Click the Tray button on the front panel to move the autosampler to the front position. 11. With the tab facing you and the RFID tag to the right, install the CBC on the autosampler. When properly installed, the CBC tabs will click as you snap them into place on the autosampler. A B 12. Click the Tray button to retract the autosampler, then close the instrument door to initialize. 13. In the Dashboard, click Refresh, then check the Quick View section for updated status. Replenish, change, flush, and store polymer IMPORTANT! Note the following: · Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids. · Use only the parts listed in Appendix D, “Catalog numbers”. · To minimize background fluorescence, use clean, powder-free, silicone-free latex gloves whenever you handle the pump assembly or any item in the polymer path. 262 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Replenish, change, flush, and store polymer 10 Precautions for use • Do not reuse a polymer pouch that has been installed on another type of instrument. For example, if you remove a partially used polymer pouch from an 8-capillary instrument, do not reuse that polymer on a 24-capillary instrument. • If you remove a polymer pouch for storage (2–8°C), place a pouch cap (Cat. No. 4412619) onto the pouch, then place an empty pouch (or conditioning reagent) on the connector to prevent desiccation of any residual polymer on the connector. Follow the instructions in the wizard to ensure proper operation of the pouch and the instrument. Replenish polymer or change polymer type 1. Check the expiration date on the label to ensure that the polymer is not expired and will not expire during intended use. IMPORTANT! Do not use if the product is expired, if the pouch or label is damaged, or if the top seal is missing or damaged. 2. Allow the refrigerated polymer to equilibrate to room temperature (15–30°C) before use. 3. In the Dashboard, click Wizards, then click Replenish Polymer (requires 10–20 minutes) or Change Polymer Type (requires 60–70 minutes). Note: If you are changing the polymer type, you also need Conditioning Reagent. 4. Follow the prompts in the Wizard window. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 263 10 Chapter 10 Maintain the instrument Replenish, change, flush, and store polymer 5. When instructed to install the polymer, peel off the seal at the top of the pouch fitting. Note: You may notice a tiny droplet of polymer inside the fitting (residual from the pouch filling process). This is not expected to cause any performance issues. 6. With the RFID label facing the instrument, slide the pouch fitting onto the slot of the lever assembly. Push the lever up to snap the pouch into the connector end of the instrument pump. Note: The RFID label must face the instrument (away from you) to ensure that the RFID information is read accurately by the instrument. 7. In the Dashboard, click Refresh, then check the Quick View section for the updated polymer status. Store partially used polymer If you remove a polymer pouch for storage (2–8°C), place a pouch cap (Cat. No. 4412619) onto the pouch. Install an empty pouch or conditioning reagent pouch on the instrument to prevent desiccation of any residual polymer on the connector. Follow the instructions in the maintenance wizards to ensure proper installation of the polymer pouch. 264 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Change and store a capillary array 10 Fill capillary array with fresh polymer 1. In the Dashboard, click Wizards. 2. In the Maintenance wizards screen, click Fill Array with Polymer. 3. Follow the prompts in the Fill Array wizard window. 4. Click Refresh in the Dashboard to update the screen. 5. Check the Quick View section of the Dashboard for updated status after filling of the capillary array with fresh polymer. Change and store a capillary array WARNING! SHARP. The electrodes on the load-end of the capillary array have small, blunt ends that can lead to piercing injury. IMPORTANT! Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 265 10 Chapter 10 Maintain the instrument Change and store a capillary array Install or change the capillary array IMPORTANT! Before installing a capillary array, examine the loading-end header to ensure that the capillary tips are not crushed or damaged. Note: The Install Capillary Array wizard takes 15–45 minutes to complete. 1. In the Dashboard, click Wizards. 2. In the Maintenance wizards screen, click Install Capillary Array. 3. Follow the prompts in the Install Capillary Array wizard window. 4. Check the Quick View section of the Dashboard for updated status of the capillary array. 266 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Maintain the pump 10 Store a capillary array WARNING! SHARP. The electrodes on the load-end of the capillary array have small, blunt ends that can lead to piercing injury. If you remove a capillary array for storage, insert the loading-end of the capillary array in distilled water to prevent the polymer from drying in the capillaries. Check periodically and add distilled water as needed. Maintain the pump IMPORTANT! Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids. IMPORTANT! To minimize background fluorescence, use clean, powder-free, silicone-free latex gloves whenever you handle the pump assembly or any item in the polymer path. Avoiding damage to the pump assembly The polymer delivery pump can be irreversibly damaged in the following situations: • Polymer dries in the polymer channels of the pump assembly, which can scratch the channels in the pump, and can cause blockage. • The pump assembly is exposed to organic solvent, which can cause cracking and clouding of the acrylic pump material. • The pump assembly is exposed to temperatures greater than 40°C, which can damage the pump components. • Arcing occurs in the pump assembly, which can damage the acrylic pump material. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 267 10 Chapter 10 Maintain the instrument Maintain the pump Remove bubbles from the polymer pump Remove bubbles from the polymer pump fluid path before each run. See “Daily instrument maintenance tasks” on page 252 for more information. Note: The Bubble Remove wizard takes 5–15 minutes to complete. 1. In the Dashboard, click Wizards. 2. In the Maintenance wizards screen, click Remove Bubbles. 3. Follow the prompts in the Bubble Remove wizard window. 4. Check the Quick View section of the Dashboard for updated status of the polymer pouch after removing bubbles from the polymer pump fluid path. Wash the pump chamber and channels In the following situations, use the Polymer Delivery Pump Cleaning Kit (Cat. No. 4414007 ) in addition to the Wash Pump Chamber and Channels wizard to thoroughly clean the polymer delivery pump: • Polymer has dried in the channels of the lower polymer block. Mechanical malfunctions may cause dried polymer to appear in the polymer delivery pump. Washing with either the Wash Pump Chamber and Channels wizard or this kit may not remove dried polymer – the lower polymer block may need to be replaced by Thermo Fisher Scientific. • A contaminant in the polymer delivery pump is suspected of causing problems. The check valve fitting might be clogged or contaminated. The Wash Pump and Channels wizard takes >40 minutes to complete. 268 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Maintain the pump 10 1. In the Dashboard, click Wizards. 2. In the Maintenance wizards screen, click Wash Pump and Channels. 3. Follow the prompts in the wizard window. Flush the water trap (pump trap) Flush the water trap monthly to prolong the life of the pump and to remove diluted polymer from the pump. Flush with distilled or deionized water and ensure that the water flows into the overflow container. Dispose of the excess water (inside the overflow container). See “Chemical safety” on page 341. Note: Leave the trap filled with either distilled or deionized water. 1. Fill the supplied 20 mL, all-plastic Luer lock syringe (in the Polymer Delivery Pump Cleaning Kit, Cat. No. 4414007 ) with distilled or deionized water. Expel any bubbles from the syringe. IMPORTANT! Do not use a syringe smaller than 20 mL. Doing so may generate excessive pressure within the trap. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 269 10 Chapter 10 Maintain the instrument Maintain the pump 2. Attach the syringe to the forward-facing Luer fitting at the top of the pump block. Hold the fitting with one hand while threading the syringe onto the fitting with the other hand. 3. Open the Luer fitting by grasping the body of the fitting and turning it to loosen. 4. Grasp the attached syringe and turn counterclockwise approximately one-half turn. 5. Slowly depress the plunger. IMPORTANT! DO NOT USE EXCESSIVE FORCE when you push the syringe plunger as this may damage the trap seals. Take approximately 30 seconds to flush 5 mL of either distilled or deionized water through the trap. Note: Because the water trap volume is approximately 325 μL, a relatively small volume of water is adequate for complete flushing. However, a larger volume improves flushing as long as force and flow rate are kept within the limits given above. 6. Remove the syringe from the Luer fitting. Hold the fitting with one hand while turning the syringe counterclockwise with the other hand. 7. Close the Luer fitting by lightly turning clockwise until the fitting seals against the block. 270 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Shutdown move and reactivate the instrument 10 Shutdown move and reactivate the instrument Shutdown the instrument A conditioning reagent pouch is required for this procedure. Use the Instrument Shutdown wizard for short- and long-term shutdown. Note: The Instrument Shutdown wizard takes 60 minutes to complete. 1. In the Dashboard, click Wizards. 2. In the Maintenance wizards screen, click Shutdown the Instrument. 3. Follow the prompts in the Instrument Shutdown wizard window. Perform the appropriate shutdown procedure based on the information in the following table: IMPORTANT! Place a conditioning reagent pouch onto the instrument before performing instrument shutdown. If the instrument will be Perform this shutdown procedure ... unattended for ... < 1 week No action is required. 1 to 2 weeks Keep the load-end of the capillary array in 1X buffer to prevent the polymer from drying in the capillaries. If fluid level is low, add DI water to buffer solution. Install the new CBC when ready to resume runs. > 2 weeks 1. Run the Install Capillary wizard and store the capillary array. 2. Clean any spills or residual polymer. 3. Run the Shutdown the Instrument wizard. 4. Unplug the instrument. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 271 10 Chapter 10 Maintain the instrument Shutdown move and reactivate the instrument Move and level the instrument IMPORTANT! If you relocate the instrument, we recommend that you have an IQ OQ performed. Contact Thermo Fisher Scientific to schedule the IQ OQ service. WARNING! PHYSICAL INJURY HAZARD. Do not attempt to lift the instrument or any other heavy objects unless you have received related training. Incorrect lifting can cause painful and sometimes permanent back injury. Use proper lifting techniques when lifting or moving the instrument. Two or three people are required to lift the instrument, depending upon instrument weight. 1. Remove the following components from the instrument: • Any plate assemblies from the autosampler. • CBC from the autosampler. • Capillary array: Click Shutdown the Instrument in the Maintenance Wizards. See “Shutdown the instrument” on page 271. • Anode buffer reservoir. 2. Switch off the circuit breaker on the back of the instrument. 3. Disconnect the power cord and the Ethernet cable. IMPORTANT! While moving the instrument, avoid any shock or vibration. 4. Move the instrument. 5. Turn the instrument legs to level the instrument. To move the instrument corner ... Turn the leg ... up right (clockwise) down left (counterclockwise) 6. Have an IQ OQ performed before using the instrument. IMPORTANT! After performing a conditioning wash, ensure that the buffer level inside the ABC is at or above fill line before proceeding to the next step. 272 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Maintain the computer 10 Reactivate the instrument Note: The Instrument Reactivate wizard takes ~45 minutes to complete. 1. In the Dashboard, click Wizards. 2. In the Maintenance wizards screen, click Reactivate the Instrument. 3. Follow the prompts in the Instrument Reactivation wizard window. Maintain the computer This section lists the common tasks required to maintain the computer for your 3500 instrument in good working condition. Note: In the event of a power disruption, restart the computer (Appendix A, “Troubleshooting”). Back up the datastore during software uninstall IMPORTANT! Do not uninstall the software unless instructed to do so by Thermo Fisher Scientific. When you uninstall the software, you are prompted to back up the datastore (the directory that contains all library items you created, such as plates and protocols). Select a location other than the install directory for the datastore backup. IMPORTANT! Do not back up the datastore to the installation directory. The installation directory is deleted during the uninstall. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 273 10 Chapter 10 Maintain the instrument Maintain the computer Archive, purge, and restore data IMPORTANT! The customer is responsible for validation of archive, restore, and purge functions. • Archive—Makes a copy of the data in an external file that you can save in another location. • Purge—Allows you to delete (purge) user-created items stored in the library. Factory-provided items are not purged. You have an option to archive the items, also. • Restore—Restores archived data back to the system. IMPORTANT! These functions affect items stored in the library (datastore). These functions do not affect sample data files. Frequency We recommend that you purge the library objects once every three months. Archive library items 1. Access the Archive screen. 2. Specify the date category, specify a date range that is earlier than the date on which you made the duplicates of the library items you want to retain, then click OK. 3. Specify a location and file name for the archive (.dsz) file, then click Save. IMPORTANT! Do not specify <<install directory>>:\Applied Biosystems\3500\datastore as the archive location. If you do so, your archive can be deleted if you uninstall the software. If you specify a location to which you do not have permission to save, a warning message is displayed and gives you the option to save in another location. 274 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Maintain the computer 10 A message is displayed when the archive is complete. Archive data files 1. Use the Windows™ backup function (Start4Control Panel4Backup and Restore) to archive the data files. Note: If you export audit records for samples that are not in their original location (samples have been deleted or moved), an error message is displayed. Return sample data files to their original location, then export again. 2. Copy the archive to a network or external drive. Restore This function restores items archived from the library. To restore audit records, see “Archive, purge, and restore audit records” on page 237. 1. Access the Restore function. 2. Select the archive (.dsz) file to restore, then click Open. If the archive file contains items that exist in the system, a message is displayed. 3. Select an option to continue. A message is displayed when the restore is complete. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 275 10 Chapter 10 Maintain the instrument Maintain the computer Purge library entries This function purges (deletes) items stored in the library. To purge audit records, see “Archive, purge, and restore audit records” on page 237. 1. Access the Purge function. 2. Click Yes in the Purge warning message stating that you are about to permanently delete all files in the library. 3. Specify the date category and range, then click OK. 4. Click Yes in the Purge warning message. A message is displayed when all records are deleted. Monitor disk space Ensure that you have sufficient drive space by regularly: • Archiving data • Deleting unneeded files • Emptying the trash • Defragmenting the drives Automatic disk space check before a run Before a run, the software checks free disk space and displays a message when the hard disk is 70–75% full. At 78% full, the software will not start a run. 276 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Chapter 10 Maintain the instrument Service Log and Usage Statistics 10 Manually check hard disk space 1. Go to My Computer, right-click the drive, then select Properties4General. 2. If there is insufficient space on the hard disk: • Archive the sample files. • Delete the sample file data from the drive D and empty the contents of the Recycle Bin. Defragment the computer hard drive This option can be set as a reminder in the scheduler. The fragmentation of files decreases the performance of both the Data Collection software and the computer operating system. Programs take a longer time to access files by performing multiple search operations of the fragments. Go to Start4Programs4Accessories4System Tools4Disk Defragmenter and follow the prompts. Note: You can click Analyze to see if you should defragment or not. Service Log and Usage Statistics The Service Log and Usage Statistics functions are for use by Thermo Fisher Scientific service engineers at the time of service. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 277 A Troubleshooting ■ Restart the instrument and the computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 ■ Instrument components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 ■ Instrument troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 ■ RFID troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 ■ Error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 ■ Dashboard troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 ■ Software troubleshooting — general . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 ■ Run, re-run, or re-inject troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 ■ Data/electropherogram troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 ■ Review Results troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 ■ Link/load plate troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 ■ Assign Plate Contents troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 ■ Spatial calibration troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 ■ Spectral calibration troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 ■ Sequencing install standard troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 ■ Fragment/HID install standard troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 ■ Monitor Run troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 ■ Audit troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 ■ Electronic signature troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 ■ Manual commands troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 ■ Troubleshooting procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 If you encounter any unforeseen and potentially hazardous event while operating the instrument, turn off the power, unplug the instrument, and call your Thermo Fisher Scientific service representative. IMPORTANT! See “Safety and electromagnetic compatibility (EMC) standards” on page 338 for instrumentation and chemical safety information and guidelines. 278 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Restart the instrument and the computer A Restart the instrument and the computer When to use this procedure: • If communication errors are displayed • If the front panel indicator is blinking red or amber • At the end of spatial calibration, if Accept/Reject buttons are dimmed • If maintenance wizards are taking longer than expected • If software operations are taking longer than expected • If a "Camera frame number mismatch detected" message is displayed • If the Server Monitor icon displays When you are instructed to restart the instrument and the computer: 1. Exit the software. 2. Power off the computer. 3. Make sure the instrument door is closed, then power off the instrument. 4. When the computer is completely powered off, wait 60 seconds, then power on the computer. Wait until the Windows™ login screen is displayed. Do not log in. 5. Power on the instrument and wait until the green status light on the front panel is on and not flashing before proceeding. 6. Log in to Windows™ operating system. IMPORTANT! Use the same administrator Windows™ user account to log in to the instrument computer. Use of a non-administrator Windows™ user account, or use of different administrator Windows™ user accounts can interfere with software operation. 7. Look in the Windows™ taskbar at the bottom right of the desktop and make sure the Server Monitor icon is displayed. If it is not, go to “Step one: Start the Server Monitor” on page 34. 8. Start the software. Instrument components Figure 42, Figure 43, and Figure 44 are provided below for reference in this section. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 279 Appendix A Troubleshooting A Instrument components 1 6 7 2 8 3 4 9 5 Figure 42 Instrument interior 1 Detection cell heater block 2 Polymer delivery pump (PDP) 3 Anode buffer container (ABC) 4 Polymer or conditioning pouch 5 Cathode buffer container (CBC) 6 Oven door 7 Capillary array 8 Oven condensation reservoir 9 Autosampler 1 Upper polymer block 5 2 Buffer-pin valve 1 3 Lower polymer block 4 Anode buffer container (ABC) 5 Array locking lever 6 Water trap waste container 7 Check valve fitting 2 8 Polymer pouch lever 6 9 Polymer pouch 3 7 10 Drip tray 8 4 9 10 Figure 43 Polymer delivery pump (PDP) 280 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Instrument troubleshooting A Figure 44 Detection cell Instrument troubleshooting Symptom Possible cause Action Power failure to instrument and computer Power failure. Restart the instrument and the computer (see “Restart the instrument and the computer” on page 279). Front panel indicator: Amber light (blinking) Run paused Resume run. Door open Close the instrument door. Communication issue Restart the instrument and the between instrument and computer (see “Restart the instrument software. and the computer” on page 279). CBC septum is lifted off the container Septum was not seated See “Ensure proper installation of CBC properly when installed. septa” on page 40. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 281 Appendix A Troubleshooting A Instrument troubleshooting (continued) Symptom Possible cause Action Autosampler does not move the plate to a Array electrodes are Ensure that the plate retainer, plate higher position bent. The plate is (or tube strip), and plate base are not aligned correctly assembled correctly. Listen for a snap resulting in the array tips when the plate retainer and the plate missing center of septa. base are clipped together. See “Prepare The plate retainer may the plate assembly” on page 70. not be snapped onto the plate base. IMPORTANT! If array tips are bent, replace the array. The plate base is not The plate base should sit flat on the sitting properly on the autosampler. When placing the plate on autosampler. the autosampler, ensure that the pins in the autosampler are properly aligned with the holes at the bottom of the plate base, and that the left and right sides are latched. The plate retainer is Securely clip the plate retainer and plate lifted off the plate base base together. by array. The septum is lifted off Ensure that the septum is completely the CBC. inserted into position. Listen for the light clicking sound that occurs when the septum is pressed down firmly into position. Polymer delivery pump (PDP) is extremely The array locking lever Lock the lever in the correct position. noisy and vibrating while running any wizard is not in the correct If this is not possible contact Thermo position. Fisher Scientific. IMPORTANT! If the lever is not in the correct position, you will receive “Leak error” message. 282 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Instrument troubleshooting A (continued) Symptom Possible cause Action Polymer delivery pump (PDP) is extremely Polymer delivery pump Gently push the buffer-pin valve lever noisy and vibrating while running any wizard block is not pushed (yoke). If the lever does not move up back into position after and down freely, Restart the instrument capillary array change and the computer. (see “Restart the instrument and the computer” on page 279). After the instrument has restarted, check the lever movement. If the lever does not move up and down freely, contact Thermo Fisher Scientific. If the lever moves up and down freely, push the upper polymer block all the way back against the wall. 1 2 Figure 45 Buffer-pin valve lever (yoke) 1 Yoke 2 Buffer-pin valve Polymer is not pumping properly - wizard fails - Check Valve is clogged Run the Wash Pump and Channels filling array wizard. Crystals present in polymer delivery pump See “Flush the water trap (pump trap)” path on page 269 and “Wash the pump chamber and channels” on page 268. If the problem persists, contact Thermo Fisher Scientific. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 283 Appendix A Troubleshooting A Instrument troubleshooting (continued) Symptom Possible cause Action Polymer is not pumping properly - wizard fails - filling array 1 2 Figure 46 Pump chamber and valve fitting 1 Debris in chamber 2 Check valve fitting Buffer-pin valve does not move Polymer crystallizations If you see any crystals, leaks, and dried have formed around the residue around the buffer-pin valve, buffer-pin valve clean the valve and the array locking lever immediately. Add DI water to the buffer solution to dissolve crystals. Note: Use the lint-free swabs, included in the PDP Cleaning kit (Cat. No. 4414007 ). If leaks persist, contact Thermo Fisher Scientific. Perform maintenance tasks routinely as described in “Maintenance schedule” on page 251. If leaks persist, contact Thermo Fisher Scientific. The vent hole behind Clean the vent hole behind the buffer- the buffer-pin valve is pin valve with DI water. clogged The PDP block is not in See "Polymer delivery pump (PDP) the correct position is extremely noisy and vibrating while running any wizard" . If the problem persists, contact Thermo Fisher Scientific. 284 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Instrument troubleshooting A (continued) Symptom Possible cause Action Polymer crystals on the buffer-pin valve Buffer valve leakage Clean the buffer-pin valve. Perform maintenance tasks routinely as described in “Maintenance schedule” on page 251. Fluid does not move through the polymer Blockage in fluid path Contact Thermo Fisher Scientific. delivery pump and into the ABC from polymer or problem with polymer or conditioning pouch delivery pump Poor signal and resolution after replenishing The Check Valve is Wash the channels using the Polymer polymer clogged (see Figure 46. Delivery Pump Cleaning Kit (Cat. No. 4414007 ). If the problem persists, contact Thermo Fisher Scientific. Any of the following visual or audible The buffer level is below Verify that buffer level is at or above the conditions: the fill line. fill line. • Unstable current The buffer spilled on top • Arc-detect errors IMPORTANT! Ensure that the of the CBC. environment (humidity) is non- • A crackling noise at the beginning of The buffer spilled on top condensing. electrophoresis of the Autosampler. • A blue lightning symbol below the oven Wipe away spills, moisture, and • An error message regarding electrical Condensation on the condensation with a lint-free lab cloth. current CBC. If the problem persists, contact Thermo Fisher Scientific. • Electric discharge Condensation around the septa. Condensation on the lower part of the oven door, near the array header. Condensation inside the oven. There is not enough Pipette the buffer from the smaller fluid in larger chamber overflow chamber to the larger of ABC, or the anode chamber. Ensure that the buffer is filled buffer has spilled to within ±1 mm of the fill line. into smaller overflow When installing new ABC, tilt the chamber. container to move buffer to the larger side of the container as described in “Prepare the Anode Buffer Container (ABC)” on page 258. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 285 Appendix A Troubleshooting A RFID troubleshooting (continued) Symptom Possible cause Action When you remove the heat seal from a new The top seal of the Use a pipette tip to remove the pouch, some residual seal remains on top of pouch has become entire seal from the pouch cap before the pouch. delaminated and left the installing on the instrument. polyethylene behind on the pouch cap. RFID troubleshooting Symptom Possible cause Action Unable to read RFID Consumable package is improperly Ensure that the RFID label is not visibly information. installed or label is defective. damaged and consumable package is properly installed. “Failure to Read from Polymer/Conditioning reagent pouch RFID tag” is not positioned properly. Ensure that label is close, and parallel, to the instrument. Reposition or re-install pouch, then click Refresh on the Dashboard. Restart the instrument and the computer (see “Restart the instrument and the computer” on page 279). Install a new consumable (if available). If problem persists, contact Thermo Fisher Scientific. Malfunctioning RFID label or reader. Place a used CBC, ABC, pouch, or array on the instrument: • If the instrument can read the RFID label, install a new CBC, ABC, pouch, or array. • If the instrument cannot read the RFID label, contact Thermo Fisher Scientific. 286 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Error messages A Error messages Symptom Possible cause Action “An error has been detected Instrument monitor circuit Restart the instrument and the computer. (see from the instrument. ” failure “Restart the instrument and the computer” on page 279). “Unable to transmit Communications error. Restart the instrument and the computer. (see measurement data. Internal data “Restart the instrument and the computer” on buffer overflow.” page 279). Electric discharge message The ABC buffer may be Replace the ABC. during runs. low. Ensure that the ABC is being replaced per calendar notifications. “Leak error” message. The array locking lever is Secure the array locking lever (see Figure 43). not in the correct position. “Leak error” occurs when Debris is clogging the While wearing gloves, use a lint‑free cloth and capillary arrays are filled with fresh check valve (CV) fitting (see water to wipe the CV Fitting. polymer or when replenishing Figure 46). Note: To prevent crystals from forming around polymer, causing the wizard to fail the check valve, always install the Conditioning to complete. Reagent Pouch after removing a used or a partially used polymer pouch. Completely remove the top seal of the Polymer pouch or Conditioning Reagent Pouch before use. If the problem persists, contact Thermo Fisher Scientific. The Yoke is not seated Make sure the buffer-pin valve lever (yoke) is properly on the buffer-pin seated properly on the buffer-pin valve (see valve. Figure 45). If the lever does not move up and down freely, close the door. Restart the instrument and the computer. (see “Restart the instrument and the computer” on page 279). After the instrument has restarted, check the lever movement. If the lever does not move up and down freely, contact Thermo Fisher Scientific. If the lever moves up and down freely, push the upper polymer block all the way back against the wall. • “Leak detected during Bubbles in the polymer Run the Remove Bubbles wizard to clear polymer delivery” system. bubbles. • “Leak detected during bubble compression” 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 287 Appendix A Troubleshooting A Error messages (continued) Symptom Possible cause Action The run aborts. Leak in the polymer Check for evidence of leaks. system. If polymer leak occurred, conduct a water wash and wash the pump trap using the Polymer Delivery Pump Cleaning Kit (Cat. No. 4414007 ) supplied with the instrument. Buffer valve leakage. Check the buffer-pin valve and see if it closes correctly. Clean the buffer-pin valve. Ensure that the maintenance schedule is followed per the software notifications. Filling the array during Run Fill the Array with fresh Polymer wizard, or install array. run Change Polymer Type wizard. “Bubble” error Bubbles present Run the Remove Bubbles wizard. “Java update scheduler” error The Java updater is unable Close the Java update scheduler. message to complete the update. Note: The Java update scheduler does not affect the performance of the software or the quality and accuracy of the data collected. “Invalid Contents” message In The first row you have • Enter sample name or select an assay in the Assign Plate Contents screen selected to fill from is first row in you have selected to fill from. when you use Ctrl+D empty. • Use the table view to add the assay to the samples. “Injection failed” message after Capillary RFID cannot be Check the connection between the instrument some of the injections complete. read. and computer. Restart the instrument and the computer (see “Restart the instrument and the computer” on page 279). “Instrument is not connected” Bad connection between Check the connection between the instrument and message after you start the the computer and computer and restart both the instrument and software. instrument. computer (see “Restart the instrument and the computer” on page 279). “Internal buffer data overflow” message. "Camera frame number The communication Restart the instrument and the computer. See mismatch detected" between the instrument “Restart the instrument and the computer” on and the computer has page 279. been interrupted and cannot be re-established automatically. 288 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Dashboard troubleshooting A Dashboard troubleshooting Symptom Possible Cause Action When you click Refresh on the Dashboard, Bad connection between the Check the connection between the and consumables information is listed as computer and instrument. instrument and computer. “Unknown.” Consumables status in the Dashboard is Dashboard does not update Click Refresh. not updated. automatically. After installing new CBC or ABC, the Dashboard does not update Click Refresh after changing or consumables status in the Dashboard is not automatically. installing consumables. updated automatically. Expiration dates are displayed in red. The consumable is within the No action. following days of expiration: Pouch 7 days, Buffers 7 days, Capillary array 1 day Dashboard indicates a consumable is RFID issue. Restart the instrument and the expired, but expiry date on consumable computer (see “Restart the indicates it is not expired. instrument and the computer” on page 279). Contact Thermo Fisher Scientific Software troubleshooting — general Symptom Possible cause Action When you start the 3500 The Norton Antivirus Sonar • Disable the optional Sonar feature in Norton Series Data Collection Protection feature is enabled Antivirus software (contact your IT department for Software v3.3, “Windows on the instrument computer. assistance). cannot find 3500.exe” • Contact Thermo Fisher Scientific. message is displayed. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 289 Appendix A Troubleshooting A Software troubleshooting — general (continued) Symptom Possible cause Action Status icon is instead One or more of the services Hover the mouse pointer over the status icon. If of . are stopped. any item does not display a checkmark, select 4Programs4Applied Biosystems435004Server Monitor. Right-click the status icon, then select Services. If any item does not display a checkmark, click the item to start the service. Print dialog box is Dialog boxes are sometimes Minimize the main screen. not displayed when you displayed behind the main select or click Print. screen The Load plate for run The window is not refreshing Click OK to dismiss the message and continue. message does not display properly. correctly. Save option is not You must select a plate Go to Define Plate Properties screen4Open available (only Save As) template from the main Plate4Edit Existing Template. when you edit a plate workflow to edit it. template from the library. Specimen name and The Specimen Name Enter the Specimen name and Amplicon name in the Amplicon name are attribute is not functional. Sample Name field in the Assign Plate Contents screen, specified in File Name Even when selected, Customize Sample information section. Convention but not specimen name is not To view Specimen Name and Amplicon Name in the included in sample name. included in the file name. Customize Sample Information section, a Sequencing assay must be assigned to a well. Note: The Specimen Name and Amplicon Name fields are available in the Plate View only, not the Table View of the Assign Plate Contents screen. Software is not behaving You open the instrument Do not open the instrument door during a run. as expected. door after you start a run You restarted the instrument Restart the instrument and the computer (see “Restart only, not the computer. the instrument and the computer” on page 279). Note: Restart the instrument and the computer as part of weekly maintenance. 290 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Run, re-run, or re-inject troubleshooting A (continued) Symptom Possible cause Action Software operations are Communication problem Restart the instrument and the computer (see “Restart taking longer than between the computer and the instrument and the computer” on page 279). expected. instrument. Audit records need archiving See “Archive, purge, and restore data” on page 274. and purging Run, re-run, or re-inject troubleshooting Symptom Possible cause Action Run stops unexpectedly or will Plate or sample information IMPORTANT! If you copy/paste sample or plate not start contains invisible, non-ASCII information into the Assign Plate Contents screen characters. or into a plate import file, copy from a plain text editor such as Notepad. Do not copy from a word processing program such as Microsoft™ Word™, which may include invisible, non-ASCII characters. Non-ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting. "Camera frame number The communication between Restart the instrument and the computer. See mismatch detected" the instrument and the “Restart the instrument and the computer” on computer has been page 279. interrupted and cannot be re- established automatically. If you re-run a plate that New protocols are not Before re-running a plate, examine the protocols specifies a re-injection, and the retained for re-injections. specified for re-injections and change as needed. re-injection specifies a protocol other than the protocol used for the original injection, the new protocol for the re-injection is not used 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 291 Appendix A Troubleshooting A Data/electropherogram troubleshooting Data/electropherogram troubleshooting Symptom Possible cause Action Signal too high. Sample concentration is too Dilute the sample. high. Decrease the injection time. Too much DNA added to the Optimize reaction conditions. reaction, resulting in uneven signal distribution. No signal. Blocked capillary. Run the Fill Array with Polymer wizard. Install a new capillary array. Bent capillary array tips or Visually inspect the capillary array, including the cracked or broken capillary detector window area for signs of breakage. array. Replace the capillary array. Failed reaction Repeat reaction. Low signal. Degraded formamide. Use a fresh aliquot of Hi‑Di™ Formamide (see “Hi‑Di™ Formamide” on page 25 for storage conditions). Not enough sample: Pipetting Prepare new sample. error. Sample has high salt Dilute or desalt samples. concentration. Insufficient mixing. Vortex the sample thoroughly, and then centrifuge the tube to condense the sample to the bottom of the tube. Weak amplification of DNA. Reamplify the DNA. Check DNA quality. Sample volume is <10 µL. Check that sample volume is at least 10 µL. Autosampler out of calibration. Contact Thermo Fisher Scientific. Elevated baseline. Possible contaminant in the Run the Wash Pump and Channels wizard. polymer path. Poor spectral calibration. Perform new spectral calibration. Loss of resolution. Too much sample injected. Dilute the sample and re-inject. Poor quality water. Use distilled or deionized water. Degraded polymer. Replace polymer. Capillary array used for more Replace the capillary array. Run the Install than 160 injections. Capillary Array wizard. 292 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Data/electropherogram troubleshooting A (continued) Symptom Possible cause Action Loss of resolution. Degraded formamide. Prepare fresh Hi‑Di™ Formamide (see “Hi‑Di™ Formamide” on page 25 for storage conditions) for sample preparation. Sample has high salt Dilute or desalt samples. concentration. Poor resolution in some Insufficient filling of capillary Tighten the connectors and array locking lever. capillaries. array. Run the Fill Array with Polymer wizard and look for polymer leakage. Check for broken capillaries, run the Install Capillary Array wizard if needed. Re-inject the same samples. Poor quality samples. Check the sample preparation. Leak in system. Tighten the connectors and array locking lever. No current. Not enough buffer in ABC. Ensure that the buffer is filled up to the fill line. See “Check buffer fill levels” on page 39. Bubble(s) present in the lower Pause the run and inspect for bubbles in the polymer block and/or the array tubing connectors. Run the Remove Bubbles and/or channels. wizard. Elevated current. Degraded polymer. Run the Replenish Polymer wizard. Arcing in the lower polymer Inspect the lower polymer block for discoloration block. or damage. Contact Thermo Fisher Scientific. Fluctuating current. Bubble in polymer block. Pause run and inspect for bubbles hidden in the tubing connectors. Run the Remove Bubbles wizard. Slow leak Check polymer blocks for leaks. Tighten the connectors and array locking lever. Not enough buffer in ABC. Ensure that the buffer is filled up to the fill line. See “Check buffer fill levels” on page 39. Arcing Check for moisture in and around the septa, the CBC, the oven, and the autosampler. Wipe condensation. Poor performance of capillary Poor quality samples, possible Desalt samples. array used for fewer than 100 cleanup problems. runs. Improperly stored formamide. Prepare fresh Hi‑Di™ Formamide (see “Hi‑Di™ Formamide” on page 25 for storage conditions) for sample preparation. Leak in system. Tighten the connectors and array locking lever. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 293 Appendix A Troubleshooting A Data/electropherogram troubleshooting (continued) Symptom Possible cause Action Migration time becomes Leak in system. Tighten the connectors and array locking lever. progressively slower. Improper filling of the system Polymer delivery pump may need to be serviced. with polymer. If the issue persists, contact Thermo Fisher Scientific. Migration time becomes Buffer valve leakage. Ensure the buffer-pin valve is closed correctly. progressively faster. Extra peaks in the Data off scale. Dilute the sample and re-inject the sample. electropherogram. Possible contaminant in Re-amplify the DNA. sample. Sample re-naturation. Heat-denature the sample in properly stored formamide (see “Hi‑Di™ Formamide” on page 25 for storage conditions) and immediately place on ice. Electrophoresis current is Bubbles in the polymer Run the Remove Bubbles wizard. unstable. system. Electrophoresis failure. Buffer below fill line. Ensure that the buffer is filled up to the fill line. “Check buffer fill levels” on page 39 . There is not enough fluid in Pipette the buffer from the smaller overflow larger chamber of ABC, or the chamber to the larger chamber. Ensure that the anode buffer has spilled into buffer is filled to within ±1 mm of the fill line. smaller overflow chamber. When installing new ABC, tilt the container to move buffer to the larger side of the container as described “Prepare the Anode Buffer Container (ABC)” on page 258. Extra peaks in the Data off scale. Dilute the sample and re-inject the sample. electropherogram. Possible contaminant in Re-amplify the DNA. sample. Sample re-naturation. Heat-denature the sample in good quality formamide and immediately place on ice. 294 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Review Results troubleshooting A Review Results troubleshooting Symptom Possible Cause Action Zoom errors in electropherogram graphical displays The zoom feature does not No action. (Monitor Run, Review Results, Spectral Calibration, re‑baseline the sample data view. and Install Check): • The zoom feature does not re‑baseline the sample data view, or • The X axis of the sample plot does not stay at the bottom of the screen. It moves up toward the region the user has zoomed in on, making data difficult to review Samples are not imported when you select multiple At least one file is not in Select individual folders for import the correct format for import, folders or files for therefore no files are imported. import instead of multiple folders. Plate Owner truncated in Annotation4Run Special characters were included Use only alpha- Configuration when entering plate information. numeric characters for plate information. Special characters in plate information fields may not be correctly displayed in some software screens. Sample files are not displayed when imported You imported (HID) files and you Click HID Samples. did not click HID Samples. Peaks are not labeled when you access the screen Labels are not automatically See “Label peaks” on applied. page 119. x and y scaling plot settings are not applied when you Scaling settings are applied only Click Zoom. click Apply when you click Zoom. The sizing quality result reported in the software You imported (FSA) files instead No action. differs from the sizing quality result for reported in the of (HID) files. GeneMapper™ ID-X Software The software does not consider the presence of broad peaks when determining sizing quality for fragment analysis data, therefore the sizing quality result reported in the software will differ from the sizing quality result reported in the GeneMapper™ ID-X Software, which considers broad peaks in sizing quality. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 295 Appendix A Troubleshooting A Link/load plate troubleshooting (continued) Symptom Possible Cause Action Sizing Overlay report displays data for all capillaries Plots displayed in Sizing Overlay Select only the including those that fail sizing Plot are based on the samples capillaries that pass selected. sizing to include in the report. Dye/Sample Peak column values in an exported sizing You changed the order of Edit the exported table are split in to two separate columns, the column columns before exporting. file: Cut/paste column name containing the Sample Peak values is incorrectly headers one column Note: The exported data are named, and the column headers after Dye/Sample are to the right, enter accurate, the column headers are shifted one column to the right. correct headers for the shifted to the right. Dye and Sample Peak columns. Link/load plate troubleshooting Symptom Possible cause Action Plate was linked, If you access the Load Plates for Run screen Access the Load Plates for Run screen from but now it is from the navigation pane, a plate may not the navigation pane and click Link Plate. unlinked. be linked (indicated by the active Link Plate button). “No plate in You physically loaded plate in position B Click Link Plates and link the plate directly to position A” (plate B position) and try to link plate. position B (plate B position). message. “No plate The plate is in position B. Place the plate in position A. See “Load the detected” plate in the instrument” on page 72. message Manually link the plate to position B. See “Link the plate” on page 72. You selected Quick Start. Do not use Quick Start, instead open plate and link via the main workflow. Note: Quick Start expects the plate to be in position A. The Autosampler has not completed Wait for the green light to light on the initialization. front panel before linking the plate. It takes approximately 10 seconds for the instrument to initialize after the instrument door is closed. Malfunctioning plate sensor(s). Contact Thermo Fisher Scientific. 296 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Link/load plate troubleshooting A (continued) Symptom Possible cause Action Pre-run validation The software does not display a date if No action. check does not it is identical to the preceding date. In display a date for a the example below, the installation and consumable. recommended replacement dates for cathode buffer are identical to the dates for anode buffer. Link/Unlink Plate Listed in Details. Click Details to determine the cause of the error message. error. When the plate is successfully loaded, the Load Plates for Run screen is displayed. “No plate The plate is in position B. Place the plate in position A. detected” message Create Injection The Pause After Last Injection preference is Go to Monitor Run and resume the run. When List and Start Run set, and the instrument is paused the run is complete, Create Injection List and buttons dimmed Start Run buttons are active. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 297 Appendix A Troubleshooting A Assign Plate Contents troubleshooting Assign Plate Contents troubleshooting Symptom Possible Cause Action Error message is displayed when you export a newly Plate is not saved. Save the plate, close the plate, open created plate from the Assign Plate Contents screen. the plate, then export. Spatial calibration troubleshooting Symptom Possible cause Action “Start” Spatial Calibration button is Communication failure between Check the connection between the disabled. the Data Collection Software and instrument and computer. instrument Restart instrument and computer (see “Restart the instrument and the computer” on page 279). Unusual peaks or a flat line for the spatial Improper installation of the array Run the Install a Capillary Array calibration. window in the detection cell (see wizard to uninstall, then re-install Figure 44). the array. If the calibration fails again: • Fill the capillaries with polymer. • Repeat the spatial calibration. Broken capillary resulting in a bad Check for a broken capillary, array fill. particularly in the detection cell area. If necessary, replace the capillary array using the Install Capillary Array wizard. Persistently bad spatial calibration results. Bad capillary array. Replace the capillary array using the Install Capillary Array wizard, then repeat the calibration. If the problem persists, contact Thermo Fisher Scientific. “Spatial Calibration Error” message. Conditioning reagent is installed. Replace the conditioning reagent with polymer. The instrument cannot perform Spatial Calibration with Array fill. Spatial calibration takes >5 minutes to Communication problem between Restart the instrument and the complete, and green light goes from the computer and instrument. computer (see “Restart the blinking to solid instrument and the computer” on page 279). 298 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Spectral calibration troubleshooting A (continued) Symptom Possible cause Action Spatial calibration takes >5 minutes to Oven is on. Do not preheat the oven before complete, and green light goes from running the spatial calibration. blinking to solid Accept/Reject buttons are dimmed. Communication problem between Restart the instrument and the the computer and instrument. computer (see “Restart the instrument and the computer” on page 279). Spectral calibration troubleshooting Symptom Possible cause Action No signal Incorrect preparation of sample Replace samples with fresh samples prepared with fresh Hi‑Di™ Formamide (see “Hi‑Di™ Formamide” on page 25 for storage conditions). Bubbles in sample wells Centrifuge samples to remove bubbles. Capillaries are not aspirating sample Check that sample volume is at least 10 µL. If sample volume is adequate, contact Thermo Fisher Scientific. The capillary tips may be hitting the Contact Thermo Fisher Scientific. bottom of the wells. Autosampler not correctly aligned. Peak heights in the Spectral report The raw data electropherogram No action. are different from the values seen display in the software does not have when viewing the spectral data in the the Run Scale Divisor applied to the electropherogram display. data. The final peak height values displayed in the Spectral report have the Run Scale Divisor applied. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 299 Appendix A Troubleshooting A Spectral calibration troubleshooting (continued) Symptom Possible cause Action The Spectral peaks in the raw data Septa contamination. Replace the CBC septa. view appear to be in the wrong order or there are extraneous peaks IMPORTANT! Make sure to replace the CBC septa as part of monthly maintenance. No history is stored for a failed run. No history is stored for a failed run. To retain a history for a failed run, generate a report before you click Reject Results. To generate a report, click View Summary Report or View Detail Report. To save the report electronically, select a PDF printer. Extra peaks or spikes in the raw data Bubbles in the polymer system. Run the Remove Bubbles wizard. or “Bad dye order detected” error message. Septa contamination. Replace the CBC septa. Possible contaminant, crystal Allow the polymer to come to deposits, or precipitate. room temperature. Do not heat to bring to room temperature. 300 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Spectral calibration troubleshooting A (continued) Symptom Possible cause Action Spectral calibration fails, or “No Blocked capillary Run the Fill Array with Polymer spectral files found” message is wizard to clear blockage. displayed. Insufficient filling of array. Check for broken capillaries. Run the Fill Array with Polymer wizard. Expired calibration standards or old Check the expiration date reagents. and storage conditions of the calibration standards and/or reagents. If necessary, replace with a fresh lot. Data Error - One or more peaks One or more peaks fall below the Rerun the spectral standards. fall below the minimum required minimum required amplitude of 750. amplitude of 750. Elevated baseline. Poor spectral calibration. Perform new spectral calibration. Pull-down (mirror image) peaks (see The first time you perform a spectral No action. the following figure) calibration (for each dye set) after installing a new capillary array, you may notice pull-down peaks (or mirror image peaks). These pull-down peaks will eventually correct themselves once the run completes. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 301 Appendix A Troubleshooting A Sequencing install standard troubleshooting (continued) Symptom Possible cause Action AnyDye Set Spectral Calibration fails. Problem with spectral calibration See “Perform a spectral calibration” on page 133. AnyDye dye set is not set up correctly. See “Create a new dye set using the AnyDye template” on page 195. Sequencing install standard troubleshooting Symptom Possible cause Action No signal Incorrect preparation of Replace samples with fresh standard samples prepared with fresh Hi‑Di™ Formamide (see “Hi‑Di™ Formamide” on page 25 for storage conditions). Bubbles in sample wells Centrifuge samples to remove bubbles. Capillaries are not aspirating Check that sample volume is at sample least 10 µL. If sample volume is adequate, contact Thermo Fisher Scientific. The capillary tips may be Contact Thermo Fisher Scientific. hitting the bottom of the wells. Autosampler not correctly aligned. The Sequencing install check fails: Failed Blocked capillary Run the Fill Array with Polymer capillaries wizard. Install a new capillary • One or more (for 8-capillary). array. • Three or more (for 24-capillary). Insufficient filling of array. Check for broken capillaries. Run the Fill Array with Polymer Accept button is not active, Reject button is wizard. active. Expired sequencing standard Check the expiration date or old reagents. and storage conditions of the sequencing standard and/or reagents. If necessary, replace with a fresh lot. Bubbles in the polymer Run the Remove Bubbles system. wizard. 302 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Fragment/HID install standard troubleshooting A (continued) Symptom Possible cause Action The Sequencing install check fails: Failed Possible contaminant or Properly bring the polymer to capillaries crystal deposits in the polymer. room temperature; do not heat. • One or more (for 8-capillary). • Three or more (for 24-capillary). Accept button is not active, Reject button is active. The starting well value you set reset to A01 If you navigate away from the No action. after you start the install check. Install Check screen after you start the install check, the starting well may be reset to A01. This is a display issue only; the starting well you specify is used for the install check. Fragment/HID install standard troubleshooting Symptom Possible cause Action Fragment/HID report All dyes are not selected before you Select all dyes, then generate the report. contains blank pages or generate the report. incomplete information. No signal Incorrect preparation of sample Replace samples with fresh samples prepared with fresh Hi‑Di™ Formamide. Bubbles in sample wells Centrifuge samples to remove bubbles. The capillary tips may not be touching Check the volume of your samples. If no the samples. results, call your Thermo Fisher Scientific representative. The capillary tips may be hitting the Call your Thermo Fisher Scientific. bottom of the wells. Autosampler not representative. correctly aligned. Fragment/HID install check Blocked capillary Refill capillary array. You may have to install fails. a fresh array or consider that capillary non- usable for purposes of planning your runs. Insufficient filling of array. Check for broken capillaries and refill the capillary array. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 303 Appendix A Troubleshooting A Monitor Run troubleshooting (continued) Symptom Possible cause Action Fragment/HID install check Expired matrix standards or old Check the expiration date and storage fails. reagents. conditions of the matrix standards and/or reagents. If necessary, replace with a fresh lot. Bubbles in the polymer system. Select the Bubble Remove wizard to clear the bubbles. Possible contaminant or crystal Properly bring the polymer to room deposits in the polymer. temperature; do not heat. The starting well value you If you navigate away from the Install No action. set reset to A01 after you Check screen after you start the install start the install check. check, the starting well may be reset to A01. This is a display issue only; the starting well you specify is used for the install check. Monitor Run troubleshooting Symptom Possible Cause Action The instrument run RFID read/write error. Click Refresh in the Dashboard. unexpectedly pauses. If consumables status does not refresh, restart the instrument and the computer (see “Restart the instrument and the computer” on page 279). Only some injections from Bad connection between the Check the connection between the instrument a series of injections are instrument and computer and the computer. Restart the instrument and the completed. computer (see “Restart the instrument and the computer” on page 279). The autosampler does not move on to the next injection Estimated Time Remaining Estimated Time Remaining is To view time remaining per injection, scroll to the in Monitor Run is longer the time remaining in the Time Remaining column in the Injection List Details. than expected. instrument run. This estimate is adjusted after the completion of every step in an injection. Contents of tooltip in Flag Special characters were Use only alpha-numeric characters for sample list is truncated included when entering sample information. Special characters in sample information information fields may not be correctly displayed in other software screens. 304 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Audit troubleshooting A (continued) Symptom Possible Cause Action Re-inject button is dimmed Injection contains samples with Select in the injection list the injection with the when you select an injection assays that specify more than instrument protocol of interest, select in the array one instrument protocol. view the capillary that corresponds to the well of interest, then click Re-inject. QV flag for sequencing Contiguous Read Length of • If the expected read length of the amplicon is data, but data quality is the amplicon is less than the <300, adjust the Contiguous Read Length Pass good Contiguous Read Length Pass value. value specified in Basecalling • If the expected read length of the Protocol QV settings or Trace amplicon ³300, review the sample quality Quality preference settings. throughout the entire trace. QV flag for sequencing Run Time in Instrument Protocol Adjust Run Time. data is too short for the amplicon. Incorrect Mobility file for Select the correct Mobility file for dye/polymer in dye/polymer is selected in Basecalling Protocol, then re‑inject. Basecalling Protocol. Apply correct Basecalling Protocol in secondary sequence analysis software. Audit troubleshooting Symptom Possible Cause Action “Export did not complete You exported records for samples that are not Return sample data files to their successfully” in their original location (samples have been original location, then export deleted or moved). again. Audit report does not print after Font settings are not activated until you close Close the report, reopen it, then you change font settings. the report. print. Electronic signature troubleshooting Symptom Possible Cause Action The dye set calibrated is not The e‑signature function creates a record To include the dye set listed in a spectral calibration when a spectral calibration is performed, but calibrated in the e‑signature e‑signature record. does not record the dye set calibrated. record, enter the dye set in the Comments field. Electronic signature prompt is Electronic signature prompt is displayed No action. displayed when you edit sample for sample comments, regardless of the comments. electronic signature setting. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 305 Appendix A Troubleshooting A Manual commands troubleshooting Manual commands troubleshooting Symptom Possible Cause Action When you select Tools4Manual Commands, The feedback from Consumables See the Dashboard for Set defined command for Consumables, then Read Tag commands does not consumables RFID tag select a Read Command, the information display valid information. information. displayed is not readable. Troubleshooting procedures Run Troubleshooting Utility.bat If instructed to do so by a Thermo Fisher Scientific representative: 1. Navigate to x:\AppliedBiosystems\3500\Troubleshoot. 2. Double-click TroubleshootingUtility.bat. 3. Navigate to s:\TroubleshootData, then email the data-<yyyy-mm-dd-hh-mm-ss-mmm>.tslog file to your Thermo Fisher Scientific representative. View the log files Use a text editor (such as Wordpad) to view the software-generated log files: Log file Description Location 3500UsageStatistics.txt Provides a summary of the <<install drive>>:\Applied number of plates run and Biosystems\3500\LogFiles number of run types You can also view this log from the Maintenance workflow under Planned Maintenance4Usage Statistics. 3500ConsumableUpdates.txt Provides a summary of <<install drive>>:\Applied consumables installation Biosystems\3500\LogFiles information and dates 306 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix A Troubleshooting Troubleshooting procedures A View instrument sensor details Click View Instrument Sensor Details in the Dashboard to display instrument information. Figure 47 Instrument sensor details Run status of the instrument is displayed while a run is in progress. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 307 Appendix A Troubleshooting A Troubleshooting procedures Review error message details Error messages in the software include a Details button. Click Details to display more information about an error message. Reset the instrument Resetting powers off, then powers on, the instrument. Reset the instrument when: • There is a fatal error as indicated by the red status light • The instrument does not respond to the Data Collection software 1. Shut down the computer. 2. Close the instrument doors. 3. Reset the instrument with the Reset button, as shown. Note: The Reset button is accessible through a small hole to the left of the Tray button. Reset button 308 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 B Run modules and dye sets Run modules Table 5 Sequencing analysis run modules Perfor- Configuration 23 hours Throughput[1] mance Run module type Run module name Contig. Cap. Run Polymer 3500 3500xL Read length time type (8‑cap.) (24-cap.) Length (cm) (min) (CRL)[2] Short read ShortReadSeq50_POP7 50 POP-7™ £30 ³368 ³1104 ³300 sequencing ShortReadSeq50_POP7xl Short read BDxShortReadSeq50_POP7 50 POP-7™ £30 ³368 ³1104 ³300 sequencing BDxShortReadSeq50_POP7xl BigDye XTerminator™ Rapid sequencing RapidSeq50_POP6 50 POP-6™ £65 ³168 ³504 ³450 RapidSeq50_POP6xl RapidSeq50_POP7 50 POP-7™ £40 ³280 ³840 ³500 RapidSeq50_POP7xl Rapid sequencing RapidSeq36_POP4 36 POP-4™ £45 ³240 ³720 ³400 RapidSeq36_POP4xl RapidSeq36_POP6 36 POP-6™ £65 ³168 ³504 ³600 RapidSeq36_POP6xl RapidSeq36_POP7 36 POP-7™ £30 ³368 ³1104 ³600 RapidSeq36_POP7xl Rapid sequencing BDxRapidSeq50_POP6 50 POP-6™ £65 ³168 ³504 ³450 BigDye BDxRapidSeq50_POP6xl XTerminator™ BDxRapidSeq50_POP7 50 POP-7™ £40 ³280 ³840 ³500 BDxRapidSeq50_POP7xl 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 309 Appendix B Run modules and dye sets B Run modules Table 5 Sequencing analysis run modules (continued) Perfor- Configuration 23 hours Throughput[1] mance Run module type Run module name Contig. Cap. Run Polymer 3500 3500xL Read length time type (8‑cap.) (24-cap.) Length (cm) (min) (CRL)[2] Rapid sequencing BDxRapidSeq36_POP4 36 POP-4™ £45 ³240 ³720 ³400 BigDye BDxRapidSeq36_POP4xl XTerminator™ BDxRapidSeq36_POP6 36 POP-6™ £66 ³164 ³494 ³600 BDxRapidSeq36_POP6xl BDxRapidSeq36_POP7 36 POP-7™ £30 ³368 ³1104 ³600 BDxRapidSeq36_POP7xl Fast sequencing FastSeq50_POP6 50 POP-6™ £90 ³122 ³368 ³600 FastSeq50_POP6xl FastSeq50_POP7 50 POP-7™ £65 ³168 ³504 ³700 FastSeq50_POP7xl Fast sequencing FastSeq36_POP7 36 POP-7™ £60 ³184 ³552 ³750 FastSeq36_POP7xl Fast sequencing BDxFastSeq50_POP6 50 POP-6™ £90 ³122 ³368 ³600 BigDye BDxFastSeq50_POP6xl XTerminator™ BDxFastSeq50_POP7 50 POP-7™ £65 ³168 ³504 ³700 BDxFastSeq50_POP7xl Fast sequencing BDxFastSeq36_POP7 36 POP-7™ £60 ³240 ³552 ³750 BigDye BDxFastSeq36_POP7xl XTerminator™ Standard StdSeq50_POP6 50 POP-6™ £135 ³80 ³240 ³600 sequencing StdSeq50_POP6xl StdSeq50_POP7 50 POP-7™ £125 ³88 ³264 ³850 StdSeq50_POP7xl Standard BDxStdSeq50_POP6 50 POP-6™ £140 ³80 ³240 ³600 sequencing BDxStdSeq50_POP6xl BigDye XTerminator™ BDxStdSeq50_POP7 50 POP-7™ £125 ³88 ³264 ³850 BDxStdSeq50_POP7xl 310 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix B Run modules and dye sets Run modules B Table 5 Sequencing analysis run modules (continued) Perfor- Configuration 23 hours Throughput[1] mance Run module type Run module name Contig. Cap. Run Polymer 3500 3500xL Read length time type (8‑cap.) (24-cap.) Length (cm) (min) (CRL)[2] Microbial MicroSeq50_POP6 50 POP-6™ £135 ³80 ³240 ³600 sequencing MicroSeq50_POP6xl Microbial MicroSeq50_POP7 50 POP-7™ £125 ³88 ³264 ³850 sequencing MicroSeq50_POP7xl [1] Throughput (Samples / Day): The total number of samples run in 23 hours (0.5 hour for user interaction and 0.5 hour for warm-up time). [2] The maximum number of contiguous bases in the analyzed sequence with an average QV ³20, calculated over a sliding window 20 base pairs wide from an AB Long Read Standard sequencing sample. This calculation starts with base number 1. The read length is counted from the middle base of the 1st good window to the middle base of the last good window, where a “good” window is one in which the average QV ³20. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 311 Table 6 Fragment and HID analysis run modules 312 B Configuration 23 hours throughput[1] Performance Run modules Appendix B Run modules and dye sets Run module Cap. Run Sizing Precision[3] Run module name 3500 3500xL type length Pol. type time Range[2] (8-cap.) (24-cap.) 50bp- (cm) (min) 401bp-600bp 601bp-1200bp 400bp Frag. analysis FragmentAnalysis50_POP7 50 POP-7™ £45 ³280 ³840[4] £40 <0.15 <0.30 NA[5] to ³520 FragmentAnalysis50_POP7xl FragmentAnalysis50_POP6 50 POP-6™ £100 ³112 ³336 £20 <0.15 <0.30 NA[5] to ³550 FragmentAnalysis50_POP6xl FragmentAnalysis36_POP4 36 POP-4™ £35 ³312 ³936 £60 <0.15 NA[5] NA[5] to ³400 FragmentAnalysis36_POP4xl 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 FragmentAnalysis36_POP7 36 POP-7™ £30 ³368 ³1104 £60 <0.15 NA[5] NA[5] to ³400 FragmentAnalysis36_POP7xl Frag. analysis FragAnalysis36_POP6 36 POP-6™ £60 ³184 ³552 £60 <0.15 <0.30 NA[5] to ³400 FragAnalysis36_POP6xl Long frag. LongFragAnalysis50_POP7 50 POP-7™ £130 ³84 ³254 £40 <0.15 <0.30 <0.45 analysis to ³700 LongFragAnalysis50_POP7xl HID HID36_POP4 36 POP-4™ £35 ³312 ³936 £60 <0.15 NA[5] NA[5] to ³400 HID36_POP4xl SNaPshot™ SNaPshot50_POP7 50 POP-7™ £30 ³376 ³1104 £40 <0.50 NA[5] NA[5] to ³120 SNaPshot50_POP7xl [1] Throughput (Samples / Day): The total number of samples run in 23 hours (0.5 hour for user interaction and 0.5 hour for warm-up time). [2] Resolution Range: The range of bases over which the resolution (peak spacing interval divided by the peak width at half-max in a GS600 or GS1200 LIZ size standard sample sized with a third order fit) is ³1. The table shows the resolution range in ³90% of samples. [3] Sizing Precision: Standard deviation of sizes for one allele in the DS-33 install standard sized with the GS600 LIZ size standard across multiple capillaries in the same run. For one injection to pass, 100% of the alleles in that injection must meet the intra-run sizing precision specifications. The table shows the sizing precision of 100% of alleles in ³90% of samples. [4] Throughput is an estimate extrapolated from average run time. [5] Not applicable because of the size of the fragments collected in the run. Appendix B Run modules and dye sets Dye sets B Dye sets Sequencing analysis dye sets Table 7 Sequence analysis dye sets Dye Set Application Name E (v1.1 BigDye™ Terminator) Rapid DNA sequencing Z (v3.1 BigDye™ Terminator) DNA sequencing Fragment analysis dye sets for all applications Table 8 Fragment analysis dye sets Application E5 SNaPshot™ kit G5 DNA sizing for 5-dye chemistry J6 or J6-T DNA sizing for 6-dye chemistry F DNA sizing for 4-dye chemistry D DNA sizing for 4-dye chemistry AnyDye DNA sizing HID analysis dye sets Table 9 AmpFℓSTR™ Kit Table AmpFℓSTR™ Kits Dye set (use with HID Fragment Analysis 36_POP4 run module) 4-dye: F • COfiler™ • Profiler Plus™ • Profiler Plus™ID • SGM Plus™ • Other 4-dye kits 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 313 Appendix B Run modules and dye sets B Dye sets Table 9 AmpFℓSTR Kit Table (continued) AmpFℓSTR™ Kits Dye set (use with HID Fragment Analysis 36_POP4 run module) 5-dye: G5 • Identifiler™ • Identifiler™ Direct • Identifiler™ Plus • MiniFiler™ • NGM™ • NGM SElect™ • NGM SElect™ Express • SEfiler Plus™ • Sinofiler™ • Yfiler™ • Yfiler™ Direct • Other 5-dye kits 6-dye: J6 • GlobalFiler™ • GlobalFiler™ Express • NGM Detect™ 314 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 C Instrument specifications Instrument specifications Table 10 Applied Biosystems™ 3500/3500xL Genetic Analyzer physical dimensions, weight, and power consumption Parameter Instrument footprint Recommended clearance Depth 61 cm (24 in.) 25.4 cm (10 in.)[1] Width 61 cm (24 in.) (closed door) 158 cm (62 in.)[2] 122 cm (48 in.) (open door) Height 72 cm (28.3 in.) 31 cm (12 in.) Weight »82 kg (180 lbs) [1] At the rear of the instrument to ensure adequate airflow and cooling [2] For the instrument, computer, and computer monitor. Table 11 Computer dimensions and weight Parameter Computer Monitor Keyboard Depth 41.7 cm (16.42 in.) 19.3 cm (7.6 in.) 44.7 cm (17.5 in.) Width 17.5 cm (6.89 in.) 44.7 cm (17.5 in.) 15.25 cm (6 in.) Height 37.4 cm (14.7 in.) 36.6 cm (14.4 in.) 5 cm (2 in.) Weight 9.6. kg (21 lbs) 6.9 kg (15.2 lbs) 0.09 kg (0.2 lbs) Table 12 Applied Biosystems™ 3500/3500xL Genetic Analyzer operating specifications Component Specification Laser • Long-life, single-line 505 nm, solid-state laser excitation source • Laser Output power 20mW • Beam divergence 1.4 mrad LED • Emitting color Natural White • Luminous Intensity 250 Cd Electrophoresis Voltage Up to 20 kV Oven Temperature Active temperature control from 18°C to 70°C 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 315 Appendix C Instrument specifications C Environmental requirements Table 12 Applied Biosystems 3500/3500xL Genetic AnalyzerRUO operating specifications (continued) Component Specification Minimum Computer Requirements • Hardware: – OptiPlex™ XE, E8400, 3 GHZ Processor or – OptiPlex™ XE2, with Intel™ Core I7-47705, 3.1 GHz Processor • Operating system: Windows™ 10 64‑bit (IOT Enterprise) • Installed RAM: 16 GB • Hard drive: 500 GB SATA 3.0 Gb/s and 8 MB Data Burst Cache Environmental requirements Table 13 Environmental requirements Condition Requirement Installation site Indoor use only, Professional Healthcare Environment Electromagnetic Do not use this device in close proximity to sources of strong interference electromagnetic radiation (for example, unshielded intentional RF sources). Strong electromagnetic radiation may interfere with the proper operation of the device. This equipment has been designed and tested to CISPR 11 Class A. The emissions characteristics of this equipment make it suitable for use in industrial areas and hospitals (CISPR 11 Class A). If it is used in a residential environment (for which CISPR 11 Class B is normally required), this equipment might not offer adequate protection to radio-frequency communication services. The user might need to take mitigation measures, such as relocating or re-orienting the equipment. Altitude Safety tested up to 2,000 m (6,562 ft) Electrical ratings Power cord with ground pin required • Instrument—AC 100–240 V ±10%, 50/60 Hz, power rated 320 VA • Computer—AC 100–240 V ±10%, 50/60 Hz, power rated 125 VA • Monitor—AC 100–240 V ±10%, 50/60 Hz, power rated 65 VA Mains AC line voltage Up to ±10 percent of nominal voltage tolerances Transient category Installation categories II Pollution degree 2 316 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix C Instrument specifications Environmental requirements C Table 13 Environmental requirements (continued) Condition Requirement Liquid waste collection Dispose of the polymer, buffer, reagents and any liquid waste as hazardous waste in compliance with local and national regulations. Operating conditions 15–30°C (59–86°F) (Room temperature should not fluctuate ±2°C during an instrument run) 20–80% relative humidity, noncondensing Transport and storage –30 to +60°C ( –22 to +140°F) Minimum 20% relative humidity, maximum 85% conditions (non-condensing) WARNING! Portable RF communications equipment (including peripherals such as antenna cables and external antennas) should be used no closer than 30 cm (12 inches) to any part of the 3500/3500xL Genetic Analyzer, including cables specified by the manufacturer. Otherwise, degradation of the performance of this equipment could result. WARNING! Use of this equipment adjacent or stacked with other equipment should be avoided because it could result in improper operation. WARNING! Use of accessories, transducers, and cables other than those specified or provided by the manufacturer of this equipment could result in increased electromagnetic emissions or decreased electromagnetic immunity of this equipment and result in improper operation. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 317 Appendix C Instrument specifications C Power and communication connections Power and communication connections 1 2 3 1 Ethernet port for computer instrument connection 2 Circuit breaker 3 Connector for instrument power cable 318 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 D Catalog numbers Plates bases retainers and septa Table 14 Plates and caps Part Description General purpose supply, obtain from any laboratory supplier 96-well General purpose supply, obtain from any laboratory supplier: 96-well PCR microtiter plate, standard or optical-grade polypropylene, 0.1 mL or 0.2 mL, half- or semi-skirted design, with or without barcode. 8-tube strips General purpose supply, obtain from any laboratory supplier: • 8-strip PCR tubes, standard- or optical-grade polypropylene, 0.1 mL • 8-strip full-height PCR tubes, standard- or optical-grade polypropylene, 0.2 mL Tube caps 8-tube PCR strip caps, domed, standard- or optical-grade polypropylene, for 0.1 mL or 0.2 mL 8-strip PCR tubes Plate, 384-well General purpose supply, obtain from any laboratory supplier: 384-well PCR microtiter plate, standard or optical-grade polypropylene, 0.02 mL, fully-skirted design, with or without barcode Table 15 Bases, retainers, and septa Part Description Cat. No. Retainer and base, 8-tube RUO 4410231 Retainer and base (Fast), 8-tube RUO 4410233 Retainer and base (Standard), 96-well 4410227 Retainer and base (Standard), 96-well RUO 4410228 Retainer and base (Fast), 96-well RUO 4409530 Retainer and base (Standard), 384-well, RUO 4410235 Septa, 8-strip RUO 4410701 Septa, 96-well 4410700 Septa, 384-well RUO 4412520 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 319 Appendix D Catalog numbers D Instrument consumables Instrument consumables Name Cat. No. Anode Buffer Container 4393927 Capillary array, 8‑Capillary, 36‑cm 4404683 Capillary array, 24‑Capillary, 36‑cm 4404687 Capillary array, 8‑Capillary, 50‑cm 4404685 Capillary array, 24‑Capillary, 50‑cm 4404689 Cathode Buffer Container 4408256 Conditioning reagent 4393718 Polymer, POP-6™(960) 4393712 Polymer, POP-6™ (384) 4393717 Polymer, POP-6™ (96) A26071 Polymer, POP-7™ (960) 4393714 Polymer, POP-7™ (384) 4393708 Polymer, POP-7™ (96) A26073 Polymer, POP-4™ (960)[1] 4393710 Polymer, POP-4™ (384)[1] 4393715 Polymer, POP-4™ (96) A26070 Polymer pouch cap 4412619 Hi‑Di™ Formamide Four 5-mL bottles 4440753 [1] Validated for HID applications. Sequencing analysis reagents and consumables Name Cat. No. BigDye™ Terminator v1.1 3500/3500xL Sequencing Standards, BigDye™ Terminator v1.1 4404314 BigDye™ Terminator v1.1 Matrix Standards Kit 4336824 BigDye™ Terminator v1.1 Cycle Sequencing Kit 4337449 320 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix D Catalog numbers Fragment and HID analysis reagents D (continued) Name Cat. No. BigDye™ Terminator v3.1 3500/3500xL Sequencing Standards, BigDye™ Terminator v3.1 4404312 BigDye™ Terminator v3.1 Matrix Standards Kit 4336974 BigDye™ Terminator v3.1 Cycle Sequencing Kit 4337454 BigDye™ Direct BigDye™ Direct Cycle Sequencing Kit 4458689 Fragment and HID analysis reagents Name Cat. No. DS-02 Matrix Standard Kit (Dye Set E5) 4323014 DS-32 Matrix Standard Kit (Dye Set F) 4345831 DS-33 Matrix Standard Kit (Dye Set G5) 4345833 DS‑36 Matrix Standard Kit (Dye set J6, 6‑dye) 4425042 DS-37 Matrix Standard Kit (Dye set J6‑T, 6‑dye) A31234 DS‑33 GeneScan™ Installation Standards with GeneScan™ 600 LIZ™ Size Standard 4376911 v2.0 (G5 dye set) GeneScan™ 120 LIZ™ Size Standard 4324287 GeneScan™ 500 ROX™ Size Standard 401734 GeneScan™ 600 LIZ™ Size Standard v2.0 4408399 GeneScan™ 1200 LIZ™ Size Standard 4379950 AmpFℓSTR™ Identifiler™ Allelic Ladder (For HID install check) Contact Thermo Fisher Scientific 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 321 E Limitations The 3500 instrument contains the limitations noted below. Please ensure that any use of the instrument takes into consideration these limitations. General • IMPORTANT! Enter only alpha-numeric characters in the software. Special characters may not be correctly displayed in some software screens, may cause problems with plate, file, folder, user account, and/or library item names, and may interfere with starting a run and/or importing and exporting library items. • IMPORTANT! If you copy/paste sample or plate information into the Assign Plate Contents screen or into a plate import file, copy from a plain text editor such as Notepad. Do not copy from a word processing program such as Microsoft™ Word™, which may include invisible, non-ASCII characters. Non-ASCII characters in plate or sample information may cause a run to stop or may prevent a run from starting. • When importing a sample plate, use the format shown in “Create a plate import template” on page 95. • If you re-run a plate that specifies a re-injection, and the re-injection specifies a protocol other than the protocol used for the original injection, the new protocol for the re-injection is not used. Before re-running a plate, examine the protocols specified for re-injections and change as needed. Computer • The computer provided with the instrument contains validated software and settings. Do not update the Windows™ operating system or firewall settings. • The computer provided with the instrument does not include antivirus software because customer preferences and network requirements vary. See “Antivirus software requirements” on page 20 for antivirus software that has been tested and approved for use with the software. 322 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix E Limitations Instrument and consumables E Instrument and consumables • Instrument firmware is to be updated only by a Thermo Fisher Scientific representative. • Use only the parts listed in Appendix D, “Catalog numbers”. • Replace the capillary array after 160 injections or expiration date listed on packaging and RFID label. • Before each run, check buffer fill levels. • In the event of a power disruption, restart the computer (“Restart the instrument and the computer” on page 279). • If you observe “Unable to transmit measurement data. Internal data buffer overflow.” error, restart the computer (“Restart the instrument and the computer” on page 279). • If you observe a “Failure to Read from RFID tag” error, see “Troubleshooting” on page 328. Calibration and install checks • If an install check for the run application type (Sequencing, Fragment, or HID) has not been performed, a message is displayed and the run does not start. • When running a spatial calibration, select Perform QC Checks as described in page 125. Refer to examples of passing spatial calibration as shown in “Example spatial profiles” on page 128. • When performing a spectral calibration, select the dye set appropriate for your application as described in “Perform a spectral calibration” on page 133. • If you navigate away from the Install Check screen after you start the install check, the starting well may be reset to A01. This is a display issue only; the starting well you specify is used for the install check. • If you change font settings before you generate a report, the report may not be generated. Generate the report again. Security Audit and E-sig • Before using the instrument, configure system security as described in “Configure the security system” on page 219. • Changes to e-signature settings are not activated until you log out of the software, then log back in. • If you change font settings before you generate an Object Audit report, the report may not be generated. Generate the report again. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 323 Appendix E Limitations E Review results Review results • For sequencing data, review sample quality as described in “Review traces” on page 106. • For fragment analysis or HID data, review sample quality as described in Review sample quality (page 115). • If you change the order of columns before exporting a sizing table, Dye/Sample Peak column values are split in to two separate columns, the column name containing the Sample Peak values is incorrectly named, and the column headers after Dye/Sample are shifted one column to the right. You can edit the exported file: Cut/paste column headers one column to the right, enter correct headers for the Dye and Sample Peak columns. 324 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 F Radio Frequency Identification (RFID) technology The instrument uses four identical wireless radio frequency identification (RFID) read/write units to monitor instrument consumables (for more information, see “Instrument parts and functions” on page 18). Precautions for use WARNING! Radio frequency identification (RFID) could possibly disrupt the operation of patient- worn and/or implanted active medical devices. To minimize such effects, do not come within 10 cm of this instrument if you have a patient-worn and/or implanted active medical device. WARNING! Radio frequency identification (RFID) signals from external devices could possibly disrupt the operation of the 3500 RFID read/write units. RFID signals from the 3500 RFID read/write units could possibly disrupt the operation of external RFID devices. To minimize such effects, do not bring external RFID devices within 10 cm of this instrument during instrument operation. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 325 Appendix F Radio Frequency Identification (RFID) technology F Locations of RFID read/write units Locations of RFID read/write units 7 1 2 3 8 9 4 5 6 Figure 48 RFID read/write unit locations within instrument interior (shown with door open) 1 Polymer delivery pump (PDP) 2 Anode buffer container (ABC) 3 Polymer or conditioning pouch 4 ABC RFID read/write unit (behind reservoir) 5 Pouch read/write unit (behind pouch) 6 CBC RFID read/write unit 7 Capillary array read/write unit 8 Cathode buffer container (CBC) 9 Autosampler Function The RFID read/write units: 1. Read up to 256 bytes from the RFID consumables tags. 2. Write up to 256 bytes to the RFID consumables tags. 3. Re-read the written data on the tags to confirm that it is accurate, using a checksum to verify data integrity. The RFID read/write units perform the functions listed above at the start of each run. 326 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix F Radio Frequency Identification (RFID) technology Specifications F Specifications Table 16 RFID read/write unit specifications Component Specification RFID read/write unit • Ultra-Compact Proximal-Type RFID Reader / Writer • Model ASI4000R2X • Manufactured by ART Finex Co., Ltd. RF frequency 13.56 MHz RF output power 60 mW RFID tags Texas Instruments RI-I03-112A-03 tags, tested by the manufacturer to reliably read and write 100,000 times with zero data loss and retain written data for more than 10 years Effective range between RFID tag and internal RFID • ABC tag: 3 cm read/write units • CBC tag: 4 cm • Capillary tag: 3 cm • Polymer tag: 3 cm Typical use range between RFID tag and internal 0.5 cm RFID read/write units Minimum separation distance of the instrument 10 cm from external RFID read/write units Minimum separation distance of the instrument 10 cm from other wireless technologies such as WiFi, Bluetooth, or Cellular Minimum separation distance of the instrument 1m from other laboratory equipment such as centrifuge or thermal cycler Wireless security • RFID tag read/write/re-read with checksum • Password access for use of software • Base-64 encoding of data between the instrument and the computer 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 327 Appendix F Radio Frequency Identification (RFID) technology F Troubleshooting Troubleshooting Table 17 RFID troubleshooting Symptom Possible cause Action Unable to read RFID Consumable package is improperly Ensure that the RFID label is not visibly information. “Failure to installed or label is defective. damaged and consumable package is properly Read from RFID tag” Polymer/Conditioning reagent pouch installed. is not positioned properly. Ensure that label is close, and parallel, to the instrument. Reposition or re-install pouch, then click Refresh on the Dashboard. Restart the instrument and the computer (see “Restart the instrument and the computer” on page 279). Install a new consumable (if available). If problem persists, contact Thermo Fisher Scientific. Malfunctioning RFID label or reader. Place a used CBC, ABC, pouch, or array on the instrument: • If the instrument can read the RFID label, install a new CBC, ABC, pouch, or array. • If the instrument cannot read the RFID label, contact Thermo Fisher Scientific. 328 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 G Signal optimization and size standard normalization Signal optimization feature The signal optimization feature reduces peak height variation across capillaries in an injection. This variation is introduced by the optics of the instrument and the injection conditions used. The signal optimization feature has two components: • Spatial calibration-dependent signal optimization (24-capillary instruments only, always enabled) • Capillary-position–dependent signal optimization (8-capillary and 24-capillary instruments, requires autosampler adjustment by field service engineer. ) For maximum signal optimization on 24-capillary instruments, we suggest that you use both components of the signal optimization feature. Spatial calibration-dependent signal optimization To enable this feature: 24-capillary instruments only, always enabled. Not available on 8-capillary instruments. To disable this feature: Cannot be disabled During spatial calibration, a Signal Optimization Factor is calculated for each capillary using a fitted curve method. The fitted curve method minimizes background signal and reduces noise. The adjusted signal intensity, not the signal intensity displayed for a capillary, is used to calculate the Signal Optimization Factor for the capillary. The Signal Optimization Factor for each capillary is displayed in the Spatial Calibration screen (the adjusted signal intensity is not displayed). 1 1 Signal Optimization Factor 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 329 Appendix G Signal optimization and size standard normalization G Signal optimization feature Note: The signal intensity, average peak height, and uniformity displayed for the capillaries are used for the Perform QC Checks function in spatial calibration. The Signal Optimization Factor range is 0.5–2. Higher or lower values are rounded down or up to bring them within range. Therefore, you may observe two peaks with different intensities but with the same Signal Optimization Factor. Note: On 8-capillary instruments, the Signal Optimization Factor field displays 1.0 for all capillaries after a spatial calibration. The Signal Optimization Factor is applied to signal data for each capillary during data collection to minimize optical variation effects and increase signal uniformity between capillaries. The Signal Optimization Factor is exported or printed and included in the spatial calibration report with the other spatial calibration results. Run module-dependent signal optimization To enable this feature: • Create an "SO" instrument protocol with the HID36_POP4(xl)_SO run module and • Create an "SO" assay with the "SO" instrument protocol and • Use the "SO" assay 8-capillary and 24-capillary instruments, optional. To disable this feature: Use an assay without _SO suffix A new HID36_POP4(xl)_SO run module (SO=signal optimization) has been optimized to minimize injection variability between capillaries. Enhancements to the run module include the introduction of a minimal amount of polymer into the well before the injection and a higher capillary position in the well during the injection. These changes have been shown to improve peak uniformity across capillaries within an injection. You can create and use new SO assays to implement the second component of signal optimization. 330 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix G Signal optimization and size standard normalization Size standard normalization feature G Size standard normalization feature Overview of the normalization feature For fragment analysis applications, the software includes an optional normalization feature for use with the GeneScan™ 600 LIZ™ Size Standard v2.0 (GS600 LIZ v2). This feature attenuates signal variations associated with instrument, capillary array, sample salt load, and injection variability between capillaries and instruments. Normalization can be applied during primary analysis of the data. To use the normalization feature, prepare each sample with the GS600 LIZ v2 size standard, then specify the appropriate normalization size standard for file primary analysis. The GS600 LIZ™ v2 reagent can function as an internal standard for signal‑height normalization as well as a size standard for peak sizing. When to use the normalization feature The software provides three normalization size-standard definition files that you can specify for primary analysis of samples prepared with the GS600 LIZ v2 size standard and the G5 and J6 dye sets: • Fragment (POP-6™ and POP-7™ polymer): – GS600LIZ+Normalization – GS600(60-600)LIZ+Normalization—For applications that have primer peaks that obscure the 20 and 40-mer peaks of the GS600 LIZ v2 size standard. • Fragment (POP-4™ polymer): – GS600(80-400)LIZ+Normalization • HID (POP-4™ polymer): – GS600(80-400)LIZ+Normalization 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 331 H Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. · Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. · Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain SDSs, visit thermofisher.com/support. Symbols on this instrument Symbols may be found on the instrument to warn against potential hazards or convey important safety information. In this document, the hazard symbol is used along with one of the following user attention words: • CAUTION!—Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. • WARNING!—Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. • DANGER!—Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. Symbol English Français Caution, risk of danger Attention, risque de danger Consult the manual for further safety Consulter le manuel pour d’autres information. renseignements de sécurité. Caution, risk of electrical shock Attention, risque de choc électrique Caution, piercing hazard Attention, danger de perforation 332 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix H Safety Symbols on this instrument H (continued) Symbol English Français Caution, hot surface Attention, surface chaude Potential biohazard Danger biologique potentiel On On (marche) Off Off (arrêt) On/Off On/Off (marche/arrêt) Protective conductor terminal (main Borne de conducteur de protection (mise à la ground) terre principale) Terminal that can receive or supply Borne pouvant recevoir ou envoyer une tension alternating current or voltage ou un courant de type alternatif Do not dispose of this product in unsorted Ne pas éliminer ce produit avec les déchets municipal waste usuels non soumis au tri sélectif. CAUTION! To minimize negative MISE EN GARDE ! Pour minimiser les environmental impact from conséquences négatives sur l’environne disposal of electronic waste, do ment à la suite de l’élimination de dé not dispose of electronic waste in chets électroniques, ne pas éliminer ce unsorted municipal waste. Follow déchet électronique avec les déchets local municipal waste ordinances usuels non soumis au tri sélectif. Se for proper disposal provision conformer aux ordonnances locales sur and contact customer service les déchets municipaux pour les disposi for information about responsible tions d’élimination et communiquer avec disposal options. le service à la clientèle pour des rensei gnements sur les options d’élimination responsable. Conformity symbols Conformity mark Description Indicates conformity with safety requirements for Canada and U.S.A. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 333 Appendix H Safety H Safety alerts on this instrument (continued) Conformity mark Description Indicates conformity with European Union requirements. Indicates conformity with Australian standards for electromagnetic compatibility. Safety alerts on this instrument Additional text may be used with one of the symbols described above when more specific information is needed to avoid exposure to a hazard. See the following table for safety alerts found on the instrument. English Français CAUTION! Hazardous chemicals. MISE EN GARDE ! Produits chimiques dange Read the Safety Data Sheets (SDSs) reux. Lire les fiches signalétiques (FS) avant de before handling. manipuler les produits. CAUTION! Hazardous waste. Refer MISE EN GARDE ! Déchets dangereux. Lire les to SDS(s) and local regulations for fiches signalétiques (FS) et la réglementation locale handling and disposal. associées à la manipulation et à l’élimination des déchets. DANGER! Class 3B (III) visible and/or DANGER ! Rayonnement laser visible ou invisible invisible laser radiation present when de classe 3B (III) présent en position ouverte et open and interlocks defeated. Avoid avec les dispositifs de sécurité non enclenchés. exposure to beam. Éviter toute exposition au faisceau. Location of safety labels on this instrument English French translation Location on Instrument WARNING! Class ATTENTION! Rayonnement Detection cell cover 3B (III) visible laser visible ou invisible de and/or invisible laser classe 3B (III) présent en radiation present position ouverte et avec when open and les dispositifs de sécurité interlocks defeated. non enclenchés. Éviter toute Avoid exposure to exposition au faisceau. beam. 334 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix H Safety Instrument safety H (continued) English French translation Location on Instrument Figure 49 Front view Figure 50 Rear panel (left) oven door (right) Instrument safety General CAUTION! Do not remove instrument protective covers. If you remove the protective instrument panels or disable interlock devices, you may be exposed to serious hazards including, but not limited to, severe electrical shock, laser exposure, crushing, or chemical exposure. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 335 Appendix H Safety H Instrument safety Physical injury CAUTION! Moving Parts. Moving parts can crush, pinch and cut. Keep hands clear of moving parts while operating the instrument. Disconnect power before servicing. Electrical safety WARNING! Ensure appropriate electrical supply. For safe operation of the instrument: · Plug the system into a properly grounded receptacle with adequate current capacity. · Ensure the electrical supply is of suitable voltage. · Never operate the instrument with the ground disconnected. Grounding continuity is required for safe operation of the instrument. AVERTISSEMENT ! Veiller à utiliser une alimentation électrique appropriée. Pour garantir le fonctionnement de l’instrument en toute sécurité : · Brancher le système sur une prise électrique correctement mise à la terre et de puissance adéqua te. · S’assurer que la tension électrique est convenable. · Ne jamais utiliser l’instrument alors que le dispositif de mise à la terre est déconnecté. La continui té de la mise à la terre est impérative pour le fonctionnement de l’instrument en toute sécurité. WARNING! Power Supply Line Cords. Use properly configured and approved line cords for the power supply in your facility. AVERTISSEMENT ! Cordons d’alimentation électrique. Utiliser des cordons d’alimentation adap tés et approuvés pour raccorder l’instrument au circuit électrique du site. WARNING! Disconnecting Power. To fully disconnect power either detach or unplug the power cord, positioning the instrument such that the power cord is accessible. AVERTISSEMENT ! Déconnecter l’alimentation. Pour déconnecter entièrement l’alimentation, dé tacher ou débrancher le cordon d’alimentation. Placer l’instrument de manière à ce que le cordon d’alimentation soit accessible. 336 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix H Safety Instrument safety H Cleaning and decontamination CAUTION! Cleaning and Decontamination. Use only the cleaning and decontamination methods specified in the manufacturer's user documentation. It is the responsibility of the operator (or other responsible person) to ensure the following requirements are met: · No decontamination or cleaning agents are used that could cause a HAZARD as a result of a reaction with parts of the equipment or with material contained in the equipment. · The instrument is properly decontaminated a) if hazardous material is spilled onto or into the equipment, and/or b) prior to having the instrument serviced at your facility or sending the instrument for repair, maintenance, trade-in, disposal, or termination of a loan (decontamination forms may be requested from customer service). · Before using any cleaning or decontamination methods (except those recommended by the manufacturer), users should confirm with the manufacturer that the proposed method will not damage the equipment. MISE EN GARDE ! Nettoyage et décontamination. Utiliser uniquement les méthodes de nettoya ge et de décontamination indiquées dans la documentation du fabricant destinée aux utilisateurs. L’opérateur (ou toute autre personne responsable) est tenu d’assurer le respect des exigences sui vantes: · Ne pas utiliser d’agents de nettoyage ou de décontamination susceptibles de réagir avec certaines parties de l’appareil ou avec les matières qu’il contient et de constituer, de ce fait, un DANGER. · L’instrument doit être correctement décontaminé a) si des substances dangereuses sont renver sées sur ou à l’intérieur de l’équipement, et/ou b) avant de le faire réviser sur site ou de l’envoyer à des fins de réparation, de maintenance, de revente, d’élimination ou à l’expiration d’une période de prêt (des informations sur les formes de décontamination peuvent être demandées auprès du Service clientèle). · Avant d’utiliser une méthode de nettoyage ou de décontamination (autre que celles recomman dées par le fabricant), les utilisateurs doivent vérifier auprès de celui-ci qu’elle ne risque pas d’endommager l’appareil. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 337 Appendix H Safety H Safety and electromagnetic compatibility (EMC) standards Laser WARNING! LASER HAZARD. Under normal operating conditions, the Applied Biosystems™ 3500/3500xL Genetic Analyzer is categorized as a Class 1 laser product. However, removing the protective covers and (when applicable) defeating the interlock(s) may result in exposure to the internal Class 3 B laser. Lasers can burn the retina, causing permanent blind spots. To ensure safe laser operation: · Never look directly into the laser beam. · Do not remove safety labels, instrument protective panels, or defeat safety interlocks. · The system must be installed and maintained by a Thermo Fisher Scientific Technical Representative. · Remove jewelry and other items that can reflect a laser beam into your eyes or those of others · Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the laser protection is defeated for servicing Thermo Fisher Scientific Technical Representatives are instructed to: DO NOT operate the laser when it cannot be cooled by its cooling fan; an overheated laser can cause severe burns on contact. Safety and electromagnetic compatibility (EMC) standards The instrument design and manufacture complies with the following standards and requirements for safety and electromagnetic compatibility. Safety (compliance) Reference Description EU Directive 2014/35/EU European Union “Low Voltage Directive” IEC 61010-1 Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1: General requirements EN 61010-1 UL 61010-1 CSA C22.2 No. 61010-1 IEC 61010-2-010 Safety requirements for electrical equipment for measurement, control and laboratory use – Part 2-010: Particular requirements for laboratory EN 61010-2-010 equipment for the heating of materials IEC 61010-2-081 Safety requirements for electrical equipment for measurement, control and laboratory use – Part 2-081: Particular requirements for automatic EN 61010-2-081 and semi-automatic laboratory equipment for analysis and other purposes 338 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix H Safety Safety and electromagnetic compatibility (EMC) standards H (continued) Reference Description IEC 60825-1:2014 Safety of laser products – Part 1: Equipment classification and requirements EN 60825-1:2014 21 CFR 1040.10 and 1040.11 as U.S. FDA Health and Human Services (HHS) “Radiological health applicable performance standards for laser products” and “Radiological health performance standards for specific purpose laser products” EMC Reference Description Directive 2014/30/EU European Union “EMC Directive” EN 61326-1 Electrical Equipment for Measurement, Control and Laboratory Use – EMC Requirements – Part 1: General Requirements FCC Part 15 (47 CFR) U.S. Standard “Industrial, Scientific, and Medical Equipment” AS/NZS 2064 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial, Scientific, and Medical (ISM) Radiofrequency Equipment ICES-001, Issue 3 Industrial, Scientific and Medical (ISM) Radio Frequency Generators Environmental design Reference Description Directive 2012/19/EU European Union “WEEE Directive” – Waste electrical and electronic equipment Directive 2011/65/EU European Union “RoHS Directive” – Restriction of hazardous substances in electrical and electronic equipment Commission Delegated Directive 2015/863 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 339 Appendix H Safety H Safety and electromagnetic compatibility (EMC) standards Radio compliance Reference Description Directive European Union "RE Directive" - Radio equipment 2014/53/EU (as of June 12, 2017) Contains FCC ID: This device complies with part 15 of FCC Rules and Innovation, Science and 2AM8P-3668 Economic Development Canada’s licence-exempt RSS(s). Operation is subject to the following two conditions: (1) this device may not cause harmful interference, and (2) Contains IC: this device must accept any interference received, including interference that may 7805A-3668 cause undesired operation. Le présent appareil est conforme à la partie 15 des règles de la FCC et aux normes des CNR d’Innovation, Sciences et Développement économique Canada applicables aux appareils radio exempts de licence. L'exploitation est autorisée aux deux condi tions suivantes : (1) l'appareil ne doit pas produire de brouillage, et (2) l'appareil doit accepter tout brouillage subi, même si le brouillage est susceptible d'en compromettre le fonctionnement. Note: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense. 340 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Appendix H Safety Chemical safety H Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions: · Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document. · Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). · Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). · Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. · Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) · After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. · Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. · IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. AVERTISSEMENT ! PRÉCAUTIONS GÉNÉRALES EN CAS DE MANIPULATION DE PRODUITS CHIMIQUES. Pour minimiser les risques, veiller à ce que le personnel du laboratoire lise attentive ment et mette en œuvre les consignes de sécurité générales relatives à l’utilisation et au stockage des produits chimiques et à la gestion des déchets qui en découlent, décrites ci-dessous. Consulter également la FDS appropriée pour connaître les précautions et instructions particulières à respecter : · Lire et comprendre les fiches de données de sécurité (FDS) fournies par le fabricant avant de stocker, de manipuler ou d’utiliser les matériaux dangereux ou les produits chimiques. Pour obtenir les FDS, se reporter à la section « Documentation et support » du présent document. · Limiter les contacts avec les produits chimiques. Porter des équipements de protection appropriés lors de la manipulation des produits chimiques (par exemple : lunettes de sûreté, gants ou vête ments de protection). · Limiter l’inhalation des produits chimiques. Ne pas laisser les récipients de produits chimiques ouverts. Ils ne doivent être utilisés qu’avec une ventilation adéquate (par exemple, sorbonne). · Vérifier régulièrement l’absence de fuite ou d’écoulement des produits chimiques. En cas de fuite ou d’écoulement d’un produit, respecter les directives de nettoyage du fabricant recommandées dans la FDS. · Manipuler les déchets chimiques dans une sorbonne. 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 341 Appendix H Safety H Biological hazard safety · Veiller à utiliser des récipients à déchets primaire et secondaire. (Le récipient primaire contient les déchets immédiats, le récipient secondaire contient les fuites et les écoulements du récipient pri maire. Les deux récipients doivent être compatibles avec les matériaux mis au rebut et conformes aux exigences locales, nationales et communautaires en matière de confinement des récipients.) · Une fois le récipient à déchets vidé, il doit être refermé hermétiquement avec le couvercle fourni. · Caractériser (par une analyse si nécessaire) les déchets générés par les applications, les réactifs et les substrats particuliers utilisés dans le laboratoire. · Vérifier que les déchets sont convenablement stockés, transférés, transportés et éliminés en res pectant toutes les réglementations locales, nationales et/ou communautaires en vigueur. · IMPORTANT ! Les matériaux représentant un danger biologique ou radioactif exigent parfois une manipulation spéciale, et des limitations peuvent s’appliquer à leur élimination. Biological hazard safety WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment. · U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at: www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2020-P.pdf · World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/publications/i/item/9789240011311 342 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 I Documentation and support Related Documentation Document Publication number 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v3.3 Quick 100081365 Reference Polymer Delivery Pump Cleaning Kit RUO Product Information Sheet 4414004 Note: For additional documentation, see “Obtaining support” on page 343. Customer and technical support Visit thermofisher.com/support for the latest service and support information. • Worldwide contact telephone numbers • Product support information – Product FAQs – Software, patches, and updates – Training for many applications and instruments • Order and web support • Product documentation – User guides, manuals, and protocols – Certificates of Analysis – Safety Data Sheets (SDSs; also known as MSDSs) Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer. Obtaining support For service and technical support: • Call Toll-Free in US: 1 800 955 6288 • Email [email protected] For the latest services and support information for all locations, go to: thermofisher.com/support 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 343 Appendix I Documentation and support I Limited product warranty At the website, you can: • Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities • Search through frequently asked questions (FAQs) • Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents • Obtain information about customer training • Download software updates and patches Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/ global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support. 344 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index Special Characters Administrator user role 225 Advanced setup .annotation.txt 42, 111 preference 44 .csv 97 using 56 .fsta 42, 111 allelic ladder .html 111 internal validation 68 .pdf re-injection 88 audit reports 236 requirements for accurate genotyping 68, 88 e-sig report 243 results group for one allelic ladder per run folder install check report 153, 161 68 plate layout 66 run requirements 178 sample quality reports 122 amber indicator light 19 spatial calibration report 129, 143 amplicon, specifying 63 spectral calibration 129, 143, 153, 161 Analysis Range trace quality reports 110 fragment analysis 208 user report 227 HID analysis 214 .phd.1 42, 111 anode buffer .qual 42, 111 install 258 .scf 42, 111 location 17 .seq 42, 111 on-instrument limits 22 .txt 97, 111 antivirus software 20 .xls 97, 111 AnyDye, create dye set 195 .xml 97 archive audit records 237 Numerics data files 275 library items 274 384-well, plate assembly 72 array. See capillary array 8-tube strip array selection, assign plate contents 93 plate assembly 71 array view 99 plate base 71 as-needed instrument maintenance 254 96-well assays capillary map 67 assign to plate 95 plate assembly 70 create 169 plate base 70 defined 61, 169 instrument protocol 171 A assign plate contents amplicon and specimen 63 ABC. See anode buffer array selection (capillary-to-plate map) 93 abort run 90 assay 95 account file name convention 95 setup 220 name samples 63, 91 suspended, activate 224 overview 61 suspension 220, 248 parts of screen 61 user 222 plate view 63, 91 action log, contents 235 results group 95 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 345 Index sample type 63 Basic setup, preference 44 table view 93 BigDye Terminator kits and standards 320 template for 97 biohazard safety 342 Assume Linearity 211 borrowing audit defined 139 export settings 244 disabled 139 import settings 245 enabled 139 troubleshooting 305 preference 42 audit records Broad Peak (BD) archive or purge 237 and SQ 217 export 237 flag in view fragment/HID results 115 factory-provided items, creation date 165 threshold 217 restore 237 buffer. See anode buffer, cathode buffer view in library 165 Buffer-Pin Valve 36 audit, administrators buffers, part numbers and limits 22 action log 235 archive records 237 audit actions 233, 235 C audit history 231 calendar audit mode 230 create entries 255 audit reason settings 231 maintenance 255 audit records, object 232 review reminders 251 audited objects and actions 230 calendar reminders export records 237 complete a task 251 export settings 244 Dashboard 36, 251 import settings 245 dismiss a task 251 object audit history 232 log 256 overview 218 trigger time, setting 42 purge records 237 capillary array reports, print or view 236 change 265, 266 restore records 237 check stored 267 settings, access 228 fill with polymer 265 system configuration history 234 install 266 audit, users maintenance 265 enter reason for change 249 on-instrument limits 22 overview 246 part numbers and limits 24 view audit records 165 storage conditions 267 autoanalysis 168 store 267 Average Normalized PH 115 capillary-to-plate map average quality value (QV), monitor run 84 diagram 67 display in software 93 catalog numbers. See part numbers B cathode buffer barcode 168 install 259 basecall probability of error 108 location 17 Basecaller 203 on-instrument limits 22 basecaller version in basecalling protocol and View CBC. See cathode buffer Sequence results 102 check valve 36 basecalling protocol Clear range 203 create 201 Cloud. See Thermo Fisher Connect defined 201 computer settings 205 archive, purge, and restore data 274 basecalling protocols, in assay 171 check hard drive space 276 Basepair Accuracy 150 disk space, check 276 346 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index do not rename 20 data files. See sample data files maintain 273 data troubleshooting 292 name requirement 20, 30, 33 datastore, backup during install or uninstall 273 restart 279 date format, setting 42 start up 33 default settings, specifying 42 condition number 136 define plate properties 59 conditioning reagent detection cell location 279 described 22 disk space, computer 276 part number 22 documentation, related 343 connections duplicate injection communication 318 See also re-injection power 318 defined 78 consumables preview run 78 anode buffer 258 See also re-injection capillary array 265, 267 duplicate library item 164 cathode buffer 259 dye sets conditioning reagent 22 create 193 fragment analysis and HID 321 custom, create 195 on-instrument limits 22 fragment analysis 313 polymer 263, 264 HID analysis 313 sequencing analysis 320 overview 192 status in Dashboard 37 sequencing 313 suppress pre-expiration warning messages 42 settings 195 contiguous read length. See CRL CRL defined 104 E distribution report 110 e-sig install check 150 See also electronic signature QV settings 205 access 238 report 110 configure 239 result 104 export 244 threshold 205 export settings 244 CRL Basepair Accuracy 150 import settings 245 CRL Median and SD 150 print report 243 custom dye set. See dye sets, custom records, display 242 customize sample info 63 settings 240 troubleshoot 305 See also electronic signature D electronic signature records daily instrument maintenance 252 actions 242 Dashboard view in library 165 consumables status 36 electronic signature, administrators parts of 26 actions that allow e-sig 239 troubleshoot 289, 298 enable or disable 238 Data Collection Software export 244 Daemon 34 export settings 244 default settings 42 functions that require e-sig 239 log in 35 import settings 245 Main workflow 27 is signed field 249 overview 26 overview 218 required procedure to start 34 report 243 Server Monitor 34 troubleshoot 305 start 34 when security is disabled 238 use without an instrument 30 electronic signature, users 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 347 Index is signed field 249 Flat Profile 203 overview 246 fragment analysis signing 249 dye sets 313 electropherogram troubleshooting 292 reagents, part numbers, storage conditions 321 environmental requirements 316 results, review 112 EPT view 100 run modules 309 error messages sizecalling protocol 206 display detail 308 fragment install check instrument 287 during a run 158 RFID 286, 328 evaluate data 159 spatial calibration 298 plate, prepare 131, 145, 155 spectral calibration 299 recommended monthly schedule 254 export results, example 160 audit records 237 results, example HID 160 audit settings 244 run 153, 156 e-sig records 244 signing 249 e-sig settings 244 troubleshoot 303 library items 165 when to perform 153 plate 97, 165 front panel indicators 19 security settings 244 sequencing results, reports, traces 111 spatial calibration 128 G spectral calibration 142 green indicator light 19 user account settings 244 GS600 LIZ v2.0 size standard, part numbers 321 GS600 LIZ® v2.0 size standard 198 GS600(60-600)LIZ+Normalization 198 F GS600(80-400)LIZ+Normalization 198 factory-provided library items 163 GS600LIZ+Normalization 198 Failure to Read from RFID tag 286 file location default 44 H in file name convention 176 hard drive with file name convention 66, 173 check space 276, 277 with results group 66, 178 defragment 277 without file name convention 66, 173 Hi-Di formamide, part number and storage 25 without results group 66, 178 HID, results group example 188 file name conventions HID analysis assign to plate 95 allelic ladder re-injection 88 create 173 allelic ladder requirements 88, 178 defined 173 allelic ladder validation 68 file location 176 dye sets 313 in plate 173 QC protocol 211 settings 176 reagents, part numbers, storage conditions 321 file name format results group for one allelic ladder per run folder default format 66 68 with file name convention 173 results groups 178 without file name convention 66, 173 run modules 309 file name maximum length 176 HID install check. See fragment install check fill and fill series, sample name 93 Fill Array with Polymer maintenance wizard 265 firmware 20 I flags import display flag table in monitor run 84 audit settings 245 re-injecting samples with 86 e-sig settings 245 348 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index library items 165 instrument run plate 60, 97 defined 74 sample files, fragment 103, 115 monitor 80 security settings 245 pause after last injection 44 user account settings 245 preview 79 injection start 79 defined 74 start with existing plate 73 duplicate 78 suppress consumables 42 folder 182 suppress delete injection warning message 44 pause after last 44 instrument run name re-injection 86 See also run name replicate 78 default 74 specify new instrument protocol 86 folder name 182 injection list See also run name blank 78 instrument run views 98 columns in table 83 instrument sensor details 307 completed injections 83 Instrument Shutdown maintenance wizard 271 duplicate injection 78 is signed field 249 modify 78 monitor run 80 preview run 78 L re-injection 86 label peaks 119, 121 replicate injection 78 laser specifications 315 Install Capillary Array maintenance wizard 265 leaks and spills 38 install check, report 153, 161 library See also fragment install check AB 163 instrument access 163 clean 257 archive 274 components 17 assay 169 description 16 basecalling protocol 201 disconnect from Thermo Fisher Connect 54 create new item 164 during a run 21 delete entry 164 interior components 279 delete entry, auditing 164 load plate 72, 132, 146, 156 dye sets 195 maintenance, weekly 253 edit entry 165 move and level 272 factory-provided 163 name, specifying 42 factory-provided items 163 prepare 57, 130, 144, 154 factory-provided, create new item from 164 reactivate 273 file name conventions 173 remove user 54 filter 166 reset 308 filtering 44 restart 279 instrument protocol 189 routine cleaning 257 locked 163 shutdown 271 locked, create new item from 164 start up 33 maintenance 274 startup after storage 273 overview 162, 167 theory of operation 20 plates 166 troubleshoot 281, 287, 289 purge 274 instrument maintenance. See maintenance purge entries 276 instrument protocol QC protocol 211 create 189 restore entries 275 defined 189 results group 178 settings 190 search 166 instrument protocols in assay 171 size standard 198 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 349 Index sizecalling protocols 206 anode buffer, change 258 sort 166 capillary array, change 265 template 163 capillary array, check stored 267 template, create new item from 164 cathode buffer, change 259 view audit records 165 polymer, fill array 265 view e-sig records 165 polymer, replenish 263 limited product warranty 344 polymer, store partially used pouch 264 linearity in large-fragment size standards 211 maintenance, instrument link plate 72, 74, 76 annual planned 254 link plate troubleshooting 296 as-needed 254 load plate 72, 132, 146, 156 daily 252 load plate for run 74 monthly 253 locked library item 163 move and level the instrument 272 log files pump, flush water trap 269 search and use 306 pump, remove bubbles 268 view 306 pump, wash chamber and channels 268 logged-in user name quarterly 254 determining 225, 247 reactivate 273 display 225, 247 routine cleaning 257 in user account 222 schedule 251 login shutdown 271 reactivate suspended account 248 manual commands, troubleshoot 306 software 35, 247 matrix standard, part numbers 321 metric analysis display results 104 M flag settings 201 maintenance warnings 104 capillary array 265 Min. Peak Half Width 208, 214 computer 273 mixed base computer archive, purge, and restore data 274 color 44 library 274 QV range 44 pump 267 settings 108 pump, wash 268 threshold 203 view planned tasks 256 mobile device weekly 253 monitor a run 52 maintenance calendar view results 52 create entries 255 Mobility file 203 recommended entries 255 monitor run view 255 current run 80 maintenance wizards flag settings 201 fill array with polymer 265 injection list 80 install capillary array 265 injection list, modify 80 instrument reactivate 273 re-injections 86 instrument shutdown 271 start, stop, resume run 90 remove bubbles 268 troubleshoot 304 replenish polymer 263 monthly instrument maintenance 253 wash pump and channels 268 move the instrument 272 maintenance, computer data files, archive 275 defragment 277 N disk space, monitor 276 non-linearity in large fragment size standards 211 library items, archive, purge, and restore 274 normalization uninstall software 273 and SQ (sizing quality) 211, 217 maintenance, consumables factor 117 350 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index factor and thresholds 190 8-tube strip assembly 71 factor in secondary analysis software 117 96-well assembly 70 factor thresholds 117 assay 95 flag in view fragment/HID results 115 assembly 70 GS600 LIZ® v2.0 size standard 198 base, standard 70 how the software normalizes 117 create from template 59 review data 117 defined 167 size standards 198 edit 97 target 117, 190 export 97 notification log, review 256 file name convention 95 notifications import 60, 97 calendar reminders in Dashboard 36, 251 link 72, 74, 76 log for calendar reminders 256 load in instrument 72, 132, 146, 156 SAE module 221 load in software 74 owner 168 pending 77 O print 66 object audit history, contents 232 processed 77 offscale flag properties 59, 168 monitor run 84 results group 95 view fragment results 115 sample plate, prepare 67 on-instrument limits 22 select for run 72 oven temperature setup error 296 in instrument protocol 190 signing 249 pre-heat 57, 130, 144, 154 type 168 overlay report 122 unlink 74 owner, plate 168 plate grid report 66 plate layout .pdf 66 P print 66 part numbers saving for future use 97 buffer 320 plate library 166 capillary array 320 plate loading 72, 132, 146, 156 conditioning reagent 22, 320 plate map, print 66 fragment/HID analysis standards 321 plate properties 167 Hi-Di formamide 320 plate record. See plate plates 319 plate report polymer 320 fragment 122 sequencing analysis reagents 320 sequencing, generate 110 password plate template change your own 247 about 58 expiration 220 create 97 restrictions 220 create plate from 59 pause after last injection 44 edit 165 pause run 90 overview 167 Peak Amplitude Thresholds 208, 214 save well attributes 97 Peak Window Size 208 plate type peaks, label 119 default 44 pending plates 77 set default 98 permissions, user account 225, 247 specify 59, 168 planned maintenance schedule 254 plate view planned maintenance tasks 256 assign plate contents 63 plate customize 92 384-well assembly 72 plate-to-capillary map 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 351 Index diagram 67 spectral calibration 129, 143, 153, 161 display in software 93 trace quality reports 110 plot settings, change 118 user report 227 plots, fragment probablility of error 109 display plots 118 processed plates 77 label peaks 119 Pull-Up peak overlay 119 flag 115 scale 118 settings 211 settings 118 pump zoom 118, 138 avoid damage 267 polymer bubbles, remove 268 fill capillary array 265 flush water trap 269 handling 262 maintenance 267 how usage is calculated 38 parts of 279 location 17 remove bubbles 268 on-instrument limits 22 trap, flush 269 part numbers and handling 22 troubleshoot 281 precautions for use 263 wash chamber and channels 268 replenish 263 water trap, flush 269 store 264 PUP Score 104 storing partially used pouch 264 pure base Polynomial Degree 208 color 44 power requirements 315 QV range 44 pre-expiration warning messages 42 pure base settings 108 pre-heat oven 57, 130, 144, 154 purge preferences audit records 237 advanced or basic setup 44 library 274 calendar reminder time 42 library entries 276 date format 42 library items 274 file location 44 instrument name 42 library filtering 44 Q pause after last injection 44 Q and Condition 136 plate type 44 QC protocol reports 44 create 212 run logs 42 defined 211 sequencing export 42 settings 214, 217 sequencing trace 44 QC report, sequencing, generate 110 sequencing trace print 44 quality flags suppress pre-expiration warning messages 42 fragment 84, 115 system 42 sequencing 84, 104 table settings 41 quality settings warning dialogs 44 fragment analysis 206 preview run sequencing analysis 201 duplicate injection 78 quality value injection list 78 See also QV start instrument run 79 probability of error 108 primer peak, eliminating 208 spectral calibration 136 print See also QV audit reports 236 quarterly instrument maintenance 254 install check eport 153, 161 Quick Start 73 plate layout 66 QV, probability of error 109 sample quality reports 122 QV (average quality value), monitor run 84 spatial calibration report 129, 143 352 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index See also average quality value [QV (average install check 153, 161 quality value) plate grid 66 QV settings, sequence sample quality 122 basecalling protocol 201 sequencing results 110 color and ranges 44 spatial calibration 129, 143 monitor run screen 201 spectral calibration 129, 143 recommended ranges 108 system configuration history 234 QV20+ trace score 110 report 110 user 227 result 104 requirements, environmental 316 setting 205 reset button on front panel 308 restore audit records 237 R library entries 275 re-inject samples library items 274 from Monitor Run screen 86 results from Results screen 109, 122 See also review results, sequencing re-injection review for previously run samples 103, 115 See also duplicate injection view, mobile device 52 allelic ladder 88 See also review results, sequencing collect data for specific wells 86 results group defined 86 create new 179 displayed in plate view 88 defined 178 file location 182 example 183, 185, 188 samples, all in injection 86 for HID applications 188 samples, individual 86 settings 182 troubleshooting 291 results groups See also duplicate injection assign to plate 95 re-injection button dimmed 86 create 179 reactivate suspended account 248 folder 182 Reactivate the Instrument maintenance wizard 273 for HID applications 68, 178 Read Length 150 store samples by plate name 183 recent results.. See review results plates 77 resume run 90 runs 77 review results red indicator light 19 currently running plate, fragment analysis 114 related documentation 343 currently running plate, sequencing 102 Remove Bubbles maintenance wizard 268 import sample files 103, 115 rename samples in review results 120 previously run plate 103, 115 Replace with index term 124, 154 rename 120 Replenish Polymer maintenance wizard 263 show/hide columns 104 replicate injection. See injection list sort 104 report review results, fragment e-sig 243 access 113 planned maintenance 256 currently running plate, fragment 102, 114 report settings, sequence 110 label peaks 119 reports overlay plots 119 action log 235 plots 118 audit 236 quality flags 115 CRL and CRL distribution 110 sample quality 115 default settings 44 scale 118 e-sig 243 sizing curve, overlay 122 export sequencing 111 thumbnails 120 fragment results 122 troubleshoot 295 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 353 Index zoom 118, 138 sample info, enter 63 review results, fragment/HID sample name modify data in secondary analysis software assign 93 111, 123 fill and fill series 93 normalized data 117 sample plate. See plate sizing 121 sample quality review results, sequencing See also review results, fragment access 102 in monitor run 84 base colors 44 in review results 115 export defaults 42 See also review results, fragment metric analysis 104 sample type modify data in secondary analysis software plate view 63 111, 123 table view 93 QV colors and ranges 44 sample view 99 thumbnails 107 samples, review previously run 103, 115 trace defaults 44 scale plots 118 trace quality reports 110 scheduled maintenance RFID notifications 251 defined 18 tasks 251 error message 286, 328 scientist user role 225 overview 325 security required position for label 263 export settings 244 troubleshoot 286, 328 import settings 245 run security policies 220 See also instrument run security, administrators abort 90 account setup 220 pause and resume 90 enable or disable 219 terminate 90 export settings 244 troubleshooting 291 export user account settings 244 See also instrument run import settings 245 run log, number of runs to preserve 42 import user account settings 245 run module 190 notification 220, 221 run modules overview 218 fragment/HID analysis 309 security policies 220 sequencing analysis 309 spaces in user names 221 run name user accounts 222 default 74 user report 227 enter 74 user role 225 run voltage 190 username restrictions 220 security, users account suspension 248 S log in 247 SAE module, security 218 overview 246 See also audit password change 247 safety, biohazard 342 permissions 247 safety labels 334 session timeout 248 sample data file storage sequence quality file name convention location 66 See also review results, sequencing injection folder 182 in monitor run 84 instrument run name folder 182 in review results 104 results group location 66 mixed base settings 108 results group name folder 182 pure base settings 108 sample data file storage location 30 recommended ranges 108 sample data files, archive 275 trace quality reports 110 354 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index See also review results, sequencing sort tables, multi-column 94, 121, 166 sequencing analysis spatial calibration reagents, part numbers and storage conditions export 128 320 historical reports 129, 143, 153, 161 dye sets 313 perform 125 run modules 309 purpose 125 sequencing install check signing 249 evaluate data 151 troubleshoot 298 examples 152 when to perform 125 plate, prepare 131, 145, 155 specifications 315 recommended monthly schedule 254 specimen, specifying 63 run 146 spectral calibration run time 144 borrowing disabled 139 signing 249 borrowing enabled 139 thresholds 150 capillary sharing 138 troubleshoot 302 condition number 136 what occurs 149 estimated run time 130 when to perform 144 evaluate data 137 sequencing install check standard 145 examples 141 sequencing report, default settings 44 export 142 Server Monitor 34 historical reports 129, 143, 153, 161 service log 277 history 143 session timeout 220, 248 instrument, prepare 130 shutdown instrument 271 perform 133 signal strength report, sequencing 110 pull-down peaks 138 signing, electronic signature 249 quality value 136 size standard, defined 198 report 129, 143 size standard normalization 115, 117 signing 249 size standard plot 121 troubleshoot 299 size standards what occurs 138 in QC protocol 214 when to perform 130 in sizecalling protocol 208 zoom 137 large fragment, non-linearity 211 spectral calibration standard 131 normalization 198 spectral pull-up flag 115 part numbers 321 SQ (sizing quality) sizecalling protocol and broad peaks 217 create new 206 and normalization 211, 217 fragment analysis 208 flag in monitor run 84 HID analysis 214 flag in view fragment results 115 settings 208, 211 fragment analysis 211 sizecalling protocols fragment analysis, difference from GeneMapper defined 206 ID-X 211 in assay 171 HID analysis 217 sizing curve, overlay 122 monitor run 84 sizing quality. See SQ (sizing quality) weighting 217 Sizing Range SS Norm Factor 115 fragment analysis 208 stand-alone installation of software 30 HID analysis 214 start the system 32 sizing report 122 status, consumables in Dashboard 36 sizing results fragment/HID, label peaks 121 support, customer and technical 343 Slope Thresholds Peak Start and End 208, 214 support, obtaining 343 smart phone. See mobile device symbols, safety 332 smoothing 208, 214 system configuration history, contents 234 software. See Data Collection Software 3 system defaults, date format 30 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 355 Index system specifications 315 TroubleshootingUtility.bat 306 system start up 32 True Profile 203 T U table view, assign plate contents uninstall the software 273 use 93 unlink plate 74 user-defined fields 63 usage statistics 277 tables user account customize 94, 166 activate suspended 224 results, show/hide columns 104 create or edit 222 settings, default 41 delete 224 sort 94, 121, 166 export 244 technician user role 225 import 245 template inactivate 224 create plate from 59 permissions 225 library 163 user role, permissions 226 plate 97 user role, create 225 plate, edit 165 user-defined fields terminate run 90 displayed in table view only 63 terms and conditions 344 including in file name 176 theory of operation 20 specifying for samples 63 Thermo Fisher Connect, disconnect instrument 54 thumbnails fragment 120 V sequencing 107 validation, allelic ladder 68 thumbnails, view 120 view fragment results.. See review results, fragment timeout, session 220, 248 analysis results trace quality reports 110 view run results.. See review results trace report (sequencing), view 110 view sequencing results. See review results, se Trace Score 104, 205 quencing results Trace Score report 110 training, information on 343 troubleshoot W Dashboard 289, 298 warning messages, suppressing data 292 consumable pre-expiration warning 42 e-sig 305 delete injection 44 electronic signature 305 export library item 44 electropherogram 292 warnings, metric analysis 104 error messages 308 warranty 344 fragment install check 303 Wash Pump and Channels maintenance wizard 268 instrument 281, 287, 289 weekly instrument maintenance 253 link plate 296 weighting, SQ 217 log files, search and use 306 well attributes manual commands 306 assign plate contents 61 monitor run 304 save in template 97 pump 281 workflow review results, fragment 295 Advanced or Basic preference 44 RFID 286, 328 set up and run 57 sequencing install check 302 workflow diagram 32 spatial calibration 298 workflow, basic setup, preference 44 spectral calibration 299 view instrument sensor details 307 troubleshooting, audit 305 356 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 Index Z plate view 92 spectral calibration 137 zoom fragment plots 118 3500/3500xL Genetic Analyzer User Guide—Data Collection Software v3.3 357 7_3500 RUO UG 3.3_100079380 -v13-GUID-7BD194C3-E934-4F08-816D-B9DE93273707-2023/12/01 21:38:08 en 21:42:22.523Z thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 1 December 2023 NGM Detect™ PCR Amplification Kit USER GUIDE Catalog Number A31832 Publication Number 100044085 Revision D For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures. For licensing and limited use restrictions visit thermofisher.com/HIDlicensing. Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition. The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: Pub. No. 100044085 Revision Date Description D 2 January 2019 • We redesigned primers at the IQC Large and TH01 markers to correct high background seen in samples with a high soil burden, then performed subsequent validation work. Accordingly, we made the following changes to this user guide: – Chapter 1—Updated “About the primers“, Figure 1, and Figure 2. – Chapter 2—Updated Figure 3. – Chapter 6—Updated text and figures throughout. • Chaper 4—Updated analysis file references to v3. C 13 March 2017 • Added information for artifact (p 85). • Removed IQC from precision and accuracy graphs (update three figures) and tables; noted that they were not included in the study (p 60–66). • Updated wording on how stutter filters are calculated; updated minus stutter values; added plus stutter values (pp 75, 81–82). • Corrected typo—change 401 to 410 (p 88). • Updated SWGDAM references from 2012 to 2016. B 28 December 2016 • Added Chapter 6, "Experiments and results". • Added information about direct amplification to Chapter 2. A 21 September 2016 New document Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Windows and Windows Vista are trademarks of Microsoft Corporation. Whatman and FTA are trademarks of Whatman Limited. NUCLEIC-CARD and Copan are trademarks of Copan Italia S.P.A., and used by Life Technologies under their permission. Harris Micro-Punch is a trademark of Harris, Joel S. TA Shunderson Communications. VWR Scientific is a trademark of VWR International, Inc. Robbins Scientific is a trademark of Molecular Bioproducts, Inc. Agilent is a trademark of Agilent Technologies, Inc. Adobe, Acrobat, and Reader are trademarks of Adobe Systems Incorporated. ©2019 Thermo Fisher Scientific Inc. All rights reserved. Contents ■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Kit overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Internal quality control system for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Validated DNA input amounts and PCR cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 About the primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Dyes used in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Loci amplified by the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Standards and controls that are required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Allelic ladder profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 DNA Control 007 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Instruments and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 ■ CHAPTER 2 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Importance of quantification before STR analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Effect of DNA quantity on results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Methods of quantifying DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Prepare low-TE buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Thaw reagents (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Direct amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 NGM Detect™ PCR Amplification Kit User Guide 3 Contents ■ CHAPTER 3 Perform electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . 24 Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Create a 3500 QC protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . 27 Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) . . . . . 28 Electrophoresis software setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Obtain and activate 6-dye license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Perform spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . 30 ■ CHAPTER 4 Analyze data with GeneMapper™ ID‑X Software . . . . . . . 32 Overview of GeneMapper ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 ™ ™ GeneMapper ID‑X Software analysis of the NGM Detect kit . . . . . . . . . . . . . . . . . . . . . . . . 33 Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 ™ Set up the GeneMapper ID‑X Software for analysis (before first use of the kit) . . . . . . . . 35 ™ Workflow: Set up GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Check panel, bin, and stutter file versions on your computer . . . . . . . . . . . . . . . . . . . . 35 (If needed) Download newer versions of panel, bin, and stutter files . . . . . . . . . . . . . 35 Import panels, bins, and marker stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 (Optional) Define custom table or plot settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Enter Analysis Method settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 About the GS600_LIZ_ (60– 460) size standard definition file . . . . . . . . . . . . . . . . . . . . 48 Create a size standard definition file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Analyze and edit sample files with GeneMapper ID-X Software . . . . . . . . . . . . . . . . . . . . . . . 50 Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 ™ For more information on using the GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . 51 4 NGM Detect™ PCR Amplification Kit User Guide Contents ■ CHAPTER 5 Assess the PCR reaction with the Internal Quality Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Overview of the Internal Quality Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Evaluate the PCR reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Balanced profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Ski slope profile with decreased IQCL peak height . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Ski slope profile with balanced IQC peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 No sample peaks with balanced IQC peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 ■ CHAPTER 6 Experiments and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 SWGDAM guideline 2.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 SWGDAM guideline 3.9.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 SWGDAM guideline 3.9.6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Thermal cycling temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 PCR cycle number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 CE injection time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 SWGDAM guideline 3.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Accuracy observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Precision and size window description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Precision and size window observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Causes of extra peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Extra peaks: Stutter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Extra peaks: Addition of 3' A nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Extra peaks: Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 SWGDAM guideline 3.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Loci in this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Nature of polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Genetic linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 SWGDAM Guideline 3.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Nonhuman studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 NGM Detect™ PCR Amplification Kit User Guide 5 Contents Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 SWGDAM guideline 3.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Sensitivity observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 SWGDAM guideline 3.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Degraded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Effect of inhibitors: hematin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Effect of inhibitors: humic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Mixture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 SWGDAM guideline 3.8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Mixture study overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Mixture study observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Resolution of genotypes in mixed samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Limit of detection of the minor component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 SWGDAM guideline 3.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Population data overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Loci in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Population samples used in these studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Concordance studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Probability of Identity definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Probability of identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Probability of paternity exclusion observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 ■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 ■ APPENDIX B Materials required but not supplied . . . . . . . . . . . . . . . . . . 127 STR kit required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Sample preparation required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Thermal cycler required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 ™ ProFlex PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 ™ Veriti Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 ™ GeneAmp PCR System 9700 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Genetic analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 3500 Series Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 3130 Series Genetic Analyzer required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Analysis software required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 ™ GeneMapper ID‑X Software v1.5.2 patch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 ™ GeneMapper ID‑X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Miscellaneous required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Plates and tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Laboratory supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 6 NGM Detect™ PCR Amplification Kit User Guide Contents ■ APPENDIX C PCR work areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 ■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 ■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 NGM Detect™ PCR Amplification Kit User Guide 7 1 Product information ■ Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 ■ Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 ■ Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 ■ Instruments and software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix in this document. Product description Kit overview The Applied Biosystems™ NGM Detect™ PCR Amplification Kit is a 6-dye, short tandem repeat (STR) multiplex assay for the amplification of specific loci in human genomic DNA. The kit amplifies: • 16 autosomal STR loci: – The 12 loci in the extended European Standard Set (ESS): FGA, TH01, vWA, D3S1358, D8S1179, D18S51, D21S11, D12S391, D1S1656, D2S441, D10S1248, and D22S1045 – 3 additional loci that are not in the ESS, but that are present in the AmpFℓSTR™ SGM Plus™ PCR Amplification Kit: D16S539, D2S1338, and D19S433 – SE33, a highly polymorphic locus • Two internal quality control markers (IQCS and IQCL) • 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel) • Amelogenin (sex determining marker) Internal quality The NGM Detect™ kit is an Applied Biosystems™ STR kit that includes an internal control system for quality control (IQC) system for PCR. The IQC system has two synthetic targets, one low molecular weight and one high molecular weight, that are amplified with the PCR sample. The behavior of the IQC target peaks can be used to evaluate the success of the PCR reaction and give an indication of sample quality. Validated DNA The kit is validated for use with 500 pg DNA (15-µL input volume) for 30 cycles. input amounts and PCR cycles 8 NGM Detect™ PCR Amplification Kit User Guide Chapter 1 Product information Product description 1 About the primers The NGM Detect™ kit primers are manufactured using the same synthesis and purification improvements as the primers in the GlobalFiler™ kit, Identifiler™ Plus kit, and NGM SElect™ kit. These improvements enhance the assay signal-to-noise ratio and simplify the interpretation of results. The primers that are used in the NGM Detect™ kit are unique when compared with primers for corresponding STR loci in the GlobalFiler™, Identifiler™ Plus, and NGM SElect™ kits except for one D12 primer and one TH01 primer that are shared with the GlobalFiler™ and NGM SElect™ kits. Changes to the IQC Large and TH01 primers A small number of laboratories reported high background with the original formulation of the NGM Detect™ kit when samples with a high soil burden were run. After internal investigations, we determined that cross-reactivity of the IQC Large and TH01 primers with soil-associated microbial DNA caused the background artifact peaks. Accordingly, we redesigned primers for both markers. We did not make any other changes to the NGM Detect™ kit formulation, protocols, or workflow. The updated formulation of the NGM Detect™ kit was re-validated with internal and external testing. Other than correcting the soil-associated background issue, the performance of the updated formulation assay is fully comparable to that of the original kit. Unless otherwise indicated, the data in Chapter 6, “Experiments and results“ are from the re-validation study. See the Technical Note: Updated NGM Detect™ PCR Amplification Kit: Validation and Comparative Study for studies directly related to soil specificity and a direct comparison of the NGM Detect™ kit original formulation to the updated formulation. Dyes used in the Dye Color Label kit 6‑FAM™ Blue Samples, allelic ladders, and controls VIC™ Green TED™ Yellow TAZ™ Red SID™ Purple LIZ™ Orange GeneScan™–600 LIZ™ Size Standard v2.0 NGM Detect™ PCR Amplification Kit User Guide 9 1 Chapter 1 Product information Product description Loci amplified by the kit Table 1 NGM Detect™ kit loci and alleles Locus Chromosome DNA Control Alleles included in Allelic Ladder Dye label designation location 007 IQCS N/A 1, 2 6-FAM™ 2 D2S1338 2q35 11–28 20,23 SE33 6q14 4.2, 6.3, 8, 9, 11–20, 20.2, 21, 21.2, 22.2, 23.2, 17, 25.2 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2,33.2, 34.2, 35, 35.2, 36–39, 42 IQCL N/A 1, 2 2 D16S539 16q24.1 5, 8–15 VIC™ 9,10 D18S51 18q21.33 7, 9, 10, 10.2, 11–13, 13.2, 14, 14.2, 15–27 12, 15 TH01 11p15.5 4–9, 9.3, 10–13, 13.3 7, 9.3 D12S391 12p13.2 14–19, 19.3, 20–27 18, 19 D3S1358 3p21.31 9–20 TED™ 15, 16 FGA 4q28 13–30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 24, 26 46.2, 47.2, 48.2, 50.2, 51.2 Y indel Yq11.221 1, 2 TAZ™ 2 Amelogenin X p22.1– 22.3, X, Y X, Y Y: p11.2 vWA 12p13.31 11–24 14, 16 D21S11 21q11.2– q21 24, 24.2, 25–28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 28, 31 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36–38 D1S1656 1q42.2 9–14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18, 18.3, 13, 16 19.3, 20.3 D2S441 2p14 8–11, 11.3, 12–17 14, 15 D8S1179 8q24.13 5–19 SID™ 12, 13 D19S433 19q12 6–12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 14, 15 17, 17.2, 18.2, 19.2 D22S1045 22q12.3 7–20 11, 16 D10S1248 10q26.3 8–19 12, 15 10 NGM Detect™ PCR Amplification Kit User Guide Chapter 1 Product information Product description 1 Standards and For the NGM Detect™ kit, the panel of standards needed for PCR amplification, PCR controls that are product sizing, and genotyping are: required • DNA Control 007—A positive control for evaluating the efficiency of the amplification step and STR genotyping using the NGM Detect™ Allelic Ladder. DNA Control 007 is present in the kit. See “DNA Control 007“ on page 13. • GeneScan™–600 LIZ™ Size Standard v2.0—Used for obtaining sizing results. This standard, which has been evaluated as an internal size standard, yields precise sizing results for PCR products. Order the GeneScan™–600 LIZ™ Size Standard v2.0 (Cat. No. 4408399) separately. • NGM Detect™ Allelic Ladder—Developed for accurate characterization of the alleles amplified by the kit. The Allelic Ladder is present in the kit and allows automatic genotyping of most of the reported alleles for the loci in the kit. See “Allelic ladder profile“ on page 11. Allelic ladder The allelic ladder profile appears on the next page. profile NGM Detect™ PCR Amplification Kit User Guide 11 1 Chapter 1 Product information Product description Figure 1 GeneMapper™ ID‑X Software plot of the NGM Detect™ Allelic Ladder 12 NGM Detect™ PCR Amplification Kit User Guide . Chapter 1 Product information Product description 1 DNA Control 007 ™ ™ Figure 2 DNA Control 007 (500 pg) amplified with the NGM Detect kit and analyzed on an Applied Biosystems 3500xL Genetic Analyzer (Y‑axis scale 0 to 8,000 RFU). NGM Detect™ PCR Amplification Kit User Guide 13 1 Chapter 1 Product information Contents and storage Contents and storage The NGM Detect™ kit (Cat. No. A31832) contains sufficient quantities of the following reagents to perform 200 amplifications with a 25 µL total reaction volume. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. IMPORTANT! Do not refreeze kit components after thawing. Table 2 NGM Detect™ PCR Amplification Kit (Cat. No. A31832; 200 reactions) Contents Description Amount Storage NGM Detect™ Master Mix Contains enzyme, salts, 2 × 0.75 mL −25°C to −15°C on receipt. dNTPs, bovine serum 2°C to 8°C after first use, for up to albumin, enzyme, and 0.05% 6 months or up to the expiration sodium azide in buffer and date stated on the kit (whichever salt. comes first). NGM Detect™ Primer Set Contains forward and 2 × 0.25 mL −25°C to −15°C on receipt. reverse primers to amplify 2°C to 8°C after first use, for up to DNA targets. 6 months or up to the expiration date stated on the kit (whichever comes first). Store protected from light. NGM Detect™ Allelic Contains amplified alleles. 1 × 0.05 mL −25°C to −15°C on receipt. Ladder See “Allelic ladder 2°C to 8°C after first use, up to the profile“ on page 11 for expiration date stated on the kit. information. Store protected from light. DNA Control 007 Contains 0.1 ng/µL human 1 × 0.3 mL −25°C to −15°C on receipt. male genomic DNA from cell 2°C to 8°C after first use, up to the line in 0.05% sodium azide expiration date stated on the kit. and buffer[1] See “DNA Control 007“ on page 13 for information. [1] DNA Control 007 is included at a concentration that is appropriate for use as an amplification control (that is, to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype). It is not designed for use as a DNA quantification control. If you quantify aliquots of DNA Control 007, the concentration may differ from the labeled concentration. Required materials not supplied See Appendix B, “Materials required but not supplied“. 14 NGM Detect™ PCR Amplification Kit User Guide Chapter 1 Product information Instruments and software compatibility 1 Instruments and software compatibility Instrument Validated models type Thermal • ProFlex™ 96‑well PCR System (Cat. No. 4484075) cyclers • ProFlex™ 2 × 96‑well PCR System (Cat. No. 4484076) • ProFlex™ 3 × 32‑Well PCR System (Cat. No. 4484073) • Veriti™ 96‑Well Thermal Cycler (Cat. No. 4479071) • GeneAmp™ PCR System 9700, 96-Well Silver (Cat. No. N8050001) • GeneAmp™ PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878) IMPORTANT! The NGM Detect™ kit is NOT validated for use with: · ProFlex™ 2 × Flat PCR System (Cat. No. 4484078) · ProFlex™ 2 × 384‑well PCR System (Cat. No. 4484077) · Veriti™ 96‑Well Fast Thermal Cycler (Cat. No. 4375305) · GeneAmp™ PCR System 9700 with the aluminium 96-well block (Cat. No. 4314879) Genetic • 3500/3500xL Genetic Analyzer with any of the following: analyzers[1] – 3500 Data Collection Software v1 (Windows™ Vista operating system) and HID Updater 3500 Data Collection Software v2 (Cat. No. 4480670) – 3500 Data Collection v2 Software (Windows™ 7 operating system) and HID Updater 3500 Data Collection Software v2 (Cat. No. 4480670) – 3500 Data Collection v3 Software (Windows™ 7 operating system) • 3130/3130xl Genetic Analyzer with: – Data Collection Software v4 (Windows™ 7 operating system) – 3130/3730 Data Collection v4 6-Dye Module v1 Analysis GeneMapper™ ID‑X Software v1.5.2 or later[2] software Windows™ 7 operating system [1] We conducted validation studies using the 3130xl, 3500, and 3500xL configurations. [2] GeneMapper™ ID‑X Software v1.2 to v1.5 can be used to analyze NGM Detect™ PCR Amplification Kit data. However, some genotype quality assessment features of the NGM Detect™ kit are not included in earlier versions of the software. Refer to Chapter 4, “Analyze data with GeneMapper™ ID‑X Software“ for more details. NGM Detect™ PCR Amplification Kit User Guide 15 1 Chapter 1 Product information Workflow Workflow Extract DNA, see: www.thermofisher.com/hid-sampleprep ▼ Quantify DNA “DNA quantification“ on page 17 ▼ Perform PCR “Prepare the amplification kit reactions“ on page 19 ▼ “Perform PCR“ on page 21 ▼ Perform electrophoresis “Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)“ on page 24 or “Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)“ on page 28 ▼ “Prepare samples for electrophoresis (3500 Series and 3130 Series instruments)“ on page 27 ▼ Analyze data ™ “Set up the GeneMapper ID‑X Software for analysis (before first use of the kit)“ on page 35 ▼ “Create an analysis method“ on page 40 ▼ “Create a size standard definition file if needed“ on page 48 ▼ “Analyze and edit sample files with GeneMapper ID-X Software“ on page 50 ▼ “Examine or edit a project“ on page 51 ▼ Chapter 5, “Assess the PCR reaction with the Internal Quality Control System“ 16 NGM Detect™ PCR Amplification Kit User Guide 2 Perform PCR ■ DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ■ Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Prepare the amplification kit reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 ■ Direct amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 DNA quantification Importance of DNA quantification can be used to determine: quantification • If the sample contains sufficient human DNA and/or human male DNA to before STR proceed with short tandem repeat (STR) analysis. analysis • The amount of sample to use in STR analysis applications. • The relative quantities of human male and female DNA in a sample (using the Quantifiler™ Trio DNA Quantification Kit). This guides selection of the applicable STR chemistry. • The DNA quality, with respect to the inhibition level and the DNA degradation level. This metric is useful for determining the likelihood of recovery of STR loci with larger amplicon sizes. • If the sample contains highly degraded DNA. Such samples may require an alternative approach to STR analysis by capillary electrophoresis. Precision ID NGS System and Panels are optimized for degraded samples. The Precision ID Identity Panel provides discrimination of individuals similar to STR genotype match probabilities. Also, the Precision ID Ancestry Panel infers biogeographical ancestry for investigative leads. • If PCR inhibitors are present in a sample. Such that samples may require additional purification before proceeding to STR analysis. Effect of DNA If too much DNA is added to the PCR reaction, the increased amount of PCR product quantity on results that is generated can result in: • Fluorescence intensity that exceeds the linear dynamic range for detection by the capillary electrophoresis instrument ("off-scale" data). NGM Detect™ PCR Amplification Kit User Guide 17 2 Chapter 2 Perform PCR DNA quantification Off-scale data are a problem because: – Quantification (peak height and area) for off-scale peaks is not accurate. For example, an allele peak that is off-scale can cause a corresponding stutter peak to appear higher in relative intensity, therefore increasing the calculated percent stutter. – Multicomponent analysis of off-scale data are not accurate. This inaccuracy results in poor spectral separation ("pull-up"). • False signals of inhibition by the IQC system, although none is present. • A reduction in the IQCL peak height. • Incomplete +A nucleotide addition. To address these problems, rerun the amplification reaction using less DNA. If too little DNA is added to the PCR reaction, the total number of allele copies added to the PCR is extremely low. Unbalanced amplification of the alleles may occur because of stochastic fluctuation. Methods of For information on recent innovations in quantification chemistry, go to quantifying DNA thermofisher.com. Kit and user guide Detects How it works Quantifiler™ HP DNA • Total human DNA • Uses 5′ nuclease assays with multiple-copy target Quantification Kit (Cat. No. (two targets—one loci, for improved detection sensitivity:[1] 4482911) small amplicon and – The human-specific target loci are multiple one larger amplicon) copy, and dispersed on various autosomal For more information, see • Degraded DNA chromosomes. Quantifiler™ HP and Quantifiler™ Trio DNA – The primary quantification targets have Quantification Kits User relatively short amplicons (75 to 80 bases), to Guide (Pub. No. 4485354) improve the detection of degraded DNA samples. Quantifiler™ Trio DNA • Total human DNA • Uses features that maximize consistency of Quantification Kit (Cat. No. (two targets—one quantification: 4482910) small amplicon and one larger amplicon) – Genomic targets have conserved primer- and For more information, see probe-binding sites. Quantifiler™ HP and • Human male DNA – Minimal copy number variation between Quantifiler™ Trio DNA • Degraded DNA different individuals and population groups. Quantification Kits User Guide (Pub. No. 4485354) • Contains a Large Autosomal target with a longer amplicon (>200 bases) to help determine if a DNA sample is degraded. • Contains an Internal PCR control (IPC) 5′ nuclease assay which amplifies an integrated synthetic DNA sequence. The performance of this assay can be used to assess whether real-time PCR of the sample has been impacted by inhibition. [1] The detection sensitivity of the Quantifiler™ HP Kit and the Quantifiler™ Trio Kit is improved over the Quantifiler™ Duo Kit. 18 NGM Detect™ PCR Amplification Kit User Guide Chapter 2 Perform PCR Before you begin 2 Before you begin Prepare low-TE For optimal results, we recommend using low-TE buffer for sample preparation. buffer Prepare it as described in this procedure or buy it from Teknova (Cat. No. T0223). 1. Mix together: • 10 mL of 1 M Tris-HCl, pH 8.0 • 0.2 mL of 0.5 M EDTA, pH 8.0 • 990 mL glass-distilled or deionized water Note: Adjust the volumes accordingly for specific needs. 2. Aliquot, then autoclave the solutions. 3. Store the aliquots at room temperature. Thaw reagents Thaw the Master Mix and Primer Set. (before first use of IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the kit) the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are stored at 2°C to 8°C and do not require subsequent thawing. Do not refreeze the reagents. Prepare the amplification kit reactions IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. IMPORTANT! Use adhesive film for plate sealing to provide a consistent seal across all wells and prevent evaporation. Caps may not provide a consistent seal across all plate wells. 1. Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes, remove droplets from the caps by centrifuging the tubes briefly. 2. Pipette the required volumes of components into an appropriately sized clear (non-colored) polypropylene tube: Reaction component Volume per reaction Master Mix 7.5 µL Primer Set 2.5 µL Note: Include volume for additional reactions to provide excess volume for the loss that occurs during reagent transfers. NGM Detect™ PCR Amplification Kit User Guide 19 2 Chapter 2 Perform PCR Prepare the amplification kit reactions 3. Vortex the reaction mixture for 3 seconds, then centrifuge briefly. 4. Dispense 10 µL of reaction mixture into each reaction well of a MicroAmp™ Optical 96-Well Reaction Plate or each MicroAmp™ tube. 5. Adjust the sample input amount and volume as needed: Note: The optimum DNA input is 500 pg. • If total sample input amount is >500 pg of DNA, dilute with low-TE buffer to achieve a 500 pg input in a 15-µL volume. • If total sample input volume is <15 µL, bring to volume with low-TE buffer to achieve a 15-µL input volume. 6. Prepare samples as shown in the following table, then add them to the appropriate well or tube (final reaction volume is 25 µL). Sample Add Negative control 15 μL of low‑TE buffer Test sample 15 μL of DNA[1] Positive control Combine, then add to the reaction well or tube: • 5 μL DNA Control 007 (0.1 ng/μL) • 10 μL of low‑TE buffer [1] Prepared in step 5. 7. Seal the MicroAmp™ Optical 96-Well Reaction Plate with MicroAmp™ Clear Adhesive Film or MicroAmp™ Optical Adhesive Film. 8. Vortex the plate or tubes at medium speed for 3 seconds. 9. Centrifuge the tubes or plate at 3,000 rpm for approximately 30 seconds in a tabletop centrifuge (with plate holders, if using 96-well plates). 10. Amplify the samples. IMPORTANT! See “Instruments and software compatibility“ on page 15 for a list of validated thermal cyclers. 20 NGM Detect™ PCR Amplification Kit User Guide Chapter 2 Perform PCR Perform PCR 2 Perform PCR IMPORTANT! This kit is validated for use with the thermal cyclers listed in “Instruments and software compatibility“ on page 15. 1. Program the thermal cycling conditions. IMPORTANT! If you are using the: · ProFlex™ PCR System, select 9700 Simulation Mode. · GeneAmp™ PCR System 9700, select the Max ramping mode. · Veriti™ Thermal Cycler, set up the method using the Convert a Method tool and select 9700 Max Mode. Do not use 9600 emulation mode. Initial Cycle (30 cycles) Final incubation Final hold Denature Anneal Extend extension step HOLD CYCLE HOLD HOLD 95°C, 96°C, 59°C, 65°C, 60°C, 4°C[1] 1 minute 5 seconds 21 seconds 29 seconds 5 minutes [1] The infinity (∞) setting allows an unlimited hold time. 2. Load the plate into the thermal cycler, close the heated cover, then start the run. IMPORTANT! If you are using a GeneAmp™ PCR System 9700 and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp™ Optical Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during thermal cycling. The Veriti™ Thermal Cycler and the ProFlex™ PCR System do not require a compression pad. 3. When the run is complete, store the amplified DNA. If you are storing the DNA... Then place at... <2 weeks 2°C to 8°C >2 weeks –25°C to –15°C IMPORTANT! Protect the amplified DNA from light. NGM Detect™ PCR Amplification Kit User Guide 21 2 Chapter 2 Perform PCR Direct amplification Direct amplification FTA™ cards and non-chemically treated bloodstain cards are useful for the collection, storage, and processing of biological samples. A small punch disc of the card containing the sample can be placed directly into an amplification tube or 96-well plate, washed, then amplified, without transferring the disc. Our studies indicate that a 1.2-mm bloodstained disc contains approximately 5 ng– 20 ng of DNA. Because of the high quantity of DNA, a lower cycle number is required to produce on-scale data. In our testing, an appropriate cycle number for this high quantity DNA was 26 cycles. We recommend that each laboratory determine the optimum cycle number which is based on internal validation studies. Note: This kit is not fully validated for use with direct amplification. Perform your own validation for this purpose or use the NGM SElect™ Express kit for direct amplification of database samples. In the example that is shown in Figure 3, a 1.2-mm disc of a bloodstained FTA™ card was purified using one wash with FTA™ Purification Reagent and one wash with 1X low TE buffer, followed by a short drying step. The sample was then amplified directly in the well of a standard 96-well amplification MicroAmp™ plate for 26 cycles. Figure 3 Combined dyes electropherogram of a 1.2-mm FTA™ bloodstain disc amplified for 26 cycles with the NGM Detect™ kit on an Applied Biosystems™ 3500xL Genetic Analyzer (Y-axis scale 0 to 33,000 RFU). d Arrows w to use in this SVG. anced callouts or arrows e_Callout&Arrow_Libary. th, as needed. e, and unused G elements e repository. 1 1 22 NGM Detect™ PCR Amplification Kit User Guide 3 Perform electrophoresis ■ Allelic ladder requirements for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ■ Materials required for electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 ■ Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 ■ Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 ■ Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 ■ Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Allelic ladder requirements for electrophoresis To accurately genotype samples, you must run an allelic ladder with the samples. Number of One Number of samples per allelic Instrument allelic ladders injection ladder(s) to run equals 3500 1 per 3 injections 8 samples 23 samples + 1 allelic ladder 3500xl 1 per injection 24 samples 23 samples + 1 allelic ladder 3130 1 per 4 injections 4 samples 15 samples + 1 allelic ladder 3130xl 1 per injection 16 samples 15 samples + 1 allelic ladder IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between runs. Follow the guidelines in the preceding table, which should account for normal variation in run speed. Perform internal validation studies to verify the required allelic ladder injection frequency, to ensure accurate genotyping of all samples in your laboratory environment. It is critical to genotype using an allelic ladder run under the same conditions as the samples. Size values obtained for the same sample can differ between instrument platforms, because of different polymer matrices and electrophoretic conditions. NGM Detect™ PCR Amplification Kit User Guide 23 3 Chapter 3 Perform electrophoresis Materials required for electrophoresis Materials required for electrophoresis Appendix B, “Materials required but not supplied“ lists the required materials that are not supplied with this kit. IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set, amplified DNA, allelic ladder, and size standard from light when not in use. Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) Electrophoresis The following table lists the data collection software and the run modules that you software setup can use to analyze PCR products that are generated by this kit. For details on the procedures, see the documents that are listed in “Documentation and support“ on page 136. Note: We conducted validation studies for the kit using the 3130xl, 3500, or 3500xL configurations. Genetic Operating Data Collection Additional Instrument protocols, run modules, and Analyzer System Software software conditions 3500 Windows™ 3500 Data HID Updater 3500 Set up the following conditions: Vista Collection DC v2 • Run module: HID36_POP4 (HID36_POP4xl for 3500xL Software v1 (Cat. No. 4480670) 3500xL) • Injection conditions[1]: 1.2 kV/11 sec (20 sec for 3500xL) • Run conditions: 13 kV/1500 sec (13 kV/1500 sec for 3500xL) • Dye Set J6-T 3500 Windows™ 7 3500 Data HID Updater 3500 Same as 3500 Data Collection Software v1 listed Collection DC v2 above 3500xL Software v2 (Cat. No. 4480670) 3500 Windows™ 7 3500 Data None Same as 3500 Data Collection Software v1 listed Collection above 3500xL Software v3 [1] Our studies indicate that the injection conditions that are documented generate profiles from 0.5 ng of input DNA with heterozygous peak height averages between 4,000– 10,000 RFU (3500 or 3500xL) with no instances of allelic dropout and minimal occurrence of off-scale allele peaks. However, individual CE instrument signal intensities can vary, therefore changes to injection parameters may need to be explored and validated to deliver the best results on your specific system. Large deviations from the recommended injection parameters could affect the performance of the size standard and/or allelic ladder, therefore validation is recommended. 24 NGM Detect™ PCR Amplification Kit User Guide Chapter 3 Perform electrophoresis Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) 3 Create a 3500 QC The NGM Detect™ kit has been validated with data that was analyzed using the Local protocol Southern method (60–460 base pairs). 1. In the Library tab, open the QC Protocol window. 2. Create a new QC protocol : a. Name the new QC protocol according to your laboratory naming convention. b. Set the following parameters: Parameter Setting Size Standard GS600_LIZ_(60-460) Size Range Partial Sizing Start Size 60 bp Sizing Stop Size 460 bp Size Calling Method Local Southern Method After checking the "Use Baselining" 33 box: Baseline Window Pts. Peak Window Size 11 NGM Detect™ PCR Amplification Kit User Guide 25 3 Chapter 3 Perform electrophoresis Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) c. Click Save. 3. Add the QC protocol to the HID assay. Perform spectral Perform a spectral calibration using the DS-37 Matrix Standard Kit (Dye set J6-T, 6- calibration dye) (J6-T Dye Set) (Cat. No. A31234). Note: Since J6-T is a new dye set, create a new dye set in the Data Collection Software before running a spectral calibration. For instructions on creating a new dye set, see the "Create a New Dye Set" section of the 3500/3500xL Genetic Analyzer with 3500 Series 26 NGM Detect™ PCR Amplification Kit User Guide Chapter 3 Perform electrophoresis Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) 3 Data Collection Software v2 User Guide (Cat. No. 4476988). Use the J6 template to set up the J6-T dye set. The following figure is an example of a passing 6-dye spectral calibration. Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) This procedure applies to the 3500 Series and 3130 Series instruments. Prepare the samples for electrophoresis immediately before loading. 1. Pipet the required volumes of components into an appropriately sized polypropylene tube: Reagent Volume per reaction ™ ™ GeneScan –600 LIZ Size Standard v2.0 0.4 μL ™ Hi‑Di Formamide 9.6 μL Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. 2. Vortex the tube, then briefly centrifuge. 3. Into each well of a MicroAmp™ Optical 96-Well Reaction Plate, add: • 10 µL of the formamide/size standard mixture • 1 µL of PCR product or Allelic Ladder Note: For blank wells, add 10 µL of Hi-Di™ Formamide. NGM Detect™ PCR Amplification Kit User Guide 27 3 Chapter 3 Perform electrophoresis Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) 4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes. 6. Immediately place the plate on ice for 3 minutes. 7. Place the sample tray on the autosampler, then start the electrophoresis run. Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) Electrophoresis The following table lists the data collection software and the run modules that you software setup can use to analyze PCR products generated by this kit. For details on the procedures, see the documents listed in “Documentation and support“ on page 136. Data Genetic Operating Collection Additional software Run modules and conditions Analyzer System Software 3130[1] Windows™7 Data Collection 3130/3730 DC v4 6- Set up the following conditions: Software v4 Dye Module v1 • HIDFragmentAnalysis36_POP4_1 • Injection conditions[2]: 3 kV/4 sec • Run conditions: 14 kV/1600 sec • Dye Set J6-T 3130xl Set up the following conditions: • HIDFragmentAnalysis36_POP4_1 • Injection conditions[2]: 3 kV/5 sec • Run conditions: 14 kV/1600 sec • Dye Set J6-T [1] We conducted validation studies using the 3130xl, 3500, and 3500xL configurations. [2] Our studies indicate that the injection conditions that are documented generate profiles from 0.5 ng of input DNA with heterozygous peak height averages between 2,000– 4,000 RFU (3130 or 3130xl) with no instances of allelic dropout and minimal occurrence of off-scale allele peaks. However, individual CE instrument signal intensities can vary, therefore changes to injection parameters may need to be explored and validated to deliver the best results on your specific system. Large deviations from the recommended injection parameters could affect the performance of the size standard and/or allelic ladder, therefore validation is recommended. Obtain and 1. Confirm that you are running Data Collection Software v4 (Help4About). activate 6-dye license 2. Obtain a 3130 DC v4 6-Dye Module v1 License key. Contact your local Human Identification representative for information. 28 NGM Detect™ PCR Amplification Kit User Guide Chapter 3 Perform electrophoresis Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) 3 3. Ensure that all network cards in the computer are enabled. IMPORTANT! You can run the 3130 Series Data Collection Software v4 using only the network cards that are enabled when you activate the software license. For example, if you activate the software when your wireless network card is disabled, you will not be able to run the software when the wireless network card is enabled. 4. Select Tools4License Manager to display the Software Activation dialog box. 5. Request the software license file by performing steps 1a, 1b, and 1c as listed on the activation screen. The license file will be emailed to you. 6. Obtain the software license file from your email. 7. Make a copy of the software license file and keep it in a safe location. 8. Copy the software license file to the desktop of the Data Collection Software v4 computer. 9. If the Software Activation dialog box has closed, select Tools4License Manager. NGM Detect™ PCR Amplification Kit User Guide 29 ut or arrow to use in this SVG. more advanced callouts or arrows 3 Chapter 3 Perform electrophoresis m_Inkscape_Callout&Arrow_Libary. Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) line-length, as needed. 10. Click Browse, then navigate to the software license file saved on your computer. s rectangle, and unused other SVG elements SVG to the repository. 11. Click Install and Validate License. A message is displayed when the license is installed and validated. 1 1 12. Click Close. 1 Perform spectral Perform a spectral calibration using the DS-37 Matrix Standard Kit (Dye set J6-T, 6- calibration dye) (J6-T Dye Set) (Cat. No. A31234). The following figure is an example of a passing 6-dye spectral calibration. Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) This procedure applies to the 3500 Series and 3130 Series instruments. Prepare the samples for electrophoresis immediately before loading. 1. Pipet the required volumes of components into an appropriately sized polypropylene tube: Reagent Volume per reaction ™ ™ GeneScan –600 LIZ Size Standard v2.0 0.4 μL ™ Hi‑Di Formamide 9.6 μL Note: Include volume for additional samples to provide excess volume for the loss that occurs during reagent transfers. IMPORTANT! The volume of size standard indicated in the table is a suggested amount. Determine the appropriate amount of size standard based on your experiments and results. 2. Vortex the tube, then briefly centrifuge. 30 NGM Detect™ PCR Amplification Kit User Guide Chapter 3 Perform electrophoresis Prepare samples for electrophoresis (3500 Series and 3130 Series instruments) 3 3. Into each well of a MicroAmp™ Optical 96-Well Reaction Plate, add: • 10 µL of the formamide/size standard mixture • 1 µL of PCR product or Allelic Ladder Note: For blank wells, add 10 µL of Hi-Di™ Formamide. 4. Seal the reaction plate with appropriate septa, then briefly vortex and centrifuge the plate to ensure that the contents of each well are mixed and collected at the bottom. 5. Heat the reaction plate in a thermal cycler at 95°C for 3 minutes. 6. Immediately place the plate on ice for 3 minutes. 7. Place the sample tray on the autosampler, then start the electrophoresis run. NGM Detect™ PCR Amplification Kit User Guide 31 4 Analyze data with GeneMapper™ ID‑X Software ■ Overview of GeneMapper ID-X Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 ■ GeneMapper™ ID-X Software analysis of the NGM Detect™ kit . . . . . . . . . . . . 33 ■ Allelic ladder requirements for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 ■ File names and versions used in this section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 ■ Set up the GeneMapper™ ID-X Software for analysis (before first use of the kit) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 ■ Create an analysis method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 ■ Create a size standard definition file if needed . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 ■ Analyze and edit sample files with GeneMapper ID-X Software . . . . . . . . . . . . 50 ■ Examine or edit a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 ■ For more information on using the GeneMapper™ ID-X Software . . . . . . . . . . 51 Overview of GeneMapper ID-X Software GeneMapper™ ID-X Software is an automated genotyping software application for forensic casework, databasing, and paternity data analysis. GeneMapper™ ID-X Software v1.5.2 or later analyzes 4-dye, 5-dye, and 6-dye data and is required to correctly analyze data that is generated using the NGM Detect™ kit. After electrophoresis, the data collection software stores information for each sample in a .fsa or .hid file. The GeneMapper™ ID-X Software v1.5.2 or later allows you to analyze and interpret the data from the .fsa or .hid files. Prior versions of GeneMapper™ ID-X Software (v1.2 and above) are capable of analyzing data that is generated using the NGM Detect™ kit. However, some data quality assessment tools are not fully functional on versions prior to v1.5.2. Details on the data assessment functionality added to specific versions of GeneMapper™ ID-X Software are found in the release notes for each version. To summarize: • In versions prior to v1.4, samples show a red flag for the CGQ if the Y-indel peak is missing, for example, with a female DNA sample. This occurs because the absence of the Y-indel peak triggers the Allele Number Rule. • In versions prior to v1.5.2, when the sample type is set as "Negative Control" the software will assign red CGQ flags. This occurs even if the negative control sample is valid, that is, it contains no peaks above the PAT in the STR, AMEL, and Y-indel marker ranges. The red flags appear because the presence of the IQC peaks triggers the Allele Number Rule for this sample type (no peaks are expected in a valid negative control sample). 32 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software GeneMapper™ ID‑X Software analysis of the NGM Detect™ kit 4 GeneMapper™ ID‑X Software analysis of the NGM Detect™ kit GeneMapper™ ID-X Software allows you to designate four sample types: • Sample • Positive Control • Allelic Ladder • Negative Control GeneMapper™ ID-X Software has logic for Negative Control sample type assignments, whereby the software generates red flags on the Genotype Quality (GQ) indicators when peaks are present above the Peak Amplitude Threshold (PAT) designated in the analysis method. The NGM Detect™ PCR Amplification Kit includes the internal quality control (IQC) system for PCR. In this system, two synthetic targets in the primer mix are amplified with the sample under test. The IQC system allows you to determine whether PCR has been successful in the absence of peaks from the sample. In versions prior to v1.5.2, when the sample type is set as "Negative Control" the software will assign red CGQ flags. This occurs even if the negative control sample is valid, that is, it contains no peaks above the PAT in the STR, AMEL, and Y-indel marker ranges. The red flags appear because the presence of the IQC peaks triggers the Allele Number Rule for this sample type (no peaks are expected in a valid negative control sample). Thermo Fisher Scientific has developed the NGM Detect™ kit using version 1.5.2 of the GeneMapper™ ID-X Software. This version includes updated logic for the Negative Control sample type. It allows the IQC marker GQ to flag green in the presence of the IQC peaks. A software patch that updates GeneMapper™ ID-X Software v1.5 to v1.5.2 is available at thermofisher.com/us/en/home/technical-resources/software-downloads/ genemapper-id-x-software.html. NGM Detect™ PCR Amplification Kit User Guide 33 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Allelic ladder requirements for data analysis Allelic ladder requirements for data analysis • HID analysis requires at least one allelic ladder sample per run folder. Perform the appropriate internal validation studies before you use multiple allelic ladder samples in an analysis. For multiple allelic ladder samples, the GeneMapper™ ID-X Software calculates allelic bin offsets by using an average of all allelic ladders that use the same panel in a run folder. • Allelic ladder samples in an individual run folder are considered to be from a single run. When the software imports multiple run folders into a project, only the ladders in their respective run folders are used for calculating allelic bin offsets and subsequent genotyping. • Allelic ladder samples must be labeled as "Allelic Ladder" in the Sample Type column in a project. Analysis will fail if the Allelic Ladder Sample Type is not specified. • Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples, to ensure proper allele calling. • Alleles that are not in the allelic ladders do exist. Off-ladder (OL) alleles can contain full and/or partial repeat units. An off-ladder allele is an allele that occurs outside the bin window of any known allelic ladder allele or virtual bin. Note: If a sample allele peak is called as an off-ladder allele, verify the sample result according to your laboratory protocol. File names and versions used in this section The file names and version numbers of panel, bin, and stutter files that are shown in this section may differ from the file names that you see when you download or import files. If you need help to determine the correct files to use, contact your local Human Identification representative, or go to thermofisher.com/support. 34 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) 4 Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) Workflow: Set up Before you use GeneMapper™ ID-X Software to analyze data for the first time, you GeneMapper™ must do the following: ID‑X Software “Check panel, bin, and stutter file versions on your computer“ on page 35 ▼ “(If needed) Download newer versions of panel, bin, and stutter files“ on page 35 ▼ “Import panels, bins, and marker stutter“ on page 36 ▼ “(Optional) Define custom table or plot settings“ on page 39 Check panel, bin, 1. Start the GeneMapper™ ID-X Software , then log in with the appropriate user and stutter file name and password. versions on your 2. Select Tools4Panel Manager. computer 3. Check the version of files that are currently available in the Panel Manager: a. Select Panel Manager in the navigation pane. b. Expand the Panel Manager folder and any subfolders to identify the analysis file version that is already installed for your kit choice. 4. Check the version of files available for import into the Panel Manager: a. Select Panel Manager, then select File4Import Panels to open the Import Panels dialog box. b. Navigate to, then open the Panels folder, then check the version of panel, bin, and stutter files installed. 5. Check for newer versions of the files as described in the next procedure. (If needed) 1. Go to thermofisher.com/us/en/home/technical-resources/software-downloads/ Download newer genemapper-id-x-software.html. versions of panel, 2. If the file versions listed are newer than the versions on your computer, bin, and stutter download the file NGM Detect Analysis Files. files Note: When downloading new versions of analysis files, see the associated Read Me file for details of changes between software file versions. Perform the appropriate internal validation studies before using new file versions for analysis. 3. Unzip the file. NGM Detect™ PCR Amplification Kit User Guide 35 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) Common Callouts and Arrows Import panels, To import the latest 1. Copy-paste panel, a callout bin set, or arrow and to use marker in this SVG. stutter from the website into the bins, and marker GeneMapper™ ID-X Software database: Note: If you need more advanced callouts or arrows stutter 1. Start use the GeneMapper™ ID-X Software, then log in with the appropriate user the TechComm_Inkscape_Callout&Arrow_Libary. name and password. 2. Edit number and/or line-length, as needed. 2. Select Tools4Panel Manager. 3. Delete this text, this rectangle, and unused 3. Find, thenarrows, callouts, openorthe folder other containing the panels, SVG elements bins, and before marker adding stutter: this SVG to the repository. 1 a. Select Panel Manager, 1 then1 select File4Import Panels to open the Import Panels dialog box. 1 b. Navigate to, then open the NGM Detect Analysis Files folder that you unzipped in the previous procedure. 4. Select NGM_Detect_Panels.txt, then click Import. Note: Importing this file creates a new folder in the navigation pane of the Panel Manager, NGM_Detect_Panel. This folder contains the panel and associated markers. uts and Arrows t or arrow to use in this SVG. more advanced callouts or arrows _Inkscape_Callout&Arrow_Libary. line-length, as needed. rectangle, and unused other SVG elements VG to the repository. 1 1 1 36 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) 4 5. Import the bins file: a. Select the NGM_Detect_Panel folder in the navigation pane. mmon Callouts and Arrows py-paste a callout or arrow to use in this SVG. e: If you need more advanced callouts or arrows the TechComm_Inkscape_Callout&Arrow_Libary. t number and/or line-length, as needed. ete this text, this rectangle, and unused outs, arrows, or other SVG elements ore adding this SVG to the repository. b. Select File4Import Bin Set to open the Import Bin Set dialog box. c. Navigate to, then open the NGM Detect Analysis Files folder. 1 1 1 d. Select NGM_Detect_Bins.txt, then click Import. Note: Importing this file associates the bin set with the panels in the NGM_Detect_Panel folder. mon Callouts and Arrows py-paste a callout or arrow to use in this SVG. e: If you need more advanced callouts or arrows the TechComm_Inkscape_Callout&Arrow_Libary. number and/or line-length, as needed. ete this text, this rectangle, and unused outs, arrows, or other SVG elements ore adding this SVG to the repository. 1 1 1 NGM Detect™ PCR Amplification Kit User Guide 37 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) 6. (Optional) View the imported panels and bins in the navigation pane: Double- click the NGM_Detect_Panel folder. The panel information is displayed in the right pane and the markers are displayed below it. ts and Arrows or arrow to use in this SVG. ore advanced callouts or arrows Inkscape_Callout&Arrow_Libary. ne-length, as needed. ectangle, and unused ther SVG elements VG to the repository. 7. Import the stutter file: a. Select the NGM_Detect_Panel folder in the navigation panel. 1 1 b. Select File4Import Marker Stutter to open the Import Marker Stutter 1 dialog box. c. Navigate to, then open the NGM Detect Analysis Files folder. 38 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) 4 d. Select NGM_Detect_Stutter.txt, then click Import. Note: Importing this file associates the marker stutter ratio with the bin set in the NGM_Detect_Panel folder and overwrites any existing stutter ratios associated with the panels and bins in that folder. mmon Callouts and Arrows py-paste a callout or arrow to use in this SVG. te: If you need more advanced callouts or arrows e the TechComm_Inkscape_Callout&Arrow_Libary. t number and/or line-length, as needed. lete this text, this rectangle, and unused louts, arrows, or other SVG elements ore adding this SVG to the repository. 8. View the imported marker stutters in the navigation pane: 1 1 a. Double-click the NGM_Detect_Panel folder to display the folder. 1 b. Double-click the folder to display its list of markers below it. c. Double-click a marker to display the Stutter Ratio & Distance view for the marker in the right pane. 9. Click Apply, then click OK to add the panel, bin set, and marker stutter to the mmon Callouts and Arrows GeneMapper™ ID-X Software database. Copy-paste a callout or arrow to use in this SVG. IMPORTANT! If you close the Panel Manager without clicking Apply, the Note: If you need more advanced callouts or arrows panels, bin sets, and marker stutter are not imported into the GeneMapper™ ID-X se the TechComm_Inkscape_Callout&Arrow_Libary. Software database. dit number and/or line-length, as needed. (Optional) Define Default views for table and plot settings are provided with the software. For custom table or information on defining custom views, see GeneMapper™ ID-X Software Getting Started elete this text, this rectangle, and unused Guide— Basic Features. plot allouts, arrows, or other SVG settings elements efore adding this SVG to the repository. NGM Detect™ PCR Amplification Kit User Guide 39 1 1 1 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Create an analysis method Create an analysis method Create an analysis IMPORTANT! Analysis methods are version-specific, so you must create an analysis method method for each version of the software. For example, an analysis method that is created in GeneMapper™ ID-X Software version 1.2 is not compatible with analysis methods that are created in earlier versions of software, or with GeneMapper™ Software v3.2.1. 1. Select Tools4GeneMapper® ID-X Manager to open the GeneMapper ID-X Manager. n Callouts and Arrows te a callout or arrow to use in this SVG. ou need more advanced callouts or arrows echComm_Inkscape_Callout&Arrow_Libary. er and/or line-length, as needed. s text, this rectangle, and unused rrows, or other SVG elements ding this SVG to the repository. 2. Click the Analysis Methods tab, then click New to open the Analysis Method 1 1 Editor with the General tab selected. 1 3. Enter the settings shown in the figures on the following pages. Note: The Analysis Method Editor closes when you save your settings. To complete this step quickly, do not save the analysis method until you finish entering settings in all of the tabs. 4. After you enter the settings on all tabs, click Save. 40 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Create an analysis method 4 Enter Analysis Enter General tab settings Method settings 1. Enter a Name and select the Security Group appropriate for your software configuration. louts and Arrows llout or arrow to use in this SVG. d more advanced callouts or arrows mm_Inkscape_Callout&Arrow_Libary. /or line-length, as needed. this rectangle, and unused or other SVG elements is SVG to the repository. 1 1 1 2. (Optional) Enter a Description and Instrument. NGM Detect™ PCR Amplification Kit User Guide 41 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Create an analysis method Enter Allele tab settings IMPORTANT! Perform appropriate internal validation studies to determine the appropriate settings to use. 1. Select the NGM_Detect_Bins_v3 bin set. ts and Arrows or arrow to use in this SVG. ore advanced callouts or arrows Inkscape_Callout&Arrow_Libary. ne-length, as needed. ectangle, and unused ther SVG elements VG to the repository. 1 1 1 Figure 4 Settings used in developmental validation of the kit 2. (Optional) To apply the stutter ratios contained in the NGM_Detect_Stutter.txt, select the Use marker-specific stutter ratio and distance if available checkbox (selected by default). 42 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Create an analysis method 4 3. If using GeneMapper™ ID-X Software v1.0.1 or later, enter values for the 4 Marker Repeat Types. 4. Enter the appropriate filter settings. Enter Peak Detector tab settings Enter the appropriate values: Field Values to enter or select Additional information Ranges Enter the values shown in Figure 5. — Note: The read region for the NGM Detect™ kit is 64 to 458 bp. Peak Detection Enter the appropriate settings. The software uses these parameters to IMPORTANT! Perform appropriate internal specify the minimum peak height, in order to validation studies to determine the limit the number of detected peaks. appropriate peak amplitude thresholds for Although GeneMapper™ ID‑X Software interpretation of data. displays peaks that fall below the specified amplitude in electropherograms, the software does not label or determine the genotype of these peaks. NGM Detect™ PCR Amplification Kit User Guide 43 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Create an analysis method Field Values to enter or select Additional information Smoothing and Enter the values shown in Figure 5, or adjust — Baseline as needed dependent on the polymer you are using. Size Calling Method Select Local Southern Method, or another — method if validated by your internal validation. Normalization (Optional) Select the Normalization A Normalization checkbox is available on checkbox. this tab in GeneMapper™ ID‑X Software for use in conjunction with data run on the 3500 Series Genetic Analyzers. 44 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Create an analysis method 4 Note: The read region for the NGM Detect™ kit is 64 to 458 bp. Figure 5 Settings used in developmental validation of the kit NGM Detect™ PCR Amplification Kit User Guide 45 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Create an analysis method Enter Peak Quality tab settings IMPORTANT! Perform the appropriate internal validation studies to determine the heterozygous and homozygous minimum peak height thresholds, maximum peak height threshold, and the minimum peak height ratio threshold for interpretation of data. Enter the following values: 46 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Create an analysis method 4 Enter SQ and GQ tab settings IMPORTANT! The values that are shown are the software defaults and are the values we used during developmental validation. Perform appropriate internal validation studies to determine the appropriate values to use. Enter the following values: allouts and Arrows callout or arrow to use in this SVG. eed more advanced callouts or arrows Comm_Inkscape_Callout&Arrow_Libary. nd/or line-length, as needed. xt, this rectangle, and unused ws, or other SVG elements this SVG to the repository. 1 1 1 Note: Set the ACC GQ Weighting according to the values you determine during internal validation studies of the ACC PQV. For example, set the ACC GQ Weighting to 0.3 or higher to flag samples in which the Amelogenin result is anything other than X, X or X, Y, or does not agree with the results for the Y indel marker. NGM Detect™ PCR Amplification Kit User Guide 47 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Create a size standard definition file if needed Create a size standard definition file if needed If you cannot use the default settings that are provided, create a new size standard definition file. About the The GS600_LIZ_(60– 460) size standard definition that is provided with GeneMapper™ GS600_LIZ_ (60– ID-X Software and used with the Local Southern size calling method contains the following peaks: 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 460) size standard 300, 314, 320, 340, 360, 380, 400, 414, 420, 440, and 460. definition file This size standard definition has been validated for use with this kit on the genetic analyzers listed in “Instruments and software compatibility“ on page 15. If you need to create your own size standard definition, see “Create a size standard definition file“ on page 48. Create a size 1. Select Tools4GeneMapper ID-X Manager to open the GeneMapper ID-X standard Manager. definition file 2. Click the Size Standards tab, then click New. n Callouts and Arrows te a callout or arrow to use in this SVG. ou need more advanced callouts or arrows echComm_Inkscape_Callout&Arrow_Libary. ber and/or line-length, as needed. s text, this rectangle, and unused arrows, or other SVG elements 3. Specify settings in the Size Standard Editor: ding this SVG to the repository. a. Enter a name as shown in the following figure or enter a new name. 1 1 b. In the Security Group field, select the Security Group appropriate for your software configuration. 1 c. In the Size Standard Dye field, select Orange. 48 NGM Detect™ PCR Amplification Kit User Guide Chapter 4 Analyze data with GeneMapper™ ID‑X Software Create a size standard definition file if needed 4 d. In the Size Standard Table, enter the peak sizes that correspond to your size standard. NGM Detect™ PCR Amplification Kit User Guide 49 Chapter 4 Analyze data with GeneMapper™ ID‑X Software 4 Analyze and edit sample files with GeneMapper ID-X Software Analyze and edit sample files with GeneMapper ID-X Software 1. In the Project window, select Edit4Add Samples to Project, then navigate to the disk or directory that contains the sample files. 2. Apply analysis settings to the samples in the project. Parameter Settings Sample Type Select the sample type. Analysis Method Select NGM Detect Analysis Method (or the name of the analysis method you created). Panel Select NGM_Detect_Panel. Size Standard Use a size range of 60– 460 bp for Local Southern size calling method [1] [1] The NGM Detect™ kit was originally validated with the GeneScan™–600 LIZ™ Size Standard v2.0. If you use a different size standard, perform the appropriate internal validation studies to support the use of this size standard with the NGM Detect™ kit. 3. Click Analyze, enter a name for the project (in the Save Project dialog box), then click OK to start analysis. • The status bar displays the progress of analysis as a completion bar. • The table displays the row of the sample currently being analyzed in green (or red if analysis failed for the sample). • The Analysis Summary tab is displayed, and the Genotypes tab is available when the analysis is complete. uts and Arrows ut or arrow to use in this SVG. more advanced callouts or arrows m_Inkscape_Callout&Arrow_Libary. line-length, as needed. s rectangle, and unused other SVG elements SVG to the repository. 50 NGM Detect™ PCR Amplification Kit User Guide 1 1 Chapter 4 Analyze data with GeneMapper™ ID‑X Software Examine or edit a project 4 Examine or edit a project Display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data. For more information on using the GeneMapper™ ID‑X Software See “Related documentation“ on page 136 for a list of available documents. NGM Detect™ PCR Amplification Kit User Guide 51 5 Assess the PCR reaction with the Internal Quality Control System ■ Overview of the Internal Quality Control System . . . . . . . . . . . . . . . . . . . . . . . . . 52 ■ Evaluate the PCR reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Overview of the Internal Quality Control System The Internal Quality Control System (IQC) is a tool that helps you to assess the PCR reaction and, in conjunction with the STR marker data properties, infer whether a sample is showing signs of degradation or inhibition. The primers for the two IQC markers, IQCS and IQCL, amplify synthetic DNA targets included in the primer mix. IQCS is a low molecular weight amplicon, 70 nt in length. Whereas, IQCL is a high molecular weight amplicon, 456 nt in length. Note: The allele designation for both IQCS and IQCL will always be 2 (as shown in Figure 2) for all samples. Two alleles for each of the IQC markers are present in the NGM Detect™ Allelic Ladder (designated 1 and 2, see Figure 1). This is because the software dictates that in order for a ladder to be valid, it must have more than one allele per locus. The IQC system enables you to: • Confirm the success or failure of the PCR reaction, by looking for the presence or absence of the IQCS and IQCL primer peaks on the electropherogram. • Determine if PCR inhibitors are present in the PCR reaction, or if PCR reaction conditions are not optimal, by comparing the relative peak heights of IQCS and IQCL. Evaluate the PCR reaction To evaluate the PCR performance of the samples, review the relative peak heights of the IQCS and IQCL. Under ideal PCR conditions, the peak height of IQCL is approximately the same or slightly higher than IQCS (termed "balanced" in Table 3). Under sub optimal PCR conditions (for example inhibition), the height of the IQCL is substantially reduced relative to the ICQS. Note that when high inputs of DNA are amplified (greater than 2 ng) some suppression of the IQC peaks may also be seen. 52 NGM Detect™ PCR Amplification Kit User Guide Chapter 5 Assess the PCR reaction with the Internal Quality Control System Evaluate the PCR reaction 5 See the following table for outcome scenarios. Table 3 IQC peak interpretation Sample DNA profile IQC peaks Interpretation Example Balanced Balanced PCR performance is See Figure 6 optimal, no sample degradation or inhibition Ski-slope IQCL peak height Inhibition See Figure 7 and significantly Figure 8 decreased or not present Ski-slope Balanced Degraded sample See Figure 9 Common Callouts and Arrows DNA . Copy-paste a callout or arrow to use in this SVG. No peaks Balanced No DNA or very little See Figure 10 Note: If you need more advanced callouts or arrows sample DNA use the TechComm_Inkscape_Callout&Arrow_Libary. No peaks No peaks PCR failure — . Edit number and/or line-length, as needed. Balanced profile . Delete this text, this rectangle, and unused callouts, arrows, or other SVG elements In this example of a balanced profile, the IQC peaks and the DNA profile peaks are before adding this SVG to the repository. balanced. This indicates that PCR has occurred optimally. 1 1 1 Figure 6 Combined dyes electropherogram showing IQCS and IQCL peaks with 0.5 ng DNA (scaled to 9,000 RFU). The IQCS peak is highlighted by the red box and the IQCL peak is highlighted by the green box. NGM Detect™ PCR Amplification Kit User Guide 53 5 Chapter 5 Assess the PCR reaction with the Internal Quality Control System Evaluate the PCR reaction Ski slope profile Figure 7 shows a significantly lower IQCL peak height relative to the IQCS peak with decreased height. This indicates that the PCR reaction has been compromised by inhibition. If high levels of PCR inhibition occur, lower IQCL peaks exhibiting some shouldering IQCL peak height due to incomplete +A nucleotide addition may be observed. Figure 8 shows the complete absence of an IQCL peak, indicating a high level of inhibition. Figure 7 Combined dyes electropherogram for the NGM Detect™ kit in the presence of 140 ng/µL humic acid. The IQCS peak is highlighted by the red box and the IQCL peak is highlighted by the green box. The arrow indicates the ski slope peak pattern observed in the DNA profile. Figure 8 Combined dyes electropherogram for the NGM Detect™ kit in the presence of 250 ng/µL humic acid. The IQCS peak is highlighted by the red box and the IQCL peak is absent. The arrow indicates the ski slope peak pattern observed in the DNA profile. 54 NGM Detect™ PCR Amplification Kit User Guide Chapter 5 Assess the PCR reaction with the Internal Quality Control System Evaluate the PCR reaction 5 Ski slope profile The presence of both the IQCS and IQCL with balanced relative peak heights with balanced IQC indicates that PCR has occurred optimally. peaks Figure 9 Combined dyes electropherogram from degraded DNA. The IQCS peak is highlighted by the red box and the IQCL peak is highlighted by the green box. The ski slope nature of the DNA profile is highlighted by the arrow. No sample peaks Although there are no DNA profile peaks in the following figure, the presence of both with balanced IQC the IQCS and IQCL peaks with balanced relative peak heights indicates that PCR has occurred optimally. peaks Figure 10 Combined dyes electropherogram showing IQCS and IQCL peaks with 0 ng DNA (scaled to 2,000 RFU). The IQCS peak is highlighted by the red box and the IQCL peak is highlighted by the green box. NGM Detect™ PCR Amplification Kit User Guide 55 6 Experiments and results ■ Importance of validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 ■ Experiment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 ■ Laboratory requirements for internal validation . . . . . . . . . . . . . . . . . . . . . . . . . . 57 ■ Developmental validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 ■ Accuracy, precision, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 ■ Extra peaks in the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 ■ Characterization of loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 ■ Species specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 ■ Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 ■ Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 ■ Mixture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 ■ Population data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Importance of validation Validation of a DNA typing procedure for human identification applications is an evaluation of the efficiency, reliability, and performance characteristics of the procedure. By challenging the procedure with samples that are commonly encountered in forensic and parentage laboratories, the validation process uncovers attributes and limitations that are critical for sound data interpretation (Sparkes, Kimpton, Watson, 1996; Sparkes, Kimpton, Gilbard, 1996; Wallin, 1998). Experiment conditions We conducted developmental validation experiments according to the updated and revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, December 2016). Based on these guidelines, we conducted experiments that comply with guidelines 2.0 and 3.0 and its associated subsections. This DNA methodology is not novel. (Moretti et al., 2001; Frank et al., 2001; Wallin et al., 2002; and Holt et al., 2000). We used conditions that produced optimum PCR product yield and that met reproducible performance standards. It is our opinion that while these experiments are not exhaustive, they are appropriate for a manufacturer of STR kits intended for forensic and/or parentage testing use. 56 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Laboratory requirements for internal validation 6 Laboratory requirements for internal validation Each laboratory using this kit must perform internal validation studies. Performance of this kit is supported when used according to the following developmentally validated parameters. Modifications to the protocol should be accompanied by appropriate validation studies performed by the laboratory. Developmental validation Except where noted, all developmental validation studies were performed using the GeneAmp™ PCR System 9700 96-Well thermal cycler or the ProFlex™ PCR System according to the protocol described in the Perform PCR chapter. Unless otherwise indicated, the data in this chapter are from the re-validation of the NGM Detect™ kit after redesigning the IQCL and TH01 markers. The performance of the updated kit formulation assay is fully comparable to that of the original kit. See the Technical Note: Updated NGM Detect™ PCR Amplification Kit: Validation and Comparative Study for studies directly related to soil specificity and a direct comparison of the NGM Detect™ kit original formulation to the updated formulation. SWGDAM “Developmental validation is the acquisition of test data and determination of guideline 2.2.1 conditions and limitations of a new or novel DNA methodology for use on forensic, database, known or casework reference samples.” (SWGDAM, December 2016) SWGDAM “The reaction conditions needed to provide the required degree of specificity and guideline 3.9.2 robustness should be determined. These include, but are not limited to, thermal cycling parameters, the concentration of primers, magnesium chloride, DNA polymerase, and other critical reagents.” (SWGDAM, December 2016) SWGDAM “Criteria for detection of amplified product should be determined based on the guideline 3.9.6 platform and/or method. ” (SWGDAM, December 2016) NGM Detect™ PCR Amplification Kit User Guide 57 6 Chapter 6 Experiments and results Developmental validation PCR components We examined the concentration of each component of the kit. The concentration of each component was in the range where data indicated that the amplification met the required performance criteria for specificity, sensitivity, and reproducibility. For example, 0.5 ng of DNA Control 007 was amplified in the presence of varying concentrations of magnesium chloride, and the results were analyzed on an Applied Biosystems™ 3500xL Genetic Analyzer (Figure 11). The performance of the multiplex is most robust within ±20% of the optimal magnesium chloride concentration. +20% +10% Optimal -10% s y. -20% ™ Figure 11 DNA Control 007 (0.5 ng) amplified with the NGM Detect kit in the presence of varying concentrations of magnesium chloride and analyzed on a 3500xL Genetic Analyzer (Y‑axis scale 0 to 12,000 RFU). Thermal cycling Thermal cycling parameters were optimized using a Design of Experiments (DOE) temperatures approach that seeks to identify the combination of temperatures and hold times that produce the best assay performance. Optimal assay performance was determined through evaluation of several factors, including; evaluation of assay sensitivity, peak- 1 height balance, and resistance to PCR inhibitors. For example, annealing/extension temperatures of 57, 58, 59, 60, and 61°C were tested using a GeneAmp™ PCR System 9700 (Figure 12). The PCR products were analyzed using a 3500xL Genetic Analyzer. 58 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Developmental validation 6 Of the tested annealing temperatures, 57°C to 61°C produced robust profiles. The optimal combination of specificity, sensitivity, and resistance to PCR inhibition was observed at 59°C. Thermal cycler temperature is critical to assay performance; therefore routine, regularly scheduled thermal cycler calibration is recommended. 57ºC 58ºC 59ºC nd Arrows ow to use in this SVG. 60ºC vanced callouts or arrows pe_Callout&Arrow_Libary. gth, as needed. 61ºC gle, and unused VG elements Figure 12 Electropherograms obtained from amplification of 0.5 ng of DNA Control 007 at he repository. annealing temperatures of 57, 58, 59, 60, and 61°C, analyzed on a 3500xL Genetic Analyzer (Y-axis scale 0 to 16,000 RFU). 1 1 NGM Detect™ PCR Amplification Kit User Guide 59 6 Chapter 6 Experiments and results Developmental validation PCR cycle number Reactions were amplified for 28, 29, 30, 31, and 32 cycles on the GeneAmp™ PCR System 9700 using 0.5 ng of DNA Control 007. As expected, the amount of PCR product increased with the number of cycles. A full profile was generated for all numbers of thermal cycles (28–32) and off-scale data were collected for several allele peaks at 32 cycles (Figure 13). We recommend using 30 cycles to optimize signal peak height and minimize artifact or other undesirable peaks. ™ Figure 13 Representative NGM Detect kit profiles obtained from amplification of 0.5 ng of DNA Control 007 using 28, 29, 30, 31, and 32 cycles, analyzed on a 3500xL Genetic Analyzer (Y-axis scale 0 to 30,000 RFU). ™ Note: The data in this figure are from the original formulation of the NGM Detect kit. CE injection time The effect of injection times on heterozygous peak heights observed for NGM Detect™ assays was studied on three capillary electrophoresis instruments; the 3500 (8- capillary), 3500xL (24-capillary), and 3130xl (16-capillary). As shown in Figure 14 through Figure 16, increasing or decreasing injection times affected profile peak heights in an approximately linear manner. All genomic DNA samples were amplified for 30 cycles. Our developmental validation studies indicate that the injection conditions that are recorded in Chapter 3 generate profiles from 0.5 ng of input DNA with heterozygous peak height averages between 4,000–10,000 RFU (3500 or 3500xL) or 2,000–4,000 RFU (3130 or 3130xl). This is with no instances of allelic dropout and minimal occurrence of off-scale allele peaks. However, individual CE instrument signal intensities can vary, therefore laboratories are encouraged to optimize injection times. Optimized injection times provide the most efficacious level of assay sensitivity, minimize the 60 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Developmental validation 6 occurrence of off-scale peaks or undesirable artifacts, and do not adversely impact the performance of the size standard and allelic ladder. Figure 14 Box plots show the effect of varying injection times (X-axis) on peak ™ heterozygous heights (Y-axis) observed for NGM Detect assays with 0.5 ng of human male 007 genomic DNA input on a 3500 Genetic Analyzer. ™ Note: The data in this figure are from the original formulation of the NGM Detect kit. NGM Detect™ PCR Amplification Kit User Guide 61 6 Chapter 6 Experiments and results Developmental validation Figure 15 Box plots show the effect of varying injection times (X-axis) on heterozygous ™ peak heights (Y-axis) observed for NGM Detect assays with 0.5 ng of human male 007 genomic DNA input on a 3500xL Genetic Analyzer. ™ Note: The data in this figure are from the original formulation of the NGM Detect kit. Figure 16 Box plots show the effect of varying injection times (X-axis) on heterozygous ™ peak heights (Y-axis) observed for NGM Detect assays with 0.5 ng of human male 007 genomic DNA input on a 3130xl Genetic Analyzer. ™ Note: The data in this figure are from the original formulation of the NGM Detect kit. 62 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 Accuracy, precision, and reproducibility SWGDAM “Precision and accuracy of the assay should be demonstrated: Precision characterizes guideline 3.5 the degree of mutual agreement among a series of individual measurements, values and/or results. Precision depends only on the distribution of random errors and does not relate to the true value or specified value. The measure of precision is usually expressed in terms of imprecision and computed as a standard deviation of the test results. Accuracy is the degree of conformity of a measured quantity to its actual (true) value. Accuracy of a measuring instrument is the ability of a measuring instrument to give responses close to a true value.” (SWGDAM, December 2016) Accuracy Laser-induced fluorescence detection of length polymorphism at short tandem repeat observation loci is not a novel methodology (Holt et al., 2000; Wallin et al., 2002). However, accuracy and reproducibility of profiles have been determined from various sample types. Figure 17, Figure 18, and Figure 19 show the size differences that were observed between sample alleles and allelic ladder alleles on the Applied Biosystems™ 3130 xl, 3500, and 3500xL Genetic Analyzers with POP-4™ Polymer. The X-axis in the following figures represents the nominal nucleotide sizes for the NGM Detect™ Allelic Ladder. The dashed lines parallel to the X-axis represent the ±0.25-nt windows. The y-axis represents the deviation of each sample allele size from the corresponding Allelic Ladder allele size. All sample alleles are within ±0.5 nt from a corresponding allele in the Allelic Ladder, irrespective of the capillary electrophoresis platforms. nd Arrows w to use in this SVG. Figure 17 Allele size vs. Allelic Ladder sizing for 81 samples analyzed on a 3130 xl Genetic ™ Analyzer. Size and ladder sizing for the NGM Detect kit were calculated using the anced callouts or arrows ™ ™ GeneScan –600 LIZ Size Standard v2.0. pe_Callout&Arrow_Libary. Note: Except for the TH01 marker data, the data in this figure are from the original formulation ™ th, as needed. of the NGM Detect kit. le, and unused VG elements he repository. NGM Detect™ PCR Amplification Kit User Guide 63 1 1 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility ws n this SVG. louts or arrows t&Arrow_Libary. Figure 18 Allele size vs. Allelic Ladder sizing for 81 samples analyzed on a 3500 Genetic ™ eded. Analyzer. Size and ladder sizing for the NGM Detect kit were calculated using the ™ ™ GeneScan –600 LIZ Size Standard v2.0. nused Note: Except for the TH01 marker data, the data in this figure are from the original formulation ™ nts of the NGM Detect kit. ory. 1 1 ows in this SVG. allouts or arrows Figure 19 Allele size vs. Allelic Ladder sizing for 81 samples analyzed on a 3500xL Genetic ut&Arrow_Libary. ™ Analyzer. Size and ladder sizing for the NGM Detect kit were calculated using the ™ ™ GeneScan –600 LIZ Size Standard v2.0. eeded. Note: Except for the TH01 marker data, the data in this figure are from the original formulation ™ of the NGM Detect kit. unused ents itory. 1 64 1 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 Precision and size Sizing precision enables the determination of accurate and reliable genotypes. The window recommended method for genotyping is to use a ±0.5-nt “window” around the size obtained for each allele in the allelic ladder. A ±0.5-nt window allows for the detection description and correct assignment of alleles. Any sample allele that sizes outside the specified window could be either: • An “off-ladder” allele, that is, an allele of a size that is not represented in the allelic ladder. • An allele that does correspond to an allele in the allelic ladder, but whose size is just outside a window because of measurement error. The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times. Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument. Precision and size Table 4 lists typical precision results obtained from multiple runs of the NGM Detect™ window Allelic Ladder using the GeneScan™–600 LIZ™ Size Standard v2.0. The results were obtained within a set of injections on a single capillary array. The number of repeated observation injections for each genetic analyzer platform is shown in the following table: CE platform Capillaries # Injections Sizing method 3130xl 16/injection 5 Local Southern, 60– 460 bp 3500 8/injection 12 Local Southern, 60– 460 bp 3500xL 24/injection 4 Local Southern, 60– 460 bp The mean sizes and the standard deviation for the allele sizing were calculated for all the alleles in each run (Table 4). The mean range and the standard deviation range show the lowest and highest values obtained across multiple runs. Sample alleles can occasionally size outside of the ±0.5-nt window for a respective Allelic Ladder allele because of measurement error. The frequency of such an occurrence is lowest in detection systems with the smallest standard deviations in sizing. The figures in “Accuracy observation“ on page 63 illustrate the tight clustering of allele sizes obtained on the Applied Biosystems™ genetic analyzers, where the standard deviation in sizing is typically less than 0.15 nt. The instance of a sample allele sizing outside the ±0.5-nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0.15 nt or less (Smith, 1995). For sample alleles that do not size within a ±0.5-nt window, the PCR product must be rerun to distinguish between a true off–ladder allele versus measurement error of a sample allele that corresponds to an allele in the Allelic Ladder. Repeat analysis, when necessary, provides an added level of confidence in the final allele assignment. GeneMapper™ ID-X Software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele by labeling the allele as OL (off-ladder). Maximum sizing precision is obtained within the same set of capillary injections. Cross–platform sizing differences occur due to several factors including type and concentration of polymer, run temperature, and electrophoresis conditions. Variations NGM Detect™ PCR Amplification Kit User Guide 65 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors. IMPORTANT! To minimize the variation in sizing between runs and to ensure accurate genotyping, follow the guidelines in “Allelic ladder requirements for data analysis“ on page 34 and use allelic ladders obtained from the same run as samples to analyze the samples. For more information on precision and genotyping, see (Lazaruk et al., 1998; Mansfield et al., 1998). Note: The IQCS and IQCL markers were omitted from this study because they are not used for genotyping. Table 4 Precision results of multiple runs of the NGM Detect™ Allelic Ladder Note: Except for the TH01 marker data, the data in this table are from the original formulation of the NGM Detect™ kit. 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation D2S1338 11 90.76– 90.87 0.023– 0.034 91.05– 91.13 0.003– 0.040 90.96– 91.03 0.028– 0.035 12 94.90– 95.00 0.021– 0.031 95.17– 95.24 0.021– 0.041 95.10– 95.16 0.024– 0.034 13 99.01– 99.13 0.023– 0.035 99.31– 99.38 0.001– 0.041 99.22– 99.28 0.027– 0.034 14 103.16– 103.28 0.020– 0.036 103.45– 103.52 0.028– 0.044 103.38– 103.44 0.031– 0.038 15 107.09– 107.19 0.025– 0.037 107.31– 107.38 0.015– 0.058 107.23– 107.28 0.028– 0.037 16 111.29– 111.38 0.028– 0.033 111.55– 111.60 0.005– 0.043 111.45– 111.50 0.025– 0.041 17 115.34– 115.43 0.022– 0.035 115.56– 115.62 0.001– 0.032 115.49– 115.52 0.011– 0.031 18 119.29– 119.36 0.012– 0.033 119.52– 119.57 0.024– 0.042 119.43– 119.47 0.015– 0.040 19 123.27– 123.36 0.020– 0.034 123.50– 123.56 0.004– 0.041 123.42– 123.47 0.034– 0.036 20 127.26– 127.35 0.026– 0.041 127.51– 127.57 0.019– 0.054 127.41– 127.46 0.028– 0.042 21 131.28– 131.39 0.024– 0.039 131.53– 131.62 0.017– 0.051 131.46– 131.51 0.029– 0.039 22 135.34– 135.44 0.023– 0.038 135.56– 135.63 0.011– 0.046 135.50– 135.53 0.027– 0.035 23 139.39– 139.49 0.020– 0.040 139.62– 139.69 0.001– 0.048 139.54– 139.59 0.002– 0.033 24 143.51– 143.61 0.020– 0.040 143.77– 143.82 0.008– 0.043 143.68– 143.74 0.026– 0.042 25 147.63– 147.74 0.017– 0.031 147.89– 147.94 0.016– 0.048 147.79– 147.85 0.024– 0.043 26 151.76– 151.85 0.023– 0.029 152.02– 152.06 0.018– 0.041 151.92– 151.97 0.030– 0.036 27 155.91– 156.01 0.018– 0.031 156.15– 156.20 0.028– 0.043 156.07– 156.11 0.026– 0.040 66 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 28 160.45– 160.49 0.008– 0.033 160.51– 160.57 0.007– 0.042 160.45– 160.47 0.037– 0.040 SE33 4.2 188.38– 188.44 0.024– 0.037 188.22– 188.26 0.018– 0.044 188.17– 188.18 0.034– 0.036 6.3 197.70– 197.76 0.022– 0.038 197.54– 197.6 0.005– 0.045 197.49– 197.50 0.027– 0.038 8 202.76– 202.80 0.022– 0.032 202.58– 202.63 0.011– 0.043 202.53– 202.55 0.022– 0.033 9 206.78– 206.85 0.023– 0.040 206.60– 206.65 0.017– 0.044 206.56– 206.56 0.030– 0.036 11 214.87– 214.92 0.031– 0.036 214.65– 214.69 0.035– 0.051 214.59– 214.62 0.029– 0.038 12 219.05– 219.12 0.021– 0.037 218.83– 218.88 0.033– 0.053 218.78– 218.80 0.035– 0.042 13 223.16– 223.20 0.032– 0.047 222.92– 222.97 0.021– 0.050 222.88– 222.90 0.033– 0.043 14 227.22– 227.29 0.032– 0.046 227.01– 227.06 0.011– 0.054 226.96– 226.98 0.033– 0.049 15 231.26– 231.34 0.028– 0.047 231.06– 231.09 0.018– 0.070 231.01– 231.03 0.037– 0.045 16 235.40– 235.46 0.024– 0.041 235.16– 235.22 0.013– 0.053 235.14– 235.14 0.039– 0.044 17 239.50– 239.56 0.029– 0.045 239.27– 239.32 0.031– 0.048 239.23– 239.24 0.039– 0.045 18 243.65– 243.71 0.020– 0.031 243.38– 243.43 0.004– 0.044 243.35– 243.36 0.027– 0.042 19 247.78– 247.84 0.030– 0.037 247.47– 247.54 0.009– 0.049 247.47– 247.48 0.026– 0.035 20 251.90– 251.95 0.026– 0.036 251.58– 251.65 0.009– 0.049 251.57– 251.59 0.033– 0.037 20.2 253.86– 253.92 0.021– 0.041 253.56– 253.63 0.025– 0.045 253.55– 253.56 0.028– 0.036 21 255.86– 255.93 0.028– 0.038 255.57– 255.63 0.015– 0.048 255.55– 255.57 0.028– 0.040 21.2 257.83– 257.90 0.026– 0.042 257.55– 257.60 0.031– 0.043 257.52– 257.54 0.021– 0.030 22.2 261.81– 261.88 0.019– 0.043 261.53– 261.58 0.005– 0.052 261.51– 261.53 0.030– 0.039 23.2 265.91– 265.98 0.025– 0.047 265.62– 265.68 0.027– 0.055 265.60– 265.62 0.030– 0.036 24.2 269.97– 270.02 0.026– 0.041 269.66– 269.72 0.025– 0.055 269.64– 269.66 0.035– 0.041 25.2 273.95– 274.00 0.025– 0.036 273.65– 273.69 0.034– 0.053 273.62– 273.64 0.031– 0.045 26.2 278.10– 278.16 0.029– 0.043 277.8– 277.84 0.007– 0.058 277.78– 277.80 0.033– 0.038 27.2 282.16– 282.22 0.027– 0.047 281.84– 281.89 0.005– 0.058 281.81– 281.84 0.026– 0.050 28.2 286.14– 286.21 0.022– 0.045 285.83– 285.86 0.023– 0.054 285.80– 285.82 0.027– 0.038 29.2 290.15– 290.22 0.028– 0.046 289.85– 289.89 0.016– 0.052 289.82– 289.83 0.031– 0.036 30.2 294.20– 294.24 0.028– 0.039 293.87– 293.92 0.029– 0.066 293.85– 293.85 0.037– 0.045 31.2 298.16– 298.24 0.028– 0.045 297.84– 297.92 0.001– 0.050 297.82– 297.83 0.032– 0.043 NGM Detect™ PCR Amplification Kit User Guide 67 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 32.2 302.17– 302.23 0.027– 0.041 301.83– 301.90 0.004– 0.051 301.81– 301.83 0.042– 0.045 33.2 306.14– 306.20 0.025– 0.050 305.81– 305.86 0.007– 0.043 305.76– 305.80 0.030– 0.033 34.2 310.16– 310.22 0.033– 0.045 309.81– 309.88 0.007– 0.048 309.78– 309.80 0.034– 0.043 35 312.20– 312.27 0.025– 0.050 311.84– 311.89 0.005– 0.047 311.82– 311.83 0.034– 0.036 35.2 314.24– 314.30 0.027– 0.041 313.87– 313.94 0.001– 0.050 313.83– 313.87 0.037– 0.048 36 316.37– 316.43 0.024– 0.042 316– 316.04 0.010– 0.038 315.97– 315.99 0.024– 0.048 37 320.60– 320.66 0.032– 0.048 320.21– 320.27 0.035– 0.053 320.19– 320.23 0.037– 0.048 38 324.76– 324.83 0.035– 0.054 324.35– 324.41 0.009– 0.064 324.35– 324.38 0.042– 0.048 39 328.85– 328.92 0.027– 0.047 328.45– 328.51 0.029– 0.055 328.45– 328.48 0.029– 0.039 42 341.00– 341.06 0.031– 0.048 340.58– 340.65 0.001– 0.064 340.57– 340.59 0.037– 0.044 D16S539 5 72.32– 72.38 0.032– 0.041 72.00– 72.05 0.009– 0.051 71.93– 71.98 0.024– 0.031 8 84.95– 84.99 0.024– 0.040 84.64– 84.70 0.008– 0.044 84.58– 84.61 0.027– 0.040 9 89.15– 89.18 0.020– 0.034 88.84– 88.9 0.004– 0.045 88.78– 88.82 0.027– 0.041 10 93.30– 93.33 0.022– 0.036 92.99– 93.05 0.004– 0.040 92.94– 92.97 0.028– 0.040 11 97.45– 97.49 0.020– 0.039 97.15– 97.22 0.007– 0.045 97.11– 97.14 0.025– 0.035 12 101.63– 101.67 0.026– 0.033 101.33– 101.39 0.030– 0.046 101.28– 101.33 0.034– 0.039 13 105.83– 105.86 0.021– 0.043 105.53– 105.59 0.009– 0.047 105.47– 105.52 0.038– 0.039 14 109.97– 110.01 0.027– 0.042 109.66– 109.74 0.032– 0.048 109.63– 109.67 0.030– 0.043 15 114.08– 114.11 0.024– 0.036 113.77– 113.85 0.028– 0.051 113.72– 113.78 0.027– 0.038 D18S51 7 127.11– 127.18 0.030– 0.038 127.52– 127.58 0.014– 0.046 127.44– 127.47 0.028– 0.042 9 134.69– 134.79 0.022– 0.047 135.21– 135.27 0.010– 0.051 135.12– 135.17 0.028– 0.045 10 138.52– 138.63 0.025– 0.051 139.07– 139.15 0.001– 0.041 139.00– 139.06 0.037– 0.042 10.2 140.66– 140.77 0.028– 0.054 141.21– 141.30 0.001– 0.042 141.15– 141.21 0.035– 0.043 11 142.41– 142.53 0.023– 0.047 143.00– 143.10 0.008– 0.052 142.93– 142.99 0.020– 0.038 12 146.33– 146.45 0.023– 0.050 146.95– 147.03 0.020– 0.044 146.88– 146.95 0.029– 0.038 13 150.27– 150.39 0.027– 0.042 150.90– 151.01 0.024– 0.045 150.83– 150.91 0.031– 0.038 13.2 152.41– 152.53 0.027– 0.050 153.05– 153.16 0.013– 0.046 153.00– 153.07 0.033– 0.044 68 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 14 154.21– 154.33 0.030– 0.048 154.86– 154.96 0.024– 0.046 154.80– 154.87 0.032– 0.036 14.2 156.35– 156.48 0.021– 0.040 157.02– 157.14 0.004– 0.047 156.96– 157.03 0.035– 0.043 15 158.15– 158.27 0.032– 0.051 158.83– 158.94 0.001– 0.043 158.76– 158.83 0.040– 0.045 16 162.05– 162.18 0.027– 0.049 162.76– 162.86 0.004– 0.043 162.69– 162.77 0.030– 0.037 17 165.92– 166.07 0.027– 0.054 166.64– 166.77 0.003– 0.039 166.58– 166.66 0.029– 0.046 18 169.80– 169.93 0.034– 0.049 170.54– 170.66 0.016– 0.044 170.49– 170.57 0.030– 0.039 19 173.66– 173.81 0.031– 0.049 174.44– 174.55 0.004– 0.044 174.39– 174.47 0.029– 0.045 20 177.54– 177.69 0.035– 0.051 178.34– 178.48 0.003– 0.047 178.28– 178.36 0.032– 0.040 21 181.47– 181.63 0.039– 0.049 182.30– 182.45 0.006– 0.044 182.26– 182.35 0.035– 0.044 22 185.33– 185.52 0.035– 0.047 186.21– 186.37 0.013– 0.042 186.17– 186.26 0.033– 0.046 23 189.25– 189.44 0.030– 0.067 190.17– 190.32 0.022– 0.046 190.12– 190.22 0.032– 0.042 24 193.19– 193.37 0.032– 0.054 194.13– 194.26 0.021– 0.050 194.07– 194.17 0.029– 0.055 25 197.14– 197.32 0.030– 0.064 198.11– 198.25 0.005– 0.048 198.05– 198.15 0.033– 0.047 26 200.98– 201.15 0.038– 0.065 201.92– 202.07 0.024– 0.048 201.88– 201.98 0.034– 0.043 27 204.81– 204.98 0.035– 0.067 205.76– 205.91 0.020– 0.040 205.72– 205.83 0.038– 0.046 TH01 4 222.50– 222.65 0.035– 0.073 223.26– 223.39 0.004– 0.049 223.19– 223.31 0.035– 0.041 5 226.48– 226.64 0.029– 0.076 227.24– 227.39 0.011– 0.060 227.19– 227.32 0.031– 0.040 6 230.47– 230.65 0.034– 0.057 231.25– 231.39 0.029– 0.049 231.19– 231.31 0.027– 0.044 7 234.46– 234.65 0.028– 0.059 235.27– 235.39 0.008– 0.048 235.18– 235.31 0.033– 0.041 8 238.47– 238.65 0.033– 0.068 239.25– 239.40 0.005– 0.050 239.19– 239.32 0.029– 0.043 9 242.55– 242.71 0.028– 0.065 243.34– 243.47 0.027– 0.053 243.26– 243.38 0.025– 0.040 9.3 245.61– 245.78 0.035– 0.058 246.38– 246.51 0.005– 0.049 246.31– 246.43 0.034– 0.047 10 246.63– 246.81 0.033– 0.074 247.40– 247.54 0.024– 0.050 247.33– 247.45 0.031– 0.041 11 250.70– 250.86 0.036– 0.066 251.43– 251.54 0.003– 0.046 251.36– 251.47 0.034– 0.043 12 254.62– 254.79 0.031– 0.069 255.36– 255.48 0.016– 0.046 255.29– 255.40 0.030– 0.044 13 258.53– 258.71 0.036– 0.063 259.28– 259.39 0.003– 0.045 259.19– 259.31 0.033– 0.040 13.3 261.46– 261.65 0.042– 0.065 262.21– 262.34 0.005– 0.038 262.15– 262.27 0.027– 0.042 NGM Detect™ PCR Amplification Kit User Guide 69 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation D12S391 14 281.70– 281.76 0.022– 0.037 281.71– 281.75 0.039– 0.054 281.68– 281.70 0.033– 0.041 15 285.72– 285.76 0.026– 0.033 285.73– 285.75 0.010– 0.048 285.69– 285.71 0.030– 0.043 16 289.68– 289.72 0.023– 0.040 289.68– 289.71 0.001– 0.049 289.65– 289.67 0.030– 0.038 17 293.71– 293.77 0.018– 0.037 293.71– 293.76 0.001– 0.046 293.68– 293.69 0.027– 0.041 18 297.70– 297.75 0.026– 0.040 297.7– 297.74 0.001– 0.050 297.67– 297.69 0.036– 0.040 19 301.69– 301.74 0.020– 0.042 301.70– 301.75 0.024– 0.048 301.65– 301.67 0.032– 0.040 19.3 304.64– 304.68 0.021– 0.033 304.62– 304.67 0.011– 0.047 304.58– 304.61 0.031– 0.037 20 305.62– 305.66 0.025– 0.037 305.61– 305.65 0.007– 0.048 305.56– 305.59 0.030– 0.039 21 309.65– 309.70 0.022– 0.043 309.65– 309.71 0.012– 0.054 309.60– 309.64 0.036– 0.044 22 313.76– 313.81 0.028– 0.041 313.77– 313.79 0.031– 0.050 313.71– 313.74 0.034– 0.040 23 318.00– 318.04 0.022– 0.037 317.97– 318.00 0.032– 0.051 317.92– 317.96 0.026– 0.039 24 322.19– 322.22 0.026– 0.046 322.13– 322.18 0.004– 0.056 322.11– 322.13 0.016– 0.047 25 326.3– 326.33 0.022– 0.041 326.24– 326.28 0.011– 0.070 326.21– 326.23 0.034– 0.052 26 330.39– 330.42 0.027– 0.033 330.34– 330.38 0.029– 0.067 330.31– 330.34 0.037– 0.048 27 334.42– 334.46 0.025– 0.039 334.38– 334.40 0.016– 0.060 334.34– 334.38 0.027– 0.042 D3S1358 9 112.46– 112.49 0.019– 0.047 112.54– 112.59 0.038– 0.046 112.50– 112.53 0.017– 0.035 10 116.39– 116.42 0.021– 0.027 116.47– 116.51 0.001– 0.036 116.43– 116.45 0.024– 0.032 11 120.26– 120.29 0.018– 0.035 120.33– 120.37 0.028– 0.044 120.29– 120.30 0.022– 0.037 12 124.05– 124.08 0.023– 0.034 124.11– 124.15 0.007– 0.049 124.08– 124.09 0.023– 0.044 13 128.11– 128.15 0.024– 0.038 128.17– 128.22 0.025– 0.049 128.14– 128.15 0.031– 0.037 14 132.04– 132.09 0.016– 0.035 132.12– 132.16 0.026– 0.050 132.09– 132.10 0.029– 0.038 15 135.93– 135.96 0.024– 0.034 136.01– 136.04 0.006– 0.037 135.98– 135.98 0.030– 0.035 16 140.07– 140.11 0.017– 0.035 140.16– 140.20 0.001– 0.046 140.13– 140.13 0.039– 0.043 17 144.27– 144.29 0.020– 0.036 144.33– 144.37 0.034– 0.052 144.31– 144.32 0.027– 0.037 18 148.33– 148.36 0.017– 0.031 148.4– 148.44 0.029– 0.049 148.38– 148.38 0.028– 0.033 19 152.35– 152.37 0.019– 0.030 152.40– 152.45 0.027– 0.045 152.38– 152.39 0.032– 0.039 20 156.66– 156.70 0.023– 0.037 156.70– 156.76 0.008– 0.038 156.69– 156.71 0.020– 0.037 70 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation FGA 13 165.50– 165.58 0.029– 0.039 165.95– 166.04 0.015– 0.047 165.93– 165.97 0.023– 0.040 14 169.35– 169.45 0.030– 0.036 169.84– 169.91 0.003– 0.043 169.81– 169.86 0.030– 0.035 15 173.21– 173.31 0.031– 0.039 173.72– 173.83 0.009– 0.043 173.70– 173.74 0.028– 0.038 16 177.06– 177.17 0.032– 0.041 177.59– 177.71 0.004– 0.047 177.58– 177.63 0.031– 0.041 17 180.94– 181.05 0.026– 0.042 181.51– 181.61 0.005– 0.047 181.48– 181.55 0.027– 0.047 18 184.85– 184.97 0.032– 0.045 185.45– 185.56 0.013– 0.051 185.43– 185.50 0.030– 0.039 19 188.76– 188.89 0.024– 0.040 189.38– 189.51 0.021– 0.039 189.38– 189.45 0.031– 0.042 20 192.67– 192.80 0.032– 0.042 193.34– 193.44 0.010– 0.049 193.31– 193.39 0.028– 0.049 21 196.56– 196.70 0.030– 0.050 197.23– 197.38 0.004– 0.046 197.24– 197.32 0.035– 0.043 22 200.46– 200.59 0.034– 0.054 201.15– 201.27 0.027– 0.054 201.13– 201.22 0.026– 0.043 23 204.28– 204.39 0.033– 0.049 204.97– 205.11 0.020– 0.044 204.96– 205.05 0.031– 0.050 24 208.09– 208.22 0.037– 0.048 208.83– 208.93 0.011– 0.056 208.81– 208.89 0.035– 0.048 25 211.94– 212.05 0.027– 0.048 212.67– 212.82 0.004– 0.048 212.67– 212.74 0.039– 0.050 26 215.83– 215.95 0.029– 0.061 216.58– 216.74 0.023– 0.041 216.59– 216.69 0.037– 0.043 27 219.83– 219.97 0.034– 0.059 220.62– 220.79 0.001– 0.050 220.64– 220.73 0.036– 0.050 28 223.71– 223.86 0.035– 0.058 224.55– 224.68 0.011– 0.053 224.54– 224.64 0.037– 0.040 29 227.55– 227.70 0.034– 0.063 228.41– 228.58 0.016– 0.061 228.43– 228.53 0.030– 0.056 30 231.34– 231.50 0.034– 0.056 232.23– 232.40 0.015– 0.057 232.25– 232.36 0.035– 0.056 30.2 233.71– 233.86 0.030– 0.054 234.60– 234.76 0.014– 0.050 234.62– 234.73 0.039– 0.052 31.2 237.59– 237.74 0.029– 0.062 238.50– 238.68 0.003– 0.050 238.52– 238.64 0.040– 0.051 32.2 241.50– 241.66 0.038– 0.053 242.46– 242.62 0.034– 0.050 242.47– 242.59 0.038– 0.047 33.2 245.48– 245.64 0.035– 0.049 246.43– 246.61 0.022– 0.053 246.47– 246.56 0.041– 0.052 42.2 280.49– 280.68 0.034– 0.067 281.59– 281.80 0.001– 0.048 281.61– 281.75 0.038– 0.052 43.2 284.32– 284.52 0.041– 0.073 285.46– 285.67 0.016– 0.047 285.47– 285.62 0.032– 0.052 44.2 288.33– 288.51 0.041– 0.070 289.46– 289.66 0.016– 0.053 289.48– 289.62 0.033– 0.055 45.2 292.23– 292.41 0.040– 0.070 293.35– 293.57 0.001– 0.055 293.38– 293.52 0.033– 0.048 46.2 295.73– 295.93 0.037– 0.065 296.95– 297.17 0.004– 0.051 296.98– 297.12 0.038– 0.059 47.2 299.50– 299.7 0.037– 0.073 300.72– 300.95 0.007– 0.059 300.76– 300.90 0.036– 0.065 NGM Detect™ PCR Amplification Kit User Guide 71 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 48.2 303.40– 303.60 0.034– 0.075 304.64– 304.87 0.018– 0.053 304.67– 304.82 0.040– 0.055 50.2 311.14– 311.37 0.048– 0.081 312.47– 312.69 0.007– 0.063 312.46– 312.64 0.043– 0.057 51.2 314.87– 315.12 0.052– 0.083 316.30– 316.56 0.021– 0.066 316.34– 316.51 0.039– 0.055 Yindel 1 90.97– 91.00 0.021– 0.031 91.04– 91.10 0.028– 0.044 90.97– 90.98 0.030– 0.033 2 96.09– 96.12 0.022– 0.033 96.18– 96.22 0.001– 0.035 96.10– 96.11 0.022– 0.035 AMEL X 108.18– 108.22 0.024– 0.039 108.08– 108.16 0.007– 0.046 108.05– 108.09 0.030– 0.035 Y 113.96– 114.01 0.001– 0.035 113.94– 113.99 0.028– 0.042 113.91– 113.94 0.025– 0.035 vWA 11 125.73– 125.77 0.017– 0.040 125.79– 125.85 0.015– 0.046 125.74– 125.78 0.024– 0.038 12 129.63– 129.66 0.021– 0.036 129.72– 129.76 0.026– 0.046 129.67– 129.69 0.026– 0.036 13 133.62– 133.65 0.023– 0.039 133.74– 133.75 0.017– 0.050 133.67– 133.70 0.031– 0.036 14 137.82– 137.86 0.021– 0.035 137.96– 137.99 0.004– 0.045 137.92– 137.93 0.025– 0.040 15 141.72– 141.75 0.024– 0.036 141.87– 141.90 0.005– 0.045 141.81– 141.83 0.025– 0.030 16 145.81– 145.83 0.022– 0.035 145.96– 146.01 0.026– 0.043 145.92– 145.93 0.027– 0.036 17 149.88– 149.92 0.024– 0.035 150.05– 150.10 0.028– 0.042 150.02– 150.03 0.034– 0.040 18 153.94– 153.97 0.027– 0.035 154.12– 154.16 0.021– 0.046 154.07– 154.08 0.028– 0.040 19 158.03– 158.07 0.023– 0.040 158.23– 158.27 0.004– 0.046 158.18– 158.20 0.031– 0.038 20 162.08– 162.13 0.022– 0.048 162.29– 162.32 0.024– 0.045 162.24– 162.27 0.023– 0.037 21 166.07– 166.11 0.024– 0.033 166.26– 166.33 0.026– 0.045 166.24– 166.25 0.022– 0.038 22 170.08– 170.11 0.016– 0.036 170.29– 170.34 0.020– 0.041 170.25– 170.27 0.032– 0.035 23 173.97– 174.03 0.020– 0.032 174.20– 174.26 0.010– 0.047 174.17– 174.18 0.031– 0.039 24 178.37– 178.42 0.029– 0.037 178.59– 178.64 0.003– 0.046 178.56– 178.57 0.029– 0.038 D21S11 24 197.75– 197.85 0.024– 0.039 198.20– 198.26 0.032– 0.046 198.16– 198.20 0.034– 0.045 24.2 199.77– 199.87 0.033– 0.040 200.23– 200.30 0.001– 0.046 200.19– 200.24 0.023– 0.042 25 201.76– 201.85 0.023– 0.045 202.20– 202.25 0.031– 0.043 202.16– 202.2 0.027– 0.035 26 205.74– 205.81 0.020– 0.046 206.16– 206.22 0.024– 0.050 206.12– 206.16 0.031– 0.044 72 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 27 209.74– 209.81 0.025– 0.043 210.16– 210.23 0.031– 0.047 210.12– 210.17 0.037– 0.046 28 213.69– 213.75 0.025– 0.037 214.11– 214.16 0.031– 0.048 214.08– 214.11 0.025– 0.042 28.2 215.69– 215.75 0.029– 0.044 216.11– 216.19 0.025– 0.046 216.09– 216.13 0.028– 0.040 29 217.69– 217.77 0.024– 0.032 218.13– 218.22 0.025– 0.045 218.13– 218.16 0.029– 0.042 29.2 219.81– 219.88 0.027– 0.045 220.23– 220.31 0.001– 0.049 220.22– 220.26 0.030– 0.041 30 221.77– 221.86 0.024– 0.037 222.23– 222.3 0.005– 0.043 222.21– 222.25 0.032– 0.047 30.2 223.77– 223.84 0.027– 0.045 224.20– 224.29 0.008– 0.056 224.20– 224.24 0.034– 0.045 31 225.78– 225.86 0.027– 0.049 226.26– 226.32 0.021– 0.059 226.24– 226.28 0.038– 0.043 31.2 227.76– 227.85 0.030– 0.041 228.23– 228.32 0.011– 0.054 228.21– 228.28 0.037– 0.039 32 229.78– 229.86 0.033– 0.047 230.28– 230.35 0.022– 0.056 230.26– 230.31 0.038– 0.043 32.2 231.76– 231.86 0.030– 0.049 232.27– 232.34 0.019– 0.055 232.25– 232.29 0.027– 0.042 33 233.79– 233.89 0.027– 0.043 234.28– 234.38 0.009– 0.048 234.28– 234.33 0.036– 0.038 33.2 235.74– 235.83 0.031– 0.043 236.27– 236.33 0.011– 0.046 236.23– 236.29 0.037– 0.040 34 237.87– 237.98 0.023– 0.040 238.38– 238.47 0.007– 0.048 238.38– 238.42 0.017– 0.043 34.2 239.80– 239.89 0.036– 0.041 240.33– 240.40 0.001– 0.048 240.30– 240.37 0.033– 0.044 35 241.92– 242.02 0.029– 0.044 242.46– 242.54 0.030– 0.052 242.43– 242.49 0.036– 0.041 35.2 243.92– 244.00 0.029– 0.038 244.43– 244.51 0.024– 0.053 244.41– 244.47 0.028– 0.041 36 245.94– 246.05 0.025– 0.037 246.46– 246.55 0.004– 0.048 246.46– 246.49 0.033– 0.040 37 250.09– 250.18 0.032– 0.039 250.59– 250.67 0.001– 0.046 250.58– 250.63 0.028– 0.040 38 253.95– 254.05 0.024– 0.044 254.46– 254.53 0.018– 0.048 254.45– 254.51 0.028– 0.043 D1S1656 9 267.24– 267.33 0.029– 0.037 267.69– 267.75 0.019– 0.049 267.64– 267.68 0.035– 0.040 10 271.29– 271.37 0.027– 0.034 271.71– 271.79 0.021– 0.047 271.67– 271.72 0.035– 0.037 11 275.31– 275.40 0.026– 0.041 275.76– 275.82 0.010– 0.048 275.70– 275.76 0.031– 0.040 12 279.35– 279.42 0.021– 0.039 279.76– 279.83 0.001– 0.049 279.71– 279.76 0.035– 0.046 13 283.38– 283.45 0.017– 0.043 283.80– 283.86 0.001– 0.051 283.73– 283.79 0.031– 0.039 14 287.35– 287.40 0.022– 0.035 287.75– 287.82 0.005– 0.037 287.70– 287.74 0.030– 0.041 14.3 290.42– 290.48 0.027– 0.040 290.80– 290.86 0.013– 0.055 290.75– 290.81 0.038– 0.047 15 291.31– 291.37 0.018– 0.037 291.72– 291.79 0.005– 0.050 291.67– 291.72 0.031– 0.040 NGM Detect™ PCR Amplification Kit User Guide 73 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 15.3 294.41– 294.45 0.025– 0.037 294.80– 294.85 0.010– 0.050 294.74– 294.78 0.035– 0.047 16 295.31– 295.38 0.020– 0.038 295.75– 295.80 0.008– 0.048 295.66– 295.72 0.033– 0.039 16.3 298.41– 298.45 0.029– 0.036 298.78– 298.84 0.031– 0.049 298.71– 298.76 0.038– 0.045 17 299.24– 299.29 0.022– 0.043 299.64– 299.72 0.001– 0.052 299.56– 299.61 0.018– 0.044 17.3 302.34– 302.39 0.028– 0.040 302.71– 302.77 0.023– 0.052 302.64– 302.69 0.023– 0.042 18 303.19– 303.24 0.021– 0.037 303.62– 303.65 0.007– 0.047 303.53– 303.58 0.030– 0.036 18.3 306.24– 306.32 0.024– 0.040 306.66– 306.70 0.018– 0.048 306.57– 306.64 0.026– 0.048 19.3 310.25– 310.32 0.026– 0.044 310.65– 310.70 0.007– 0.057 310.57– 310.63 0.029– 0.036 20.3 314.22– 314.30 0.008– 0.041 314.65– 314.71 0.001– 0.048 314.56– 314.62 0.018– 0.045 D2S441 8 334.96– 335.09 0.033– 0.047 335.78– 335.91 0.031– 0.050 335.70– 335.84 0.027– 0.045 9 338.84– 338.99 0.035– 0.049 339.68– 339.78 0.031– 0.053 339.57– 339.71 0.018– 0.053 10 342.87– 343.01 0.029– 0.049 343.71– 343.87 0.032– 0.054 343.64– 343.78 0.035– 0.047 11 346.91– 347.05 0.035– 0.046 347.80– 347.91 0.016– 0.056 347.68– 347.82 0.030– 0.049 11.3 349.92– 350.06 0.032– 0.061 350.83– 350.96 0.016– 0.049 350.72– 350.87 0.026– 0.054 12 351.00– 351.15 0.026– 0.054 351.91– 352.03 0.021– 0.055 351.80– 351.94 0.035– 0.053 13 354.97– 355.12 0.032– 0.051 355.88– 356.01 0.010– 0.050 355.78– 355.94 0.027– 0.047 14 358.95– 359.10 0.036– 0.056 359.88– 360 0.001– 0.053 359.78– 359.90 0.037– 0.048 15 362.94– 363.09 0.040– 0.058 363.86– 363.99 0.010– 0.051 363.77– 363.92 0.040– 0.044 16 366.93– 367.07 0.037– 0.049 367.83– 367.97 0.024– 0.051 367.77– 367.91 0.033– 0.039 17 370.94– 371.08 0.030– 0.051 371.86– 372.01 0.035– 0.049 371.79– 371.94 0.032– 0.043 D8S1179 5 95.58– 95.63 0.025– 0.030 95.79– 95.84 0.027– 0.045 95.73– 95.76 0.025– 0.035 6 99.66– 99.71 0.016– 0.032 99.88– 99.92 0.001– 0.042 99.83– 99.84 0.016– 0.034 7 103.81– 103.86 0.023– 0.033 104.04– 104.09 0.010– 0.047 103.98– 104.01 0.028– 0.036 8 107.91– 107.96 0.023– 0.037 108.15– 108.19 0.007– 0.044 108.09– 108.10 0.033– 0.042 9 111.97– 112.02 0.017– 0.034 112.21– 112.26 0.007– 0.045 112.17– 112.18 0.025– 0.035 10 115.96– 116.00 0.017– 0.033 116.19– 116.24 0.001– 0.039 116.15– 116.16 0.019– 0.026 11 119.87– 119.91 0.024– 0.031 120.12– 120.16 0.001– 0.051 120.07– 120.08 0.014– 0.024 74 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Accuracy, precision, and reproducibility 6 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation 12 123.91– 123.96 0.027– 0.033 124.17– 124.22 0.007– 0.052 124.13– 124.15 0.026– 0.040 13 127.72– 127.79 0.022– 0.038 128.01– 128.06 0.021– 0.043 127.96– 127.98 0.030– 0.041 14 131.64– 131.70 0.021– 0.038 131.94– 132.00 0.019– 0.050 131.91– 131.93 0.031– 0.041 15 135.62– 135.69 0.024– 0.040 135.95– 136.00 0.033– 0.046 135.90– 135.93 0.023– 0.036 16 139.62– 139.69 0.031– 0.037 139.95– 140.01 0.001– 0.041 139.92– 139.94 0.023– 0.038 17 143.71– 143.78 0.018– 0.043 144.08– 144.14 0.010– 0.044 144.04– 144.08 0.029– 0.040 18 147.81– 147.88 0.028– 0.039 148.19– 148.24 0.020– 0.042 148.14– 148.18 0.033– 0.039 19 151.93– 152.01 0.024– 0.038 152.31– 152.37 0.018– 0.048 152.27– 152.30 0.031– 0.042 D19S433 6 172.80– 172.89 0.025– 0.039 173.29– 173.39 0.013– 0.043 173.28– 173.33 0.028– 0.034 7 176.77– 176.85 0.030– 0.038 177.25– 177.35 0.003– 0.047 177.25– 177.30 0.035– 0.042 8 180.72– 180.82 0.028– 0.042 181.25– 181.35 0.004– 0.047 181.25– 181.30 0.023– 0.040 9 184.79– 184.92 0.019– 0.043 185.37– 185.47 0.013– 0.045 185.37– 185.44 0.031– 0.040 10 188.80– 188.92 0.028– 0.043 189.42– 189.51 0.021– 0.042 189.40– 189.47 0.035– 0.042 11 192.70– 192.81 0.023– 0.047 193.33– 193.42 0.010– 0.053 193.31– 193.39 0.028– 0.045 12 196.67– 196.79 0.033– 0.044 197.32– 197.44 0.007– 0.045 197.32– 197.40 0.029– 0.046 12.2 198.81– 198.94 0.025– 0.046 199.44– 199.56 0.001– 0.046 199.46– 199.53 0.038– 0.048 13 200.64– 200.75 0.035– 0.043 201.28– 201.40 0.005– 0.043 201.28– 201.36 0.028– 0.048 13.2 202.61– 202.71 0.030– 0.045 203.26– 203.36 0.010– 0.045 203.26– 203.33 0.027– 0.043 14 204.53– 204.64 0.033– 0.045 205.19– 205.29 0.016– 0.052 205.19– 205.26 0.037– 0.040 14.2 206.51– 206.62 0.023– 0.047 207.17– 207.27 0.019– 0.052 207.16– 207.23 0.029– 0.043 15 208.44– 208.54 0.031– 0.048 209.11– 209.21 0.008– 0.050 209.10– 209.18 0.028– 0.046 15.2 210.41– 210.50 0.029– 0.050 211.08– 211.19 0.009– 0.050 211.09– 211.16 0.020– 0.044 16 212.36– 212.45 0.027– 0.051 213.03– 213.13 0.003– 0.048 213.03– 213.11 0.037– 0.039 16.2 214.34– 214.43 0.032– 0.047 215.03– 215.14 0.031– 0.048 215.03– 215.11 0.032– 0.045 17 216.42– 216.52 0.033– 0.048 217.12– 217.25 0.023– 0.046 217.15– 217.23 0.029– 0.044 17.2 218.34– 218.44 0.027– 0.049 219.05– 219.19 0.003– 0.062 219.07– 219.15 0.035– 0.048 18.2 222.38– 222.48 0.031– 0.044 223.12– 223.25 0.017– 0.054 223.14– 223.22 0.036– 0.055 19.2 226.31– 226.42 0.036– 0.057 227.06– 227.22 0.015– 0.061 227.11– 227.19 0.034– 0.052 NGM Detect™ PCR Amplification Kit User Guide 75 6 Chapter 6 Experiments and results Accuracy, precision, and reproducibility 3130xl 3500 3500xL Allele Standard Standard Standard Mean Mean Mean deviation deviation deviation D22S1045 7 245.00– 245.10 0.024– 0.044 245.61– 245.70 0.003– 0.051 245.59– 245.66 0.031– 0.041 8 248.05– 248.14 0.026– 0.041 248.65– 248.74 0.007– 0.053 248.63– 248.69 0.039– 0.045 9 251.04– 251.15 0.025– 0.042 251.63– 251.71 0.035– 0.046 251.61– 251.67 0.022– 0.034 10 253.99– 254.08 0.021– 0.035 254.56– 254.66 0.014– 0.044 254.55– 254.61 0.032– 0.040 11 256.91– 257.00 0.031– 0.047 257.50– 257.58 0.001– 0.043 257.47– 257.53 0.030– 0.035 12 259.81– 259.90 0.034– 0.046 260.43– 260.52 0.031– 0.050 260.40– 260.47 0.039– 0.044 13 262.79– 262.89 0.031– 0.057 263.39– 263.51 0.026– 0.046 263.38– 263.45 0.031– 0.039 14 265.76– 265.87 0.031– 0.059 266.41– 266.49 0.013– 0.040 266.36– 266.44 0.030– 0.049 15 268.74– 268.85 0.032– 0.044 269.36– 269.47 0.020– 0.059 269.35– 269.42 0.027– 0.034 16 271.74– 271.85 0.029– 0.040 272.36– 272.46 0.030– 0.046 272.33– 272.41 0.032– 0.037 17 274.73– 274.83 0.028– 0.048 275.35– 275.45 0.012– 0.056 275.33– 275.40 0.033– 0.042 18 277.73– 277.83 0.026– 0.043 278.36– 278.47 0.030– 0.050 278.33– 278.41 0.034– 0.043 19 280.71– 280.81 0.015– 0.049 281.34– 281.45 0.029– 0.049 281.31– 281.39 0.033– 0.042 20 283.70– 283.78 0.027– 0.037 284.32– 284.42 0.001– 0.048 284.28– 284.37 0.026– 0.048 D10S1248 8 303.34– 303.37 0.021– 0.039 303.46– 303.49 0.012– 0.049 303.42– 303.45 0.031– 0.040 9 307.25– 307.29 0.021– 0.036 307.39– 307.43 0.010– 0.049 307.33– 307.37 0.031– 0.042 10 311.24– 311.27 0.027– 0.039 311.37– 311.40 0.005– 0.046 311.30– 311.35 0.030– 0.045 11 315.30– 315.33 0.024– 0.042 315.42– 315.46 0.024– 0.050 315.38– 315.41 0.034– 0.048 12 319.51– 319.53 0.022– 0.040 319.60– 319.64 0.035– 0.053 319.58– 319.6 0.019– 0.044 13 323.62– 323.63 0.021– 0.042 323.67– 323.72 0.007– 0.064 323.65– 323.69 0.030– 0.045 14 327.67– 327.68 0.028– 0.039 327.73– 327.76 0.016– 0.053 327.70– 327.73 0.033– 0.042 15 331.70– 331.71 0.024– 0.037 331.74– 331.80 0.023– 0.048 331.72– 331.76 0.030– 0.043 16 335.70– 335.72 0.022– 0.039 335.76– 335.79 0.012– 0.049 335.74– 335.76 0.030– 0.046 17 339.68– 339.69 0.019– 0.032 339.73– 339.78 0.041– 0.053 339.71– 339.73 0.031– 0.038 18 343.71– 343.74 0.023– 0.038 343.76– 343.8 0.011– 0.048 343.74– 343.77 0.030– 0.036 19 347.76– 347.78 0.012– 0.035 347.80– 347.85 0.014– 0.049 347.78– 347.81 0.032– 0.038 76 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Extra peaks in the electropherogram 6 Extra peaks in the electropherogram Causes of extra Peaks other than the target alleles may be detected on the electropherogram. Causes peaks for the appearance of extra peaks include stutter products, incomplete 3´ A nucleotide addition (at the n-1 position), dye artifacts, and mixed DNA samples (see DNA Advisory Board (DAB) Standard 8.1.2.2). Extra peaks: Stutter definition Stutter Stutter is a well-characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller than the target STR allele product (minus stutter), or less frequently, one repeat larger (plus stutter) (Butler, 2005; Mulero et al., 2006). Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the minus stutter product is missing a single tetranucleotide core repeat unit relative to the main allele (Walsh et al., 1996). Although plus-stutter is normally much less significant than minus-stutter in STR loci with tetranucleotide repeats, the incidence of plus-stutter may be more significant in trinucleotide repeat-containing loci. Contact HID Support for more information on plus stutter. The proportion of the stutter product relative to the main allele (percent stutter) is measured by dividing the height of the stutter peak by the height of the main allele peak. Stutter observations Plus-stutter was regularly observed and was more significant in trinucleotide repeat- containing loci (Figure 25 on page 83). Peak heights were measured for amplified samples at the loci that are used in the kit. All data were generated on the 3500xL Genetic Analyzer. Some conclusions from these measurements and observations are: • For each locus, the stutter percentage generally increases with allele length. • Each allele within a locus displays a relatively consistent average stutter percentage. • Peaks in the stutter position that are above the stutter filter percentage specified in the software are not filtered. • The measurement of stutter percentage for allele peaks that are off-scale may be unusually high due to artificial truncation of the main allele peak. • Stutter can be elevated when minus stutter and plus stutter overlap. This is typically observed when a given allele flanks another allele that is 2 repeat units away (as seen with the FGA locus in control 007 DNA). • The magnitude and/or variability of stutter may increase with low DNA input amounts. Figure 20 through Figure 24 show the stutter observed in the population study that are one repeat unit away from the alleles recorded. All data were generated on the 3500xL Genetic Analyzer. NGM Detect™ PCR Amplification Kit User Guide 77 6 Chapter 6 Experiments and results Extra peaks in the electropherogram The stutter filter settings that are derived from this data are listed in “Stutter percentage filter settings provided with GeneMapper™ ID-X Software“ on page 83. Figure 20 Stutter percentages for D1S1656, D2S441, D2S1338, and D3S1358 loci (Blue=FAM™ dye, black=TED™ dye, red=TAZ™ dye) Note: The data in this figure are from the original formulation of the NGM Detect™ kit. 78 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Extra peaks in the electropherogram 6 Figure 21 Stutter percentages for D8S1179, D10S1248, D12S391, and D16S539 (Green=VIC™ dye and purple=SID™ dye) Note: The data in this figure are from the original formulation of the NGM Detect™ kit. NGM Detect™ PCR Amplification Kit User Guide 79 6 Chapter 6 Experiments and results Extra peaks in the electropherogram Figure 22 Stutter percentages for D18S51, D19S433, and D21S11 loci (Green=VIC™ dye, red=TAZ™ dye, purple=SID™ dye) Note: The data in this figure are from the original formulation of the NGM Detect™ kit. 80 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Extra peaks in the electropherogram 6 d Arrows to use in this SVG. nced callouts or arrows _Callout&Arrow_Libary. h, as needed. , and unused Figure 23 Stutter percentages for D22S1045, TH01, and FGA loci (Purple=SID™ dye, elements Green=VIC™ dye, black=TED™ dye) repository. Note: Except for the TH01 marker data, the data in this figure are from the original formulation of the NGM Detect™ kit. 1 1 NGM Detect™ PCR Amplification Kit User Guide 81 6 Chapter 6 Experiments and results Extra peaks in the electropherogram Figure 24 Stutter percentages for SE33 and vWA loci (Blue=FAM™ dye, red=TAZ™ dye) Note: The data in this figure are from the original formulation of the NGM Detect™ kit. 82 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Extra peaks in the electropherogram 6 Non-standard stutter peaks at the D22S1045 and SE33 loci The D22S1045 locus in the NGM Detect™ kit is a trinucleotide repeat locus, and shows an elevated level of plus stutter (Figure 25). Other loci, such as THO1 and D2S1338, may also exhibit relatively elevated plus stutter. Figure 25 NGM Detect™ kit electropherogram showing minus and plus stutter associated with the D22S1045 STR locus. Data produced on a 3500xL Genetic Analyzer. Note: The data in this figure are from the original formulation of the NGM Detect™ kit. STR loci such as D1S1656 and SE33 (Figure 26) contain more complex nucleotide sequences including regions of dinucleotide repeats which can yield additional stutter peaks. If these stutter peaks exceed the peak amplitude threshold (typically 175 RFU), they may be detected as additional alleles in the profile. The stutter file that is provided with the GeneMapper™ ID-X Software for analysis of NGM Detect™ kit data contains a minus 2−nt stutter filter for SE33 and D1S1656, as well as filters for commonly observed plus stutter, to prevent these peaks from being called in normal profiles. Figure 26 Example of a –2-nt reproducible stutter artifact at the SE33 locus. Data produced on a 3500xL Genetic Analyzer. Note: The data in this figure are from the original formulation of the NGM Detect™ kit. Stutter percentage filter settings provided with GeneMapper™ ID‑X Software The settings in Table 5 and Table 6 were derived using the data that is shown in “Stutter observations“ on page 77. The proportion of the stutter product relative to the main allele (stutter percent) is measured by dividing the height of the stutter peak by the height of the main allele peak. NGM Detect™ PCR Amplification Kit User Guide 83 6 Chapter 6 Experiments and results Extra peaks in the electropherogram Analysis showed that observed stutter data points were not normally distributed. As such, at each locus a best-fit, non-parametric statistical model was applied to the data and a threshold filter level that emulated historical stutter filter levels (approximately 99.7%) was derived. IMPORTANT! The values that are shown in the table are the values that were determined during developmental validation studies using specific data sets. To determine the appropriate values to use for your applications, always perform internal validation studies. Table 5 Minus stutter percentage filter settings provided with the GeneMapper™ ID‑X Software Note: The data in this table are from the original validation study of the NGM Detect™ kit. Based on the validation study with the updated kit formulation, no changes to the stutter filter values were required. Locus [1] % Stutter D2S1338 17.61 SE33 18.63 SE33 (−2 nt) 6.41 D16S539 11.97 D18S51 16.15 TH01 7.66 D12S391 18.99 D3S1358 15.42 FGA 14.70 vWA 12.51 D21S11 16.88 D1S1656 16.68 D1S1656 (−2 nt) 3.90 D2S441 9.55 D8S1179 13.80 D19S433 13.57 D22S1045 19.02 D10S1248 14.32 [1] These percentages are used as stutter filters in NGM_Detect_Stutter.txt 84 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Extra peaks in the electropherogram 6 Table 6 Plus stutter percentage filter settings provided with the GeneMapper™ ID‑X Software Note: The data in this table are from the original validation study of the NGM Detect™ kit. Based on the validation study with the updated kit formulation, no changes to the stutter filter values were required. Locus [1] % Stutter D2S1338 11.55 SE33 9.80 D16S539 4.68 D18S51 8.19 TH01 6.14 D12S391 7.76 D3S1358 4.36 FGA 9.16 vWA 7.59 D21S11 9.14 D1S1656 5.47 D2S441 4.92 D8S1179 5.39 D19S433 6.91 D22S1045 8.22 D10S1248 2.60 [1] These percentages are used as stutter filters in NGM_Detect_Stutter.txt Extra peaks: 3′ A nucleotide addition definition Addition of 3' A Many DNA polymerases can catalyze the addition of a single nucleotide nucleotide (predominantly adenosine) to the 3′ ends of double-stranded PCR products (Clark, 1988; Magnuson et al., 1996). This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence. The PCR product with the extra nucleotide is referred to as the “+A” form. NGM Detect™ PCR Amplification Kit User Guide 85 6 Chapter 6 Experiments and results Extra peaks in the electropherogram 3′ A observations The efficiency of +A addition is related to the particular sequence of the DNA at the 3´ end of the PCR product. The NGM Detect™ kit includes two main design features that promote maximum +A addition: • The primer sequences have been optimized to encourage +A addition. • The PCR chemistry allows complete +A addition with a short final incubation at 60°C for 5 minutes. This final extension step gives the DNA polymerase additional time to complete +A addition to all double-stranded PCR products. Figure 27 shows examples of incomplete and normal +A addition. Final extension incubation for longer than the recommended time can result in double +A addition, in which two nontemplate adenosine residues are added to the PCR product. Double +A addition can cause "shoulders" on the right side of main allele peaks. 0 minutes final extension 5 minutes final extension d Arrows to use in this SVG. nced callouts or arrows _Callout&Arrow_Libary. h, as needed. , and unused Figure 27 Omitting the final extension step results in shoulders on main allele peaks due G elements to incomplete +A nucleotide addition. Examples shown are the smaller amplicons of VIC™ repository. and SID™ dye channel data from a 3500xL Genetic Analyzer using the NGM Detect™ kit. If the amount of input DNA is greater than recommended concentration, 1 "shouldering" of allele peaks can be observed. Amplification of excess input DNA can also result in off-scale data and lowered IQCL peak heights. In this situation, the IQCL 1 may also exhibit some shouldering due to incomplete +A nucleotide addition. 86 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Extra peaks in the electropherogram 6 Extra peaks: Artifact definition Artifacts Artifacts and anomalies are seen in all molecular biological systems. Artifacts are typically reproducible. Anomalies are non-reproducible, intermittent occurrences that are not observed consistently in a system (for example, spikes and baseline noise). Dye artifact observation Due to improvements in PCR primer manufacturing processes, the incidence of artifacts has been greatly reduced in the NGM Detect™ kit. Internal population studies show that kit electropherograms are free of reproducible dye artifacts in the kit read region of 64–458 nt. Two exceptions are as follows: • A low level 113–117 nt dye artifact in the VIC™ dye channel that has been detected below commonly used analytical thresholds. • A low level ~66 nt dye artifact in the TED™ dye channel. This artifact was approximately 40– 80 RFU in our studies. The peak height observed may vary depending on the sensitivity of individual CE instruments. Figure 28 shows the low baseline-level fluorescence that is observed in a typical negative control PCR. However, it is important to consider noise and other amplification-related artifacts when interpreting data. Figure 28 Examples of fluorescence background in data produced on a 3500xL Genetic Analyzer (Y-axis scale 0 to 200 RFU). and Arrows Some small PCR artifacts were occasionally observed with the human male 007 DNA arrow to use in this SVG. provided as the control DNA in the NGM Detect™ kit. The most prominent and consistent artifacts were: advanced callouts or arrows scape_Callout&Arrow_Libary. • A 170-bp artifact in the SID™ channel • Two artifacts consistent with the size of a –2 and –3 repeat unit stutter for allele 14 ength, as needed. of D2S441 In all cases, the peak heights of the artifacts were typically 1% or less than peak angle, and unused heights of the nearest true allele. If higher amounts of DNA (that is, 1 ng or more) are r SVG elements amplified, or if a lower PAT is used, then the peak heights could theoretically exceed o the repository. the analytical threshold. The peak height of the artifacts appeared to be proportional to the amount of input 007 DNA in the PCR. 1 1 NGM Detect™ PCR Amplification Kit User Guide 87 6 Chapter 6 Experiments and results Characterization of loci Characterization of loci SWGDAM “The basic characteristics of a genetic marker should be determined and guideline 3.1 documented.” (SWGDAM, December 2016) Loci in this kit This section describes basic characteristics of the 16 autosomal STR loci, Y indel locus, and sex-determining marker (Amelogenin), that are amplified with the NGM Detect™ kit. Most of these loci have been extensively characterized by other laboratories. Nature of The primers for the Amelogenin locus flank a 6-nucleotide deletion in intron 1 of the polymorphisms X homolog. Amplification generates 108-nt and 114-nt products from the X and Y chromosomes, respectively. The primers for the Y indel flank a region in the q arm of the Y chromosome (Yq11.221). Depending on the haplotype of the sample, the amplification generates either a 91-nt or a 96-nt product. (Sizes are the actual nucleotide size according to sequencing results, including 3´ A nucleotide addition, and size may not correspond exactly to allele mobility observed on capillary electrophoresis platforms.) Except for D22S1045, a trinucleotide STR, the remaining loci are tetranucleotide short tandem repeat (STR) loci. The length differences among alleles of a particular locus are caused by differences in the number of repeat units. We have sequenced all the alleles in the NGM Detect™ kit Allelic Ladder, including microvariants. In addition, other groups in the scientific community have sequenced alleles at some of these loci (Nakahori et al., 1991; Puers et al., 1993; Möller et al., 1994; Barber et al., 1995; Möller and Brinkmann, 1995; Barber et al., 1996; Barber and Parkin, 1996; Brinkmann et al., 1998; Momhinweg et al., 1998; Watson et al., 1998). Among the various sources of sequence data on the loci, there is consensus on the repeat patterns and structure of the STRs. Inheritance The Centre d'Etude du Polymorphisme Humain (CEPH) has collected DNA from families of Utah Mormon, French Venezuelan, and Amish descent. These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci. Each family set contains three generations, generally including four grandparents, two parents, and several offspring. Consequently, the CEPH family DNA sets are ideal for studying inheritance patterns (Begovich et al., 1992). Mapping The NGM Detect™ kit loci have been mapped, and the chromosomal locations have been published (Nakahori et al., 1991; Edwards et al., 1992; Kimpton et al., 1992; Mills et al., 1992; Sharma and Litt, 1992; Li et al., 1993; Straub et al., 1993; Barber and Parkin, 1996; and Lareu, et al., 1996). Genetic linkage Two sets of STR loci in the NGM Detect™ kit are located on the same chromosomes. vWA and D12S391 are located approximately 6.3 million bp apart on the p arm of chromosome 12; D2S1338 and D2S441 are located approximately 150 million bp apart on opposite arms of chromosome 2. Linkage disequilibrium analysis was conducted on the genotype results from 1,034 individuals of three ethnic groups (350 African American, 349 Caucasian, and 335 Hispanic). STR locus genotype results from the population study were analyzed using the Linkage Disequilibrium module of GenePop software version 4.0.10 (Raymond and Rousset, 1995; Rousset, 2008). See Table 7 for results. 88 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Species specificity 6 The relatively high probability values indicate that there is no statistically significant linkage disequilibrium found between the pairs of loci that are located on the same chromosome. An independent analysis of data from the same collection of population samples (Budowle, et al., 2010) also concluded that the 15 STR loci that are shared between the NGM™ and NGM SElect™ kits were independent at the population level (note that the SE33 locus was not part of this analysis). Therefore, to calculate the rarity of a profile for comparison to single-source and mixture samples, the frequencies of all loci including vWA and D12S391 could be multiplied. However, the analysis of the CEPH pedigree families demonstrated a degree of linkage between vWA and D12S391 that does not support the assumption of independence for kinship analysis. Table 7 GenePop software LD Result (p‑value for pairwise analysis of loci) Chromosome African- Chromosome Nuclear Caucasian Hispanic Locus American map position[1] Coordinates[1] (n = 350) (n = 293) (million bp) (n = 350) vWA 12p13.31 5.9 0.86 0.29 0.27 D12S391 12p13.2 12.2 D2S441 2p14 68 0.11 0.32 0.19 D2S1338 2q35 218 [1] STR locus mapping data was obtained from the NCBI Map Viewer http://www.ncbi.nlm.nih.gov/projects/ mapview/map_search.cgi?taxid=9606 or the UCSC Genome Browser (http://genome.ucsc.edu/). GenePop LD analysis probability results (p values) greater than 0.05 were considered to indicate that linkage disequilibrium between the loci within the population tested was not statistically significant. Species specificity SWGDAM “The ability to detect genetic information from non-targeted species (e.g., detection of Guideline 3.2 microbial DNA in a human assay) should be determined. The detection of genetic information from non-targeted species does not necessarily invalidate the use of the assay, but may help define the limits of the assay.” (SWGDAM, December 2016) Nonhuman The NGM Detect™ kit provides the required specificity for detecting human alleles. studies Species specificity testing was performed to ensure that there is no cross-reactivity with nonhuman DNA that may be present in forensic casework samples. The following species were tested (in the specified amounts) using standard PCR and capillary electrophoresis conditions for the NGM Detect™ kit kit: • Primates: gorilla, chimpanzee, orangutan, and cynomolgous (macaque) (0.5 ng each) • Non-primates: mouse, dog, sheep, pig, rabbit, cat, horse, hamster, rat, chicken, and cow (5.0 ng each) • Microorganisms: Candida albicans, Staphylococcus aureus, Escherichia coli, Neisseria gonorrhoeae, Bacillus subtilis, and Lactobacillus rhamnosus (pooled genomic DNAs, with approximately 50,000 copies of DNA from each species, per reaction) NGM Detect™ PCR Amplification Kit User Guide 89 6 Chapter 6 Experiments and results Species specificity Results were evaluated for the presence of any amplified peaks that would indicate cross reactivity of the NGM Detect™ kit with any of these non-human species. Figure 29 shows select electropherogram results from the species specificity tests. The chimpanzee, gorilla (data not shown), orangutan (data not shown), and macaque (data not shown) DNA samples produced partial profiles in the 70–400 nucleotide region. Human Chimp Dog Arrows Cat o use in this SVG. ed callouts or arrows Callout&Arrow_Libary. as needed. Horse and unused elements epository. 1 Pig 1 Sheep Cow ows in this SVG. llouts or arrows ut&Arrow_Libary. eded. 90 NGM Detect™ PCR Amplification Kit User Guide nused nts Chapter 6 Experiments and results Species specificity 6 Mouse Rat Rabbit Arrows Chicken o use in this SVG. ced callouts or arrows Callout&Arrow_Libary. as needed. Bacterial pool and unused elements epository. uts and Arrows Negative control 1 ut or arrow to use in this SVG. 1 more advanced callouts or arrows _Inkscape_Callout&Arrow_Libary. Figure 29 Representative electropherograms for some species tested in a species line-length, as needed. specificity study. Data produced on a 3500xL Genetic Analyzer. Table 8 shows the most significant cross-reactive peaks that were observed among s rectangle, and unused non-human, non-primate, genomic DNAs (that is, peaks over a 175 RFU Peak other SVG elements Amplitude Threshold on the 3500xL Genetic Analyzer). Peaks were observed for dog, SVG to the repository. horse, and rabbit. Most peaks did not fall into human STR locus bins or marker ranges, and would therefore not be confused with human STR alleles. This data shows that the likelihood of obtaining an allelic profile consistent with that from a 1 1 human sample, from non-primates or microorganisms, is low. 1 NGM Detect™ PCR Amplification Kit User Guide 91 6 Chapter 6 Experiments and results Sensitivity Table 8 Observed cross-reactive peaks for non-human, non-primate animals. Species Dye channel Size Peak height 401 bp 1,294 RFU B Dog 407 bp 1,046 RFU Y 366 bp 289 RFU Horse R 104 bp 915 RFU Rabbit P 260 bp 389 RFU Sensitivity SWGDAM “The ability to obtain reliable results from a range of DNA quantities, to include the guideline 3.3 upper and lower limits of the assay, should be evaluated.” (SWGDAM, December 2016) Sensitivity The recommended amount of input DNA for the NGM Detect™ kit is 0.5 ng for observation 30 cycles of amplification based on real-time PCR quantification, such as with the Quantifiler™ Trio DNA Quantification Kit or the Quantifiler™ HP DNA Quantification Kit. To determine the optimum input DNA, perform studies according to the quantification kit that you use. If the sample contains degraded or inhibited DNA, amplification of a higher amount of DNA may be beneficial. In Figure 30, DNA Control 007 was serially diluted from 0.5–0.008 ng. Full profiles (35 PCR products) were consistently obtained at 0.125 ng, but occasional partial profiles resulted at lower concentrations. 500 pg 250 pg 125 pg rrows se in this SVG. callouts or arrows 63 pg llout&Arrow_Libary. needed. d unused 92 NGM Detect™ PCR Amplification Kit User Guide ments ository. Chapter 6 Experiments and results Stability 6 32 pg 16 pg uts and Arrows t or arrow to use in this SVG. 8 pg more advanced callouts or arrows _Inkscape_Callout&Arrow_Libary. line-length, as needed. Figure 30 Electropherograms for amplifications using 500, 250, 125, 63, 32, 16, and 8 pg of DNA Control 007. Electrophoresis was performed on a 3500xL Genetic Analyzer. Note SVG. that as the DNA input is serially diluted by 2-fold, the Y-axis scale is also adjusted, as rectangle, and unused other SVG elements needed, to accommodate lower peak heights. or arrows VG to the repository. w_Libary. Stability 1 1 and Arrows SWGDAM1 “The ability to obtain results from DNA recovered from biological samples deposited guideline 3.4 on various substrates and subjected to various environmental and chemical insults row to use in this SVG. should be evaluated. In most instances, assessment of the effects of these factors on dvanced callouts or arrows new forensic DNA procedures is not required. However, if substrates and/or ape_Callout&Arrow_Libary. environmental and/or chemical insults could potentially affect the analytical process, then the process should be evaluated to determine the effects of such factors.” 1 (SWGDAM, December 2016) ngth, as needed. ngle, and unused SVG elements the repository. 1 1 NGM Detect™ PCR Amplification Kit User Guide 93 6 Chapter 6 Experiments and results Stability Degraded DNA As the average size of degraded DNA approaches the size of the target sequence, the amount of PCR product that is generated is reduced, because of the reduced number of intact templates in the size range required for amplification. Degraded DNA was prepared to examine the potential for differential amplification of loci. Approximately 10 µg of high molecular weight DNA was sonicated and subjected to digestion with 0.25 U/µL of DNase I for various lengths of time (from ~5–10 minutes) to produce a range (low, medium, and high) of degradation levels (Bender et al., 2004). The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point. Amplification of 0.5 ng of degraded DNA using the NGM Detect™ kit was performed. As the DNA became progressively degraded, the loci failed to amplify robustly in order of decreasing size. Preferential amplification was not observed. Control Low Medium d Arrows to use in this SVG. nced callouts or arrows High _Callout&Arrow_Libary. h, as needed. Figure 31 Amplification of a single donor DNA sample sonicated and incubated with , and unused 0.25 U/µL of DNase I for various lengths of time (from ~5–10 minutes). G elements repository. 1 1 94 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Stability 6 Effect of Heme compounds have been identified as PCR inhibitors in DNA samples that are inhibitors: extracted from bloodstains (DeFranchis et al., 1988; Alkane et al., 1994). It is believed that the inhibitor is co-extracted and co-purified with the DNA and then interferes hematin with PCR by inhibiting polymerase activity. To examine the effects of hematin on the performance of the NGM Detect™ kit, 0.5 ng of DNA Control 007 was amplified in the presence of increasing concentrations of hematin for 30 cycles of amplification (Figure 32). The final concentrations of hematin that is used in the reaction were 0, 450, 575, and 700 µM (Table 9). NGM Detect kit No inhibitor NGM Detect kit 575 uM Hematin NGM SElect kit No inhibitor nd Arrows ow to use in this SVG. NGM SElect kit 575 uM Hematin vanced callouts or arrows ape_Callout&Arrow_Libary. ™ ™ ngth, as needed. Figure 32 Electropherograms for the NGM Detect kit and the NGM SElect kit show the ™ improved performance of the NGM Detect kit in the presence of hematin compared with ™ previous AmpFℓSTR kits. The same set of inhibited samples was analyzed with the gle, and unused ™ ™ VG elements NGM Detect kit and the NGM SElect kit for 30 cycles of amplification. the repository. Table 9 NGM Detect™ kit performance in simulated hematin inhibition (N = 5). Average number of alleles Minimum, 1 Hematin concentration detected[1] maximum 1 0 µM 35 35, 35 450 µM 35 35, 35 575 µM 12.4 12, 13 700 µM 0 0, 0 [1] Only those peaks ≥175 RFU were counted. A complete profile with DNA Control 007 yields 35 peaks using the NGM Detect™ kit. NGM Detect™ PCR Amplification Kit User Guide 95 6 Chapter 6 Experiments and results Stability Effect of Traces of humic acid may inhibit the PCR amplification of DNA evidence that is inhibitors: humic collected from soil. Amplification of 0.5 ng of DNA Control 007 in the presence of increasing amounts of humic acid was performed using the NGM Detect™ kit for acid 30 cycles of amplification (Figure 33). The final concentrations of humic acid tested in the reactions were 0, 140, 250 and 400 ng/µL (Table 10). NGM Detect kit No inhibitor NGM Detect kit 250 ng/uL Humic acid NGM SElect kit No inhibitor Arrows to use in this SVG. NGM SElect kit 250 ng/uL Humic acid nced callouts or arrows _Callout&Arrow_Libary. ™ ™ , as needed. Figure 33 Electropherograms for the NGM Detect kit and the NGM SElect kit show the ™ improved performance of the NGM Detect kit in the presence of humic acid compared ™ and unused with previous AmpFℓSTR kits. The same set of inhibited samples was analyzed with the ™ ™ elements NGM Detect kit and the NGM SElect kit for 30 cycles of amplification. repository. Table 10 NGM Detect™ kit performance in simulated humic inhibition (N = 5). 1 Minimum, Humic acid concentration Number of alleles detected[1] maximum 1 0 ng/µL 35 35, 35 140 ng/µL 35 35, 35 250 ng/µL 26 26, 26 400 ng/µL 1 1, 1 [1] Only those peaks ≥175 RFU were counted. A complete profile with DNA Control 007 yields 35 peaks using the NGM Detect™ kit. 96 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Mixture studies 6 Mixture studies SWGDAM “The ability to obtain reliable results from mixed source samples should be guideline 3.8 determined.” (SWGDAM, December 2016) Mixture study Evidence samples may contain DNA from more than one individual. The possibility overview of multiple contributors should be considered when interpreting the results. Perform studies to determine a minimum peak height threshold to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures. Evidence samples that contain body fluids and/or tissues originating from more than one individual are an important category of forensic casework. It is essential to ensure that the DNA typing system is able to detect DNA mixtures. Typically, mixed samples can be distinguished from single-source samples by: • The presence of more than two alleles at one or more loci • The presence of a peak at a stutter position that is significantly greater in percentage than typically observed in a single-source sample • Significantly imbalanced alleles for a heterozygous genotype The peak height ratio is defined as the height of the lower peak (in RFU) divided by the height of the higher peak (in RFU), expressed as a percentage. If an unusually low peak height ratio is observed for one locus, and there are no other indications that the sample is a mixture, reamplify and reanalyze the sample to determine if the imbalance is reproducible. Possible causes of imbalance at a locus are: • Degraded DNA • Presence of inhibitors • Extremely low amounts of input DNA • A mutation in one of the primer binding sites • Presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele NGM Detect™ PCR Amplification Kit User Guide 97 6 Chapter 6 Experiments and results Mixture studies Mixture study Median, minimum, and maximum peak height ratios observed for alleles in the observation NGM Detect™ kit loci in unmixed human population database samples (N=60) are shown in Figure 34. The donor samples that were used are listed in “Population samples used in these studies“ on page 102. Arrows Figure 34 Heterozygote ratios for 0.5 ng of input DNA. The distribution of intra-locus peak use in this SVG. height ratio is expressed as a percentage, by locus (low peak height allele divided by high ed callouts or arrows peak height allele). Boxes show the middle 50% or interquartile range (IQR). Box halves allout&Arrow_Libary. below and above median show the second and third quartile, respectively. "Whiskers" indicate 1.5 IQR from the upper and lower margins of the IQR. Black dots are outlier data points more than 1.5 IQR from the median (N=60). as needed. nd unused Resolution of A sample containing DNA from two sources can comprise (at a single locus) any of ements genotypes in the seven genotype combinations (see below). pository. mixed samples • Heterozygote + heterozygote, no overlapping alleles (four peaks) • Heterozygote + heterozygote, one overlapping allele (three peaks) 1 • Heterozygote + heterozygote, two overlapping alleles (two peaks) 1 • Heterozygote + homozygote, no overlapping alleles (three peaks) • Heterozygote + homozygote, overlapping allele (two peaks) • Homozygote + homozygote, no overlapping alleles (two peaks) • Homozygote + homozygote, overlapping allele (one peak) Specific genotype combinations and input DNA ratios of the samples contained in a mixture determine whether or not it is possible to resolve the genotypes of the major and minor components at a single locus. The ability to obtain and compare quantitative values for the different allele peak heights on Applied Biosystems™ instruments provides additional valuable data to aid in resolving mixed genotypes. Ultimately, the likelihood that any sample is a mixture must be determined by the analyst in the context of each particular case, including the information provided from known reference samples. 98 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Mixture studies 6 Note: Peak detection is a complex process that involves the STR chemistry, capillary electrophoresis conditions, and the data analysis software. Contact HID Support for a Technical Note with additional information on detecting peaks in electropherograms. Limit of detection Mixtures of two DNA samples were examined at various ratios (0:1, 1:1, 1:3, 1:7, 1:15, of the minor 1:30, 30:1, 15:1, 7:1, 3:1 and 1:0 M:F ratios). The total amount of genomic input DNA mixed at each ratio was 0.5 ng. The samples were amplified in a GeneAmp™ PCR component System 9700, then electrophoresed and detected using a 3500xL Genetic Analyzer. The results of the mixed DNA samples are shown in Figure 35. The male 007 and female 9947a were mixed according to the ratios indicated. The minor component allele calls at non-overlapping loci are highlighted. Detection of full profiles for the minor contributor was possible at ratios of 7:1 (0.438 ng of 007 and 0.063 ng of 9947a) and 1:7 (0.063 ng of 007 and 0.438 ng of 9947a, with 2/3 replicates giving full profiles of the 007 minor contributor). 15:1, 30:1, 1:30, and 1:15 ratios resulted in partial profiles for the minor component. The genotypes of these samples are shown in Table 11. Table 12 shows mixture sample compositions and STR allele counts from NGM Detect™ assays. Figure 35 Amplification of DNA mixtures at various ratios. Panels show electropherograms for 1:0 (male 007 DNA only), 1:1 mixture, 7:1 mixture, 1:7 mixture, and 0:1 (female 9947a DNA only). Note: The data in this figure are from the original formulation of the NGM Detect™ kit. Table 11 Genotypes of mixed DNA samples Note: The data in this table are from the original validation study of the NGM Detect™ kit. NGM Detect™ PCR Amplification Kit User Guide 99 6 Chapter 6 Experiments and results Mixture studies Locus Male 007 alleles Female 9947a alleles IQCS — — — — D2S1338 20 23 19 23 SE33 17 25.2 19 29.2 IQCL — — — — D16S539 9 10 11 12 D18S51 12 15 15 19 TH01 7 9.3 8 9.3 D12S391 18 19 18 20 D3S1358 15 16 14 15 FGA 24 26 23 24 Y indel 2 — — — Amelogenin X Y X — vWA 14 16 17 18 D21S11 28 31 30 — D1S1656 13 16 18.3 — D2S441 14 15 10 14 D8S1179 12 13 13 — D19S433 14 15 14 15 D22S1045 11 16 11 14 D10S1248 12 15 13 15 Table 12 Mixture sample compositions and STR allele counts from NGM Detect™ assays Note: The data in this table are from the original validation study of the NGM Detect™ kit. Number of unique alleles in ng per reaction non-stutter positions per Ratio (M:F) contributor[1] Female Female Male 007 Total DNA Male 007 9947a 9947a 1:0 0.500 0.00 0.5 16.00 0.00 1:1 0.250 0.250 0.5 16.00 13.00 1:3 0.125 0.375 0.5 16.00 13.00 1:7 0.063 0.438 0.5 15.67 13.00 100 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 Number of unique alleles in ng per reaction non-stutter positions per Ratio (M:F) contributor[1] Female Female Male 007 Total DNA Male 007 9947a 9947a 1:15 0.031 0.469 0.5 12.33 13.00 1:30 0.016 0.484 0.5 8.00 13.00 30:1 0.484 0.016 0.5 16.00 8.67 15:1 0.469 0.031 0.5 16.00 11.67 7:1 0.438 0.063 0.5 16.00 13.00 3:1 0.375 0.125 0.5 16.00 13.00 0:1 0.000 0.500 0.5 0.00 13.00 [1] Mixture sample compositions and STR allele counts from NGM Detect™ assays. Average number of unique, distinct alleles (that is, alleles neither present in the other contributor genotype nor located in a stutter position) obtained in 3 replicate reactions per mixture sample. Full profiles of unique alleles in 007 and 9947a DNAs were 16 and 13, respectively. Population data SWGDAM “The distribution of genetic markers in populations should be determined in relevant guideline 3.7 population groups.” (SWGDAM, December 2016) Population data To interpret the significance of a match between genetically typed samples, you must overview know the population distribution of alleles at each locus in question. If the genotype of the relevant evidence sample is: • Different from the genotype of the reference sample for a suspect, then the suspect is excluded as the donor of the biological evidence that was tested. An exclusion is independent of the frequency of the two genotypes in the population. • The same as the genotype of the reference sample for a suspect, then the suspect is included as a possible source of the evidence sample. The probability that another, unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant populations. Loci in the kit The NGM Detect™ kit contains loci for which extensive population data are available. For additional information on the loci shared between many of the AmpFℓSTR™ kits, see the population data and additional studies section of the AmpFℓSTR™ NGM SElect™ PCR Amplification Kit Kit User Guide (Pub. No. 4458841) and the AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit User Guide (Pub. No. 4440211). NGM Detect™ PCR Amplification Kit User Guide 101 6 Chapter 6 Experiments and results Population data Population Note: The data in this section are from the original validation study of the samples used in NGM Detect™ kit. these studies The NGM Detect™ kit has high genotypic concordance (>99.5%) to the NGM SElect™ kit and by extension to the GlobalFiler™ kit. (All STR markers in the NGM SElect™ kit are included in the GlobalFiler™ kit with both the GlobalFiler™ and NGM SElect™ kits sharing the same primers for common loci.) Therefore, the existing population data from the GlobalFiler™ kit was used, as applicable, to generate the population data that is provided in this section. Whole blood samples, provided by the Interstate Blood Bank (Memphis, Tennessee) and Boca Biolistics (Coconut Creek, Florida), were collected in the United States (with no geographical preference) from randomly selected individuals of known ethnicities. Ethnicities of sample donors were: • African-American—330 samples • Asian—153 samples • Caucasian—343 samples • Hispanic—368 samples DNA was extracted using a 6100 Nucleic Acid Prep Station. In addition to the alleles that we observed and recorded in our databases, other alleles have been published or reported to us by other laboratories (see the STRBase at www.cstl.nist.gov/div831/strbase). Concordance In the validation study of the original NGM Detect™ kit formulation, a concordance studies population study with 1,165 in-house samples identified two samples that were discordant because of allele dropout or severe allele imbalance in the NGM Detect™ assay. The samples were characterized and degenerate oligos were added to the SE33 (2 oligos) and D8S1179 (1 oligo) markers in the final stages of development. The resulting concordance between the NGM SElect™ kit and the NGM Detect™ kit was greater than 99.5%. In the validation study of the updated NGM Detect™ kit formulation, we used the new primer set with 1,092 individuals from the same donor population. We observed 100% concordance between the new primer set and the original primer set at the TH01 locus. Probability of The PI value is the probability that two individuals selected at random will have an Identity definition identical genotype (Sensabaugh, 1982). 102 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 Probability of Table 13 shows the Autosomal STR allele frequencies at NGM Detect™ kit loci by identity population group. Table 14 shows the Y-specific allele frequencies by population group for NGM Detect™ kit Y indel locus. The Y-specific allele frequencies were not included in the probability of identity calculation. Table 15 shows the Probability of identity (PI) values of the NGM Detect™ kit loci individually and combined. Table 13 Autosomal allele frequencies by population group for NGM Detect™ kit STR loci. (*=Alleles not detected or not detected in significant quantities) African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) D2S1338 10 * * * * 11 * * * * 12 * * * * 13 0.15* * 0.15* * 14 * * 0.15* * 15 0.30* * 0.15* * 16 5.3 1.63 4.08 3.8 17 10 14.05 18.37 17.8 18 4.85 13.07 8.31 6.52 19 16.21 16.67 14.14 17.53 20 10.45 8.82 15.74 13.86 21 11.97 2.94 2.92 3.67 22 12.42 5.88 1.75 6.52 23 9.24 18.3 10.06 14.27 24 8.79 11.11 10.2 8.83 25 6.97 5.88 12.1 5.43 26 2.58 * 1.6 1.49 27 0.76 0.33* 0.29* 0.14* 28 * 0.98* * 0.14* 29 * 0.33* * * NGM Detect™ PCR Amplification Kit User Guide 103 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) SE33 3.2 * * * * 4.2 * * * * 5 * * * * 6 * * * * 6.3 * * 0.15* * 7 * * * * 8 * * * * 8.2 * * * * 9 * * * * 9.2 * * * * 10 * * * * 10.2 * * * * 11 * * * * 11.2 0.76 * * 0.14* 12 0.15* 0.33* 0.44* 0.14* 12.1 * * 0.15* * 12.2 0.30* * 0.15* 0.14* 13 1.36 * 0.87 1.22 13.2 0.45* * * 0.14* 14 3.33 * 3.64 1.77 14.2 0.15* * * 0.82 14.3 * * 0.15* * 15 4.24 1.31* 3.64 4.89 15.2 0.15* * 0.15* 0.14* 15.3 * * * * 16 6.97 3.59 5.39 5.57 16.2 0.30* * * 0.27* 16.3 * * * 0.14* 104 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 17 7.73 5.23 6.56 8.7 17.2 0.15* * * * 17.3 * * 0.15* * 18 10.76 4.9 7.87 10.05 18.2 0.15* * * 0.27* 19 15 9.48 8.31 8.15 19.2 0.30* * 0.29* * 19.3 * * 0.29* * 20 9.55 6.86 5.25 4.48 20.2 0.91 0.33* 0.87 0.82 21 5.76 6.21 2.04 3.12 21.1 * * * * 21.2 0.91 1.63 1.17 1.09 22 1.97 2.61 0.58* 1.09 22.2 1.36 2.29 3.35 2.31 23 0.30* * * * 23.2 0.61* 2.61 2.62 2.85 23.3 * * * 0.14* 24 0.30* 0.33* 0.15* 0.14* 24.2 1.67 6.54 4.52 2.31 25 * * * * 25.2 2.42 7.19 3.79 3.12 26 0.15* * * 0.27* 26.2 5.61 7.52 4.52 6.39 27 * * * * 27.2 5.91 3.59 6.85 7.07 28 * * * * 28.2 3.94 7.84 7.73 6.25 29 * * * * NGM Detect™ PCR Amplification Kit User Guide 105 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 29.2 2.58 8.5 7.87 5.84 30 * * * * 30.2 1.21 7.52 4.66 3.8 31 * * * 0.14* 31.2 1.06 1.63 2.77 2.31 32 * * 0.44* * 32.2 0.76 1.31* 1.6 2.04 33 * * 0.29* 0.41* 33.2 0.45* 0.33* 0.15* 0.54* 34 * * 0.29* 0.41* 34.2 0.15* * * 0.27* 35 * * 0.15* * 35.2 * 0.33* * * 36 * * 0.15* * 36.2 * * * * 37 * * * 0.14* 37.2 * * * * 38 * * * * 38.2 * * * * 39 * * * * 39.2 * * * * 40 * * * * 41 * * * * 42 * * * * 43 * * * * D16S539 4 * * * * 5 * * * * 6 * * * 0.14* 106 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 7 * * * * 8 3.33 * 1.46 2.04 8.3 * * * * 9 21.67 31.05 12.68 10.19 9.3 * * * * 10 11.52 14.05 4.08 15.76 11 30 20.59 32.22 31.79 11.3 * * * * 12 19.09 21.57 30.9 24.18 12.1 * * * * 12.2 * * * * 13 13.03 11.44 16.76 14.4 13.3 * * * * 14 1.36 1.31* 1.75 1.22 15 * * 0.15* 0.27* 16 * * * * D18S51 6 * * * * 7 * * * * 8 9 * * * 0.14* 9.2 * * * * 10 0.15* * 1.17 0.68 10.2 0.15* * * * 11 0.45* 1.31* 0.87 1.22 11.2 * * * * 12 6.21 5.56 15.01 10.46 12.2 * * * * 12.3 * * * * NGM Detect™ PCR Amplification Kit User Guide 107 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 13 3.94 17.32 11.95 11.41 13.1 * * * * 13.2 0.30* * * * 13.3 * * * * 14 5.91 22.88 17.64 16.3 14.2 0.45* * * 0.14* 15 16.52 16.99 15.31 12.23 15.2 * * * 0.14* 15.3 * * * * 16 18.18 12.42 11.95 12.91 16.1 * * * * 16.2 * * * * 17 16.36 6.54 10.79 17.39 17.2 * * * * 17.3 * * * * 18 14.09 4.9 8.31 7.74 18.1 * * * * 18.2 * * * * 19 9.7 5.23 4.08 3.53 19.2 * * * * 20 4.7 1.96 1.31 1.9 20.2 0.15* * * * 21 1.82 1.96 1.02 2.17 21.2 * * * * 22 0.61* 0.98* 0.29* 0.68 22.2 * * * * 23 0.30* 0.98* 0.29* 0.54* 23.2 * * * * 24 * 0.65* * 0.27* 108 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 25 * * * 0.14* 26 * 0.33* * * 27 * * * * 28 * * * * TH01 3 * * * * 4 * * * * 5 0.45* * 0.15* * 5.3 * * * * 6 15.45 13.07 21.72 27.17 6.3 * * * * 7 37.42 26.14 17.64 32.74 7.3 * * * * 8 20.61 3.59 11.37 8.7 8.3 * * * * 9 16.06 51.63 17.06 12.77 9.3 8.33 4.25 31.2 17.12 10 1.52 1.31* 0.87 1.49 10.3 * * * * 11 * * * * 12 * * * * 13 * * * * 13.3 * * * * 14.3 * * * * D12S391 13 * * * 0.14* 14 * * * 0.14* 15 7.58 2.61 4.37 4.08 16 5.15 0.98* 3.35 5.03 NGM Detect™ PCR Amplification Kit User Guide 109 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 17 16.52 8.17 10.35 7.34 17.1 0.45* * * 0.27* 17.3 0.61* * 1.9 1.22 18 24.55 28.43 16.18 19.7 18.1 * * * * 18.3 1.21 * 2.19 2.17 19 13.94 23.86 12.54 18.75 19.1 0.61* * * * 19.3 0.30* * 0.58* 1.22 20 11.52 16.99 9.77 17.12 20.1 * * * * 20.3 * * 0.15* * 21 7.27 10.78 13.56 8.7 21.3 0.15* * 0.15* * 22 5 3.59 10.79 6.79 23 3.64 3.27 8.16 3.67 24 0.61* 0.98* 3.64 1.9 24.3 * * * * 25 0.61* 0.33* 1.9 1.36 26 * * 0.29* 0.27* 27 * * 0.15* 0.14* D3S1358 8 * * * * 9 0.30* * * 0.14* 10 * * * * 11 * * 0.29* * 12 0.15* 0.33* * 0.14* 13 0.61* * 0.15* 0.41* 14 9.09 2.61 15.16 9.1 110 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 15 28.18 49.02 27.26 34.65 15.2 0.30* * * * 16 32.42 21.9 24.34 26.9 16.2 * * * * 17 22.27 19.61 19.68 17.93 17.1 * * * * 17.2 * * * * 18 6.06 6.54 11.66 9.92 18.2 * * * * 19 0.61* * 1.46 0.82 20 * * * * 20.1 * * * * 21 * * * * FGA 12.2 * * * * 13 * * * * 14 * * * * 15 * * * * 16 * 0.33* 0.15* * 16.1 0.30* * * * 16.2 * * * * 17 * 0.33* 0.15* * 17.2 * * * * 18 0.91 3.27 1.02 0.68 18.2 0.61* * * * 19 6.97 4.25 5.69 7.61 19.2 0.45* * * * 19.3 * * * * 20 6.82 3.92 14.87 8.7 NGM Detect™ PCR Amplification Kit User Guide 111 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 20.1 * * * * 20.2 0.30* * 0.44* 0.27* 20.3 * * * * 21 11.67 13.07 18.22 13.45 21.2 0.15* 0.33* 0.29* * 21.3 * * * * 22 17.27 14.38 19.24 14.4 22.1 * * * * 22.2 0.15* * 0.87 0.54* 22.3 * * * * 23 17.27 27.12 14.87 12.91 23.1 * * * * 23.2 * 0.65* 0.44* 0.41* 23.3 0.30* * * * 24 18.94 18.3 14.43 15.62 24.1 * * * * 24.2 * 0.33* * * 24.3 * * * * 25 9.55 9.8 6.71 13.72 25.1 * * * * 25.2 * * * * 25.3 * * * * 26 4.09 3.27 1.9 7.07 26.1 * * * * 26.2 * * * * 27 2.58 0.65* 0.58* 3.12 27.2 * * * * 28 1.21 * 0.15* 0.95 28.2 * * * * 112 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 29 * * * 0.41* 29.2 * * * * 30 0.15* * * 0.14* 30.2 0.15* * * * 31 * * * * 31.2 * * * * 32 * * * * 32.2 * * * * 33.2 * * * * 34.2 0.15* * * * 41.2 * * * * 42.2 * * * * 43.2 * * * * 44.2 * * * * 45.2 * * * * 46.2 * * * * 47.2 * * * * 48.2 * * * * 49.2 * * * * 50.2 * * * * 51.2 * * * * 52.2 * * * * vWA 10 * * * * 11 0.45* * * 0.14* 12 * * * 0.27* 13 0.91 * 0.15* 0.14* 14 7.27 23.53 8.75 6.52 15 20.91 1.63 12.24 9.78 NGM Detect™ PCR Amplification Kit User Guide 113 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 15.2 * * * * 16 27.58 15.36 22.3 30.57 17 19.85 29.74 27.41 27.17 18 13.79 19.61 17.78 18.07 18.2 * * * * 19 6.52 9.15 10.06 6.39 20 1.97 0.98* 1.31 0.82 21 0.61* * * * 22 * * * * 23 0.15* * * * 24 * * * * 25 * * * * D21S11 23 * * * * 23.2 * * * * 24 * * * * 24.2 * * * 0.27* 24.3 * * * * 25 * * * * 25.2 * * * * 25.3 * * * * 26 0.30* * 0.58* 0.41* 26.2 * * * * 27 5.91 * 2.62 1.49 27.1 * * * * 27.2 * * * * 28 25.15 4.9 16.76 11.41 28.1 * * * * 28.2 * 0.65* * 0.14* 114 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 28.3 * * * * 29 15.61 26.8 23.76 21.06 29.1 * * * * 29.2 * * 0.15* * 29.3 0.15* * 0.15* * 30 20.76 30.72 23.18 27.17 30.1 * * * * 30.2 1.67 0.65* 2.77 1.77 30.3 * * * * 31 8.79 9.48 6.85 5.16 31.1 * * * * 31.2 4.55 3.92 8.89 11.14 31.3 * 0.33* * * 32 1.36 2.61 2.33 1.36 32.1 * * * * 32.2 7.12 14.38 9.62 12.5 32.3 * * * * 33 0.91 0.98* * 0.14* 33.1 * * * * 33.2 3.18 4.58 1.9 5.3 33.3 * * * * 34 0.15* * * * 34.1 * * * * 34.2 * * 0.44* 0.14* 35 3.64 * * 0.27* 35.1 * * * * 35.2 * * * * 36 0.76 * * 0.14* 36.1 * * * * NGM Detect™ PCR Amplification Kit User Guide 115 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 36.2 * * * * 37 * * * * 37.2 * * * * 38 * * * 0.14* 38.2 * * * * 39 * * * * D1S1656 8 * * * * 9 0.15* * * 0.14* 10 1.36 * 0.29* 0.41* 11 5.3 3.59 6.27 3.94 12 8.48 4.25 15.74 9.38 13 11.06 13.73 7 7.07 13.3 * * * * 14 25 6.21 6.27 11.28 14.3 0.91 * 0.29* 0.27* 15 16.97 20.26 15.31 15.49 15.3 1.82 * 8.75 2.99 16 10 31.05 9.33 15.08 16.3 7.27 0.65* 4.96 5.16 17 2.73 14.05 4.96 6.79 17.1 * * 0.29* * 17.3 5.76 3.59 12.68 15.76 18 0.45* 0.33* 0.29* 0.82 18.3 1.82 1.63 5.98 4.48 19 0.15* * * * 19.3 0.61* 0.33* 1.6 0.68 20 * 0.33* * * 20.3 0.15* * * * 116 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 21 * * * * 21.3 * * * * D2S441 7 * * * * 8 0.15* * * * 9 * * 0.58* 0.14* 9.1 * 2.94 * * 10 9.09 20.59 19.83 30.3 11 35.61 36.27 33.09 31.93 11.3 2.88 2.61 5.1 4.62 12 20.45 20.92 4.08 3.8 12.3 0.15* * 0.29* 0.41* 13 3.48 6.21 3.35 1.9 13.3 * * * * 14 26.21 9.8 28.86 23.1 14.3 * * * * 15 1.97 0.65* 4.37 3.4 16 * * 0.44* 0.41* 17 * * * * 18 * * * * D8S1179 4 * * * * 5 * * * * 6 * * * * 7 * * * * 8 0.30* * 2.04 0.68 9 0.30* * 1.31 0.27* 10 3.33 9.8 10.5 9.51 10.2 * * * * NGM Detect™ PCR Amplification Kit User Guide 117 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 11 5.61 9.48 6.71 5.03 12 11.36 13.4 15.16 12.5 12.3 * * * * 13 18.18 24.18 33.24 33.15 13.3 * * * * 14 35.91 15.69 18.8 23.23 15 18.03 22.22 9.04 11.41 15.3 * * * * 16 5.91 4.25 2.77 3.53 17 1.06 0.98* 0.44* 0.68 18 * * * * 19 * * * * 20 * * * * D19S433 5 * * * * 5.2 * * * * 6 * * * * 6.2 * * * * 7 * * * * 8 * * * * 9 0.30* 0.33* * * 9.2 * * * * 10 1.21 * 0.15* 0.41* 10.2 0.15* * * * 11 9.85 * * 1.63 11.2 0.30* * * 0.27* 12 10.45 4.58 7.29 8.42 12.1 * * 0.15* * 12.2 3.94 0.33* 0.15* 1.49 118 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 13 27.88 28.1 27.26 18.48 13.1 * * * * 13.2 5.3 2.61 1.6 6.93 14 18.94 23.2 35.13 30.71 14.2 5.3 9.48 2.04 4.62 14.3 * * * * 15 6.67 7.52 16.18 13.04 15.2 4.39 20.26 3.5 6.79 16 1.52 0.33* 5.69 4.08 16.2 3.18 2.61 0.29* 2.17 17 * * 0.29* 0.54* 17.2 0.61* 0.65* 0.15* 0.41* 18 * * 0.15* * 18.2 * * * * 19 * * * * 19.2 * * * * 20.2 * * * * D22S1045 6 * * * * 7 * * * * 8 0.61* * * * 9 * * * * 10 4.09 * 0.44* 0.68 11 14.7 15.36 13.85 7.61 12 6.21 0.33* 0.58* 0.95 13 0.30* 0.33* 1.02 1.09 14 7.88 0.33* 3.35 2.04 15 23.33 33.66 36.3 43.48 16 20.3 23.86 36.3 34.65 NGM Detect™ PCR Amplification Kit User Guide 119 6 Chapter 6 Experiments and results Population data African U.S. Caucasian U.S. Hispanic Allele American Asian (n=153) (n=343) (n=368) (n=330) 17 20.45 24.18 7.58 8.42 18 2.12 1.96 0.58* 0.95 19 * * * * 20 * * * 0.14* 21 * * * * D10S1248 7 0.15* * * * 8 * * * 0.14* 9 0.15* * * 0.14* 10 * 0.33* * 0.14* 11 3.64 * 0.58* 0.27* 12 14.09 10.78 3.5 4.48 13 22.88 36.93 29.45 25.95 14 27.88 22.55 29.74 36.14 15 18.48 22.55 19.39 22.69 16 10.15 5.23 13.41 7.74 17 2.27 1.63 3.64 2.31 18 0.30* * 0.29* * 19 * * * * 20 * * * * Table 14 Y-specific frequencies by population group for the NGM Detect™ kit Y indel locus. (*=Alleles not detected or not detected in significant quantities) U.S. African American U.S. Hispanic Allele Asian (n = 65) Caucasian (n = 246) (n = 182) (n = 233) Y indel 1 1.22 67.69 * 0.55* 2 98.78 32.31 100 99.45 120 NGM Detect™ PCR Amplification Kit User Guide Chapter 6 Experiments and results Population data 6 Table 15 Probability of identity (PI) values for the NGM Detect™ kit STR loci African U.S. Caucasian U.S. Hispanic Locus American Asian (n = 153) (n = 343) (n = 368) (n = 330) D10S1248 0.0693 0.1045 0.0943 0.1131 D12S391 0.0377 0.0664 0.0231 0.0318 D16S539 0.0727 0.0915 0.1043 0.0809 D18S51 0.0322 0.0402 0.0311 0.0281 D19S433 0.0388 0.0663 0.0862 0.0484 D1S1656 0.034 0.0564 0.0223 0.0247 D21S11 0.0453 0.0671 0.052 0.0487 D22S1045 0.0559 0.1073 0.1309 0.1604 D2S1338 0.0225 0.0337 0.0316 0.0316 D2S441 0.103 0.0961 0.0976 0.1079 D3S1358 0.0984 0.1689 0.0749 0.0949 D8S1179 0.0762 0.0527 0.0631 0.0661 FGA 0.0322 0.0555 0.0384 0.0282 SE33 0.0118 0.0139 0.0085 0.0081 TH01 0.0949 0.175 0.0801 0.0902 vWA 0.0622 0.084 0.065 0.0926 Combined 7.96 × 10−22 2.25 × 10−19 2.29 × 10−21 3.12 × 10−21 Probability of Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match paternity Probability (MP), and p-value of each locus was calculated using a software program developed by Ge (Li et al., 2013) and shown in the following table. exclusion observation Departures from Hardy-Weinberg Equilibrium (HWE) expectations of each locus were derived using Arlequin (Excoffier et al., 2010). After Bonferroni correction (Weir, 1990), (p-value = 0.05/21 = 0.0024), no departures from HWE were observed at any locus. NGM Detect™ PCR Amplification Kit User Guide 121 Table 16 Allele frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Match probability (MP), and p-value of STR loci 122 6 African American Asian U.S. Caucasian U.S. Hispanic Population data Chapter 6 Experiments and results Marker p- p- p- p- Ho He MP Ho He MP Ho He MP Ho He MP value value value value Y-indel - - 0.976 - - - 0.556 - - - 1 - - - 1 - D3S1358 0.772 0.762 0.094 0.902 0.686 0.682 0.151 0.012 0.753 0.786 0.079 0.698 0.698 0.758 0.098 0.291 vWA 0.752 0.798 0.068 0.116 0.829 0.784 0.08 0.309 0.841 0.807 0.064 0.621 0.842 0.782 0.08 0.463 D16S539 0.772 0.797 0.071 0.026 0.8 0.764 0.092 0.457 0.801 0.749 0.103 0.047 0.77 0.774 0.084 0.835 D8S1179 0.782 0.793 0.068 0.662 0.814 0.828 0.052 0.795 0.793 0.797 0.065 0.122 0.755 0.802 0.064 0.518 D21S11 0.861 0.849 0.039 0.553 0.8 0.791 0.069 0.667 0.873 0.837 0.046 0.385 0.827 0.839 0.044 0.315 D18S51 0.931 0.868 0.031 0.324 0.829 0.853 0.038 0.572 0.873 0.872 0.03 0.962 0.849 0.87 0.031 0.945 D2S441 0.772 0.756 0.099 0.421 0.714 0.746 0.101 0.238 0.757 0.766 0.09 0.077 0.791 0.763 0.094 0.611 D19S433 0.812 0.825 0.051 0.663 0.714 0.802 0.064 0.13 0.785 0.774 0.083 0.859 0.82 0.834 0.046 0.446 TH01 0.762 0.747 0.102 0.418 0.614 0.656 0.171 0.381 0.753 0.783 0.081 0.326 0.77 0.767 0.091 0.845 FGA 0.782 0.866 0.033 0.082 0.9 0.841 0.044 0.24 0.829 0.857 0.037 0.337 0.842 0.882 0.025 0.127 D22S1045 0.842 0.822 0.055 0.062 0.743 0.742 0.112 0.966 0.705 0.714 0.131 0.026 0.698 0.672 0.162 0.064 NGM Detect™ PCR Amplification Kit User Guide SE33 0.96 0.929 0.009 0.776 0.943 0.936 0.008 0.526 0.968 0.947 0.005 0.532 0.921 0.941 0.007 0.597 D10S1248 0.792 0.789 0.075 0.823 0.757 0.764 0.091 0.928 0.785 0.769 0.09 0.63 0.691 0.724 0.124 0.336 D1S1656 0.921 0.863 0.033 0.351 0.757 0.818 0.056 0.043 0.912 0.899 0.019 0.55 0.871 0.896 0.02 0.048 D12S391 0.861 0.864 0.032 0.19 0.771 0.808 0.063 0.65 0.904 0.896 0.02 0.45 0.842 0.874 0.028 0.071 D2S1338 0.911 0.894 0.02 0.763 0.871 0.872 0.03 0.356 0.88 0.878 0.027 0.23 0.906 0.877 0.027 0.929 Chapter 6 Experiments and results Population data 6 The following table shows the Probability of paternity exclusion (PE) values of the NGM Detect™ kit STR loci individually and combined. The PE value is the probability, averaged over all possible mother-child pairs, that a random alleged father will be excluded from paternity after DNA typing using the NGM Detect™ kit STR loci (Chakraborty, Stivers, and Zhong, 1996). Table 17 Probability of paternity exclusion values for the NGM Detect™ kit STR loci Locus African American Asian (n = 153) Caucasian (n = 343) Hispanic (n = 368) (n = 330) D10S1248 0.6623 0.5353 0.5649 0.4644 D12S391 0.7401 0.631 0.8032 0.6588 D16S539 0.5548 0.6063 0.5915 0.5623 D18S51 0.7892 0.656 0.7557 0.7121 D19S433 0.6332 0.5238 0.5135 0.6431 D1S1656 0.7462 0.5703 0.8032 0.7338 D21S11 0.728 0.6063 0.7264 0.7013 D22S1045 0.7038 0.4795 0.4507 0.397 D2S1338 0.814 0.7463 0.7498 0.7392 D2S441 0.5228 0.5353 0.4986 0.5051 D3S1358 0.4918 0.3976 0.5338 0.4689 D8S1179 0.599 0.6063 0.6187 0.5381 FGA 0.728 0.8397 0.6632 0.7175 SE33 0.8639 0.88 0.9231 0.8781 TH01 0.5124 0.3424 0.5036 0.5381 vWA 0.6103 0.6186 0.6576 0.6276 PEi 7.8605 × 10−9 1.3136 × 10−7 1.2071 × 10−8 7.3910 × 10−8 Combined 0.9999999921 0.9999998686 0.9999999879 0.9999999261 NGM Detect™ PCR Amplification Kit User Guide 123 A Troubleshooting Observation Possible cause Recommended action Faint or no signal from both the The incorrect volume of Master Use the correct volume of Master Mix or DNA Control 007 and the DNA Mix or Primer Set was used. Primer Set. test samples at all loci, The DNA Polymerase was not Repeat the amplification with an initial hold at including the IQC markers activated. 95°C for 1 minute. The Master Mix was not Vortex the Master Mix thoroughly. vortexed thoroughly before aliquoting. The Primer Set was exposed to Replace the Primer Set and store it protected too much light. from light. Evaporation. Ensure that the plate is properly sealed with film and that a compression pad was used with the GeneAmp™ PCR System 9700. (A compression pad should not be used with other validated thermal cyclers.) The thermal cycler See the thermal cycler user manual and check malfunctioned. the instrument calibration. Incorrect thermal cycler Use correct thermal cycler conditions. conditions were used. A MicroAmp™ base was used Remove the MicroAmp™ base. with a tray/retainer set and tubes in GeneAmp™ PCR System 9700. The tubes or plate were not Push the tubes or plate firmly into the block seated tightly in the thermal after first cycle. cycler during amplification. The wrong PCR reaction tubes Use MicroAmp™ Reaction Tubes with Caps or or plate were used. the MicroAmp™ Optical 96‑well Reaction Plate for the GeneAmp™ PCR System 9700 or Veriti™ Thermal Cycler. Insufficient PCR product was Use correct genetic analyzer settings. electrokinetically injected. Degraded formamide was used. Check the storage of formamide. Do not thaw and refreeze multiple times. Try Hi‑Di™ Formamide. Positive signal from DNA The quantity of test DNA Quantify DNA and (when possible) add 500 pg Control 007 but partial or no sample is below the assay of DNA. For low concentration samples, add up signal from DNA test samples sensitivity. to 15 µL of the DNA sample to the reaction mix. when IQC peaks are present and balanced 124 NGM Detect™ PCR Amplification Kit User Guide Appendix A Troubleshooting Population data A Observation Possible cause Recommended action Positive signal from DNA The test sample DNA is Use the Quantifiler™ HP DNA Quantification Kit Control 007 but partial or no severely degraded. or the Quantifiler™ Trio DNA Quantification Kit signal from DNA test samples to evaluate sample quality during the when IQC peaks are present quantification step. If DNA is degraded, and balanced reamplify with an increased amount of DNA or consider using the Precision ID GlobalFiler™ NGS STR Panel. The test sample was diluted in Redilute DNA using low-TE buffer (with 0.1 mM the wrong buffer (for example, EDTA). a TE buffer with an incorrect EDTA concentration). Positive signal from DNA The test sample contains a high Quantify the DNA, then use the minimum Control 007 but partial or no concentration of PCR inhibitor necessary volume of test sample DNA. signal from DNA test samples (for example, heme Wash the sample in a Centricon™-100 when IQC peaks are present compounds, certain dyes). centrifugal filter unit. and unbalanced The test sample was diluted in Redilute DNA using low-TE buffer (with 0.1 mM the wrong buffer (for example, EDTA). a TE buffer with an incorrect EDTA concentration). Positive signal from DNA The quantity of the test sample Quantify DNA, then use 500 pg. Control 007 and elevated signal DNA is in excess of from DNA test samples when recommended input amount, IQC peaks are present and which can cause loss of balance unbalanced in IQC peaks. More than two alleles present Exogenous DNA is present in Use appropriate techniques to avoid at a locus the sample. introducing foreign DNA during laboratory handling. Stutter product (–1 repeat unit Ensure that stutter filters are applied. position) was amplified. Stutter filters apply to sample Increase the sample input above 250 pg, if input amounts equal or greater possible. (Note, the optimum sample input is than 250 pg. Samples well 500 pg.) below the recommended input amount may exhibit stutters values exceeding the filters due to the stochastic effects of the PCR. The test sample contained If a mixed profile is not expected, check that mixed DNA. laboratory protocols relating to cleanliness are followed. Incomplete 3´ A base addition Include the final extension step of 60°C for (n−1 nt position) occured. 5 minutes in the PCR. Remove amplified plate from storage (thaw if necessary) and place on thermal cycler at 60°C for 15 minutes. Check the quantity of the original sample DNA to ensure input is less than 750 pg per reaction. Adjust input as necessary during re- amplification. NGM Detect™ PCR Amplification Kit User Guide 125 A Appendix A Troubleshooting Population data Observation Possible cause Recommended action More than two alleles present If the total amount of DNA in the reaction at a locus exceeds 1 ng, adjust the final extension time to 15 minutes to minimize incomplete 3′ A base addition. The signal exceeds the dynamic Check that you are using the recommended range of the instrument and is number of PCR cycles. Repeat PCR causing signal "pull-up" into amplification using reduced input DNA amount, adjacent channels. or interpret the off-scale data according to your laboratory procedure. Check that you are using the recommended injection conditions on the instrument. Poor spectral separation Perform a spectral calibration. occurred. Confirm that Filter Set J6-T modules are installed and used for analysis. Too much DNA was present in Use the recommended amount of template the reaction. DNA: 500 pg for 30 PCR cycles. The double-stranded DNA was Use the recommended amount of Hi‑Di™ not completely denatured. Formamide and heat the sample plate at 95°C for 3 minutes. Poor peak height balance Incorrect thermal cycler Use correct thermal cycler conditions. conditions were used. Some but not all loci visible on The PCR reaction volume you Use the correct PCR reaction volume: 25 μL electropherogram of DNA Test used is lower than the volume Samples required for the amplification. STR profiles contain many off- DNA quantification was not Ensure that DNA quantification is accurate. scale alleles performed or not accurate. 126 NGM Detect™ PCR Amplification Kit User Guide B Materials required but not supplied Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier. STR kit required materials Item Source NGM Detect™ PCR Amplification Kit, 200 reactions A31832 Sample preparation required materials Item Source GeneScan™ 600 LIZ™ Size Standard v2.0, 2 × 200 µL 4408399 IMPORTANT! Do not use GeneScan™ 350 ROX™, GeneScan™ 500 ROX™, or GeneScan™ 500 LIZ™ Size Standards with this kit. Low-TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Teknova T0223 or see “Prepare low-TE buffer“ on page 19. Hi‑Di™ Formamide, 25‑mL 4311320 Thermal cycler required materials ProFlex™ PCR System Item Source ProFlex™ 96‑well PCR System 4484075 ™ ProFlex 2 × 96‑well PCR System 4484076 ProFlex™ 3 × 32‑Well PCR System 4484073 NGM Detect™ PCR Amplification Kit User Guide 127 B Appendix B Materials required but not supplied Genetic analyzer required materials Veriti™ Thermal Cycler Item Source Veriti™ 96‑Well Thermal Cycler 4479071 (Optional) Tabletop centrifuge with 96-Well Plate Adapters MLS GeneAmp™ PCR System 9700 Item Source GeneAmp™ PCR System 9700, 96-Well Silver N8050001 ™ GeneAmp PCR System 9700, 96-Well Gold-Plated 4314878 Silver 96-Well Sample Block N8050251 Gold-Plated 96-Well Block 4314443 Genetic analyzer required materials 3500 Series Genetic Analyzer Item Source 3500 Series Data Collection Software 3.1 Upgrade (RUO) A26287[1] 3500 Series Data Collection Software 3.1 (RUO) 4475183[1] HID Updater 3500 Data Collection Software v2 4480670 Anode buffer container (ABC) 4393927 Cathode buffer container (CBC) 4408256 POP-4™ Polymer (960 samples) for 3500/3500xL Genetic Analyzers 4393710 POP-4™ Polymer (384 samples) for 3500/3500xL Genetic Analyzers 4393715 DS-37 Matrix Standard Kit (Dye Set J6-T) A31234 Conditioning reagent 4393718 8-Capillary array, 36 cm for 3500 Genetic Analyzers 4404683 24-Capillary array, 36 cm for 3500xL Genetic Analyzers 4404687 96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers 4410228 8-Tube retainer & base set (Standard) for 3500/3500xL Genetic Analyzers 4410231 8-Strip Septa for 3500/3500xL Genetic Analyzers 4410701 128 NGM Detect™ PCR Amplification Kit User Guide Appendix B Materials required but not supplied Analysis software required materials B Item Source 96-Well Septa for 3500/3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container, 3500 series 4410715 [1] Contact your Thermo Fisher Scientific HID representative. 3130 Series Genetic Analyzer required materials Item Source 3130 Data Collection Software v4 4475105 3130xl Data Collection Software‑v4 4475126 3130/3730 Data Collection Software‑v4 6‑Dye Module v1 4478404 96‑Well Plate Septa 4315933 Reservoir Septa 4315932 3130/3130xl Genetic Analyzer 16‑Capillary Array, 36 cm 4315931 POP-4™ Polymer for the 3130/3130xl Genetic Analyzer 4352755 Running Buffer, 10✕ 402824 DS‑37 Matrix Standard Kit (Dye Set J6-T) A31234 MicroAmp™ Optical 96-Well Reaction Plate N8010560 Analysis software required materials GeneMapper™ In addition to the GeneMapper™ ID-X Software listed in the following table, a v.1.5.2 ID‑X Software patch is required. The patch enables full functionality of the data quality assessment tools when using the NGM Detect™ PCR Amplification Kit. v1.5.2 patch The patch is available for free download at thermofisher.com/us/en/home/ technical-resources/software-downloads/genemapper-id-x-software.html. GeneMapper™ ID‑X Software Item Source GeneMapper™ ID‑X Software v1.5 Full Installation A27884 GeneMapper™ ID‑X Software v1.5 Client Installation A27886 GeneMapper™ ID‑X Software v1.4 Full Installation 4479707 GeneMapper™ ID‑X Software v1.4 Client Installation 4479711 NGM Detect™ PCR Amplification Kit User Guide 129 B Appendix B Materials required but not supplied Miscellaneous required materials Miscellaneous required materials Plates and tubes Item Source MicroAmp™ 96-Well Tray N8010541 MicroAmp™ Reaction Tube with Cap, 0.2 mL N8010540 MicroAmp™ 8-Tube Strip, 0.2 mL N8010580 MicroAmp™ Optical 8-Cap Strips 4323032 MicroAmp™ 96-Well Tray/Retainer Set 403081 MicroAmp™ 96-Well Base N8010531 MicroAmp™ Clear Adhesive Film 4306311 MicroAmp™ Optical Adhesive Film 4311971 MicroAmp™ Optical 96-Well Reaction Plate N8010560 Laboratory supplies Item Source Various procedures Aerosol resistant pipette tips MLS[1] Microcentrifuge tubes MLS Pipettors MLS Tape, labeling MLS ™ Tube, 50-mL Falcon MLS Tube decapper, autoclavable MLS Deionized water, PCR grade MLS Vortex MLS [1] Major laboratory supplier 130 NGM Detect™ PCR Amplification Kit User Guide C PCR work areas ■ Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 ■ PCR setup work area materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 ■ Amplified DNA work area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory. If you are using this kit for: • Forensic DNA testing, see "Forensic Laboratories: Handbook for Facility Planning, Design, Construction, and Moving", National Institute of Justice, 1998 • Parentage DNA testing, see the "Guidance for Standards for Parentage Relationship Testing Laboratories", American Association of Blood Banks, 7th edition, 2004 The sensitivity of this kit (and other PCR-based tests) enables amplification of minute quantities of DNA, necessitating precautions to avoid contamination of samples yet to be amplified (Kwok and Higuchi, 1989). Process samples carefully to prevent contamination by human DNA. Wear gloves at all times and change them frequently. Close sample tubes when not in use. Limit aerosol dispersal by handling sample tubes and reagents carefully. Note: We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology. PCR setup work area materials IMPORTANT! Do not remove these items from the PCR Setup Work Area. • Calculator • Gloves, disposable • Marker pen, permanent • Microcentrifuge • Microcentrifuge tubes, 1.5-mL, or 2.0-mL, or other appropriate nuclease-free tube (for master mix preparation) • Microcentrifuge tube rack • Pipette tips, sterile, disposable hydrophobic filter-plugged • Pipettors NGM Detect™ PCR Amplification Kit User Guide 131 C Appendix C PCR work areas Amplified DNA work area • Tube decapper, autoclavable • Vortex Amplified DNA work area IMPORTANT! Place the thermal cyclers in the Amplified DNA Work Area. Use only the validated thermal cyclers listed in “Instruments and software compatibility“ on page 15. 132 NGM Detect™ PCR Amplification Kit User Guide D Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. · Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. · Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain SDSs, see the “Documentation and Support” section in this document. NGM Detect™ PCR Amplification Kit User Guide 133 D Appendix D Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions: · Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document. · Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). · Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). · Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. · Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) · After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. · Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. · IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. 134 NGM Detect™ PCR Amplification Kit User Guide Appendix D Safety Biological hazard safety D Biological hazard safety WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment. · U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at: www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf · World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf NGM Detect™ PCR Amplification Kit User Guide 135 Documentation and support Related documentation Document title Pub. No. STR kits NGM Detect™ PCR Amplification Kit – PCR Amplification and CE Quick Reference 100044088 NGM Detect™ PCR Amplification Kit – PCR Setup Quick Reference 100044087 Technical Note: Updated NGM Detect™ PCR Amplification Kit: Validation and Comparative Study — Note: Go to thermofisher.com/document-connect/document-connect.html?url=https:// assets.thermofisher.com/TFS-Assets/GSD/Technical-Notes/ngm-detect-results-tech-note.pdf to open the Technical Note. Quantification kits Quantifiler™ HP and Quantifiler™ Trio DNA Quantification Kits User Guide 4485355 Thermal cyclers ProFlex™ PCR System User Guide MAN0007697 Veriti™ Thermal Cycler User Guide 4375799 GeneAmp PCR System 9700 Base Module User Manual ™ 4303481 3500 Series Genetic Analyzer 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v1 User Guide 4401661 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software v2 User Guide 4476988 HID Updater 3500 Data Collection Software v2.0 User Bulletin NA 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3.1 User Guide 100031809 3500 Series Data Collection Software v3 User Bulletin: New Features and HID Validation MAN0010812 Summary 3500 Series Data Collection Software v3.1 User Bulletin: New Features and HID Validation MAN0014110 Summary 3130xl Series Genetic Analyzer 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide 4352716 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0 User Bulletin 4363787 3130/3130xl Genetic Analyzers Getting Started Guide 4352715 136 NGM Detect™ PCR Amplification Kit User Guide Documentation and support Related documentation Document title Pub. No. 3130/3130xl Genetic Analyzers Quick Reference Card 4362825 3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 GeneMapper™ ID‑X Software all versions GeneMapper™ ID‑X Software Bin Overlap User Bulletin 100029546 GeneMapper™ ID‑X Software v1.0 GeneMapper™ ID‑X Software v1.0 Getting Started Guide— Basic Features 4375574 GeneMapper™ ID‑X Software v1.0 Quick Reference— Basic Features 4375670 GeneMapper™ ID‑X Software v1.0 Installation Guide 4476603 GeneMapper™ ID‑X Software v1.0 Administrator Guide 4376327 GeneMapper™ ID‑X Software v1.0 Reference Guide 4375671 GeneMapper™ ID‑X Software v1.1 GeneMapper™ ID‑X Software v1.1 Getting Started Guide— Mixture Analysis Tool 4396773 GeneMapper™ ID‑X Software v1.2 GeneMapper™ ID‑X Software v1.2 Verification Experiments and Installation Procedures User 4462639 Bulletin GeneMapper™ ID‑X Software v1.2 Quick Reference— Mixture Analysis Tool 4426482 GeneMapper™ ID‑X Software v1.2 Reference Guide 4426481 GeneMapper™ ID‑X Software v1.3 GeneMapper™ ID‑X Software v1.3 Verification Experiments and Installation Procedures User 4470483 Bulletin GeneMapper™ ID‑X Software v1.4 GeneMapper™ ID‑X Software v1.4 New Features and Installation Procedures User Bulletin 4477684 GeneMapper™ ID‑X Software v1.5 GeneMapper™ ID‑X Software v1.5 New Features and Verification User Bulletin 100031708 GeneMapper™ ID‑X Software v1.5 Getting Started Guide— Basic Features 100031701 GeneMapper™ ID‑X Software v1.5 Quick Reference— Basic Features 100031702 GeneMapper™ ID‑X Software v1.5 Getting Started Guide— Mixture Analysis Tool 100031704 GeneMapper™ ID‑X Software v1.5 Quick Reference— Mixture Analysis Tool 100031705 GeneMapper™ ID‑X Software v1.5 Installation Guide 100031706 GeneMapper™ ID‑X Software v1.5 Administrator Guide 100031703 GeneMapper™ ID‑X Software v1.5 Reference Guide 100031707 NGM Detect™ PCR Amplification Kit User Guide 137 Documentation and support Customer and technical support Customer and technical support For support: • In North America—Send an email to [email protected], or call 888-821-4443 option 1. • Outside North America—Contact your local support office. For the latest services and support information for all locations, go to thermofisher.com/support to obtain the following information. • Worldwide contact telephone numbers • Product support • Order and web support • Safety Data Sheets (SDSs; also known as MSDSs) Additional product documentation, including user guides and Certificates of Analysis, are available by contacting Customer Support. Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support. 138 NGM Detect™ PCR Amplification Kit User Guide References Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. 1994. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J. Forensic Sci. 39:362–372. Barber, M.D. and Parkin, B.H. 1996. Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179. Intl. J. Legal Med. 109:62–65 Barber, M.D., Piercy, R.C., Andersen, J.F. and Parkin, B.H. 1995. Structural variation of novel alleles at the Hum vWA and Hum FES/FPS short tandem repeat loci. Int. J. Leg. Med. 108:31–35. Barber, M.D., McKeown, B.J. and Parkin, B.H. 1996. Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus. Int. J. Leg. Med. 108:180–185. Begovich A.B., McClure G.R., Suraj V.C., Helmuth R.C., Fildes N., Bugawan T.L., Erlich H.A., Klitz W. 1992. Polymorphism, recombination, and linkage disequilibrium within the HLA class II region. J. Immunol. 148:249–258. Bender, K., Farfan, M.J., Schneider, P.M. 2004. Preparation of degraded human DNA under controlled conditions. Forensic Sci. Int. 139:134–140. Brinkmann, B., Klintschar, M., Neuhuber, F., Huhne, J. and Rolf, B. 1998. Mutation rate in human microsatellites: Influence of the structure and length of the tandem repeat. Am. J. Hum. Genet. 62:1408–1415. Budowle, B. et al. 2010. Population genetic analyses of the NGM STR loci. Int. J. Legal Med. e-publication www.springerlink.com/content/p713q3w5440674u3/ Butler, J.M. 2005. Forensic DNA Typing. Burlington, MA:Elsevier Academic Press. Chakraborty, R., Stivers, D., and Zhong, Y. 1996. Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci. Mutat. Res. 354:41–48. Clark J.M. 1988. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16:9677–9686. DeFranchis, R., Cross, N.C.P., Foulkes, N.S., and Cox, T.M. 1988. A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA. Nucleic Acids Res. 16:10355. DNA Advisory Board, Federal Bureau of Investigation, U.S. Department of Justice. 1998. Quality assurance standards for forensic DNA testing laboratories. Edwards, A., Hammond, H.A., Lin, J., Caskey, C.T., and Chakraborty, R. 1992. Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12:241–253. Excoffier, L., Lischer, H.E.L. 2010. A new series of programs to perform population genetics analyses under Linux and Windows. Arleguin suite v. 3.5. Mol. Ecol. Res. 10:564–567. NGM Detect™ PCR Amplification Kit User Guide 139 References Limited product warranty Frank, W., Llewellyn, B., Fish, P., et al. 2001. Validation of the AmpFlSTR Profiler Plus PCR Amplification Kit for use in forensic casework. J. Forensic Sci. 46:642–646. Holt, C., Stauffer, C., Wallin, J., et al. 2000. Practical applications of genotypic Surveys for forensic STR testing. Forensic Sci. Int. 112:91–109. Kimpton, C., Walton, A., and Gill, P. 1992. A further tetranucleotide repeat polymorphism in the vWF gene. Hum. Mol. Genet. 1:287. Kong, X., Murphy, K., Raj, T., He, C., White, P.S., Matise, T.C. 2004. A combined linkage-physical map of the human genome. Am. J. Hum. Genet. 75:1143–1148. Lareu, M.V., Pestoni, M.C., Barros, F., Salas, A., Carracedo, A. 1996. Sequence variation of a hypervariable short tandem repeat at the D12S391 locus. Gene 182:151–153. Lazaruk, K., Walsh, P.S., Oaks, F., Gilbert, D., Rosenblum, B.B., Menchen, S., Scheibler, D., Wenz, H.M., Holt, C., Wallin, J. 1998. Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument. Electrophoresis 19:86–93. Li, H. Schmidt, L., Wei, M-H., Hustad, T. Leman, M.I., Zbar, B. and Tory, K. 1993. Three tetranucleotide polymorphisms for loci:D3S1352; D3S1358; D3S1359. Hum. Mol. Genet. 2:1327. Li, B., Ge, J., Wu, F., Ye, L., Budowle, B., Vhen, Y. 2013. Population genetic analyses of the STR loci of the AmpFlSTR NGM SElect PCR Amplification Kit for Han population in Fujian Province, China. Int J. Legal Med. 127:345–346. Magnuson, V.L., Ally, D.S., Nylund, S.J., Karanjawala, Z.E., Rayman, J.B., Knapp, J.I., Lowe, A.L., Ghosh, S., Collins, F.S. 1996. Substrate nucleotide-determined nontemplated addition of adenine by Taq DNA polymerase: implications for PCR- based genotyping and cloning. Biotechniques 21:700–709. Mansfield, E.S., Robertson, J.M., Vainer, M., Isenberg, A.R., Frazier, R.R., Ferguson, K., Chow, S., Harris, D.W., Barker, D.L., Gill, P.D., Budowle, B., McCord, B.R. 1998. Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis. Electrophoresis 19:101–107. Mills, K.A., Even, D., and Murrau, J.C. 1992. Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA). Hum. Mol. Genet. 1:779. Möller, A. and Brinkmann, B. 1994. Locus ACTBP2 (SE33): Sequencing data reveal considerable polymorphism. Int. J. Leg. Med. 106:262–267. Möller, A. and Brinkmann, B. 1995. PCR-VNTRs (PCR-Variable Number of Tandem Repeats) in forensic science. Cellular & Molec. Bio. 41(5):715-724. Momhinweg, E., Luckenbach, C., Fimmers, R., and Ritter, H. 1998. D3S1358: sequence analysis and gene frequency in a German population. Forensic Sci. Int. 95:173–178. Momhinweg, E., Luckenbach, C., Fimmers, R., and Ritter, H. 1998. D3S1358: sequence analysis and gene frequency in a German population. Forensic Sci. Int. 95:173–178. Moretti, T., Baumstark, A., Defenbaugh, D., Keys, K., Smerick, J., and Budowle, B. 2001. Validation of short tandem repeats (STRs) for forensic usage: Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J. Forensic Sci. 46(3):647–660. Mulero, J.J., Chang, C.W., and Hennessy, L.K. 2006. Characterization of N+3 stutter product in the trinucleotide repeat locus DYS392. J. Forensic Sci. 51:826–830. Nakahori, Y., Takenaka, O., and Nakagome, Y. 1991. A human X-Y homologous region encodes amelogenin. Genomics 9:264–269. 140 NGM Detect™ PCR Amplification Kit User Guide References Limited product warranty Puers C., Hammond H.A., Jin L., Caskey C.T., Schumm J.W. 1993. Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTH01[AATG]n and reassignment of alleles in population analysis by using a locus-specific allelic ladder. Am J. Hum. Genet. 53(4):953–958. Raymond M. and Rousset F. 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86:248–249. Rousset, F. 2008. Genepop'007: A complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources 8:103–106. Sensabaugh, G.F. 1982. Biochemical markers of individuality. In: Saferstein, R., ed. Forensic Science Handbook. Prentice-Hall, Inc., New York, pp. 338–415. Sharma, V. and Litt, M. 1992. Tetranucleotide repeat polymorphism at the D21S11 locus. Hum Mol. Genet. 1:67. Smith, R.N. 1995. Accurate size comparison of short tandem repeat alleles amplified by PCR. Biotechniques 18:122–128. Sparkes, R., Kimpton, C., Watson, S., Oldroyd, N., Clayton, T., Barnett, L., Arnold, J., Thompson, C., Hale, R., Chapman, J., Urquhart, A., and Gill, P. 1996a. The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies. Int. J. Legal Med. 109:186–194. Sparkes, R., Kimpton, C., Gilbard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D., Urquhart, A., and Gill, P. 1996b. The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artifacts, casework studies and success rates. Int. J. Legal Med. 109:195–204. Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J., and Gilliam, T.C. 1993. A microsatellite genetic linkage map of human chromosome 18. Genomics 15:48– 56. Scientific Working Group on DNA Analysis Methods (SWGDAM). 2016. Validation Guidelines for DNA Analysis Methods. Available at http://media.wix.com/ugd/ 4344b0_813b241e8944497e99b9c45b163b76bd.pdf Accessed 22 February 2017. Wallin, J.M., Buoncristiani, M.R., Lazaruk, K.D., Fildes, N., Holt, C.L., Walsh, P.S. 1998. SWGDAM validation of the AmpFlSTR blue PCR amplification kit for forensic casework analysis. J. Forensic Sci. 43:854–870. Wallin, J.M., Holt, C.L., Lazaruk, K.D., Nguyen, T.H., Walsh, P.S. 2002. Constructing universal multiplex PCR systems for comparative genotyping. J. Forensic Sci. 47:52–65. Walsh, P.S., Fildes, N.J., Reynolds, R. 1996. Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 24:2807– 2812. Watson, S., Kelsey, Z., Webb, R., Evans, J., and Gill, P. 1998. The development of a third generation STR multiplex system (TGM). Olaisen, B., Brinkmann, B., and Lincoln, P.J., eds. Progress in Forensic Genetics 7: Proceedings of the 17th International ISFH Congress, Oslo 2-6 September 1997. Elsevier, Amsterdam, pp. 192–194. Weir, B. 1990. Genetic Data Analysis. Sinauer Associates Sunderland, MA. NGM Detect™ PCR Amplification Kit User Guide 141 Index +A 85 references 24, 28 +A nucleotide addition 85 run module 24, 28 setup of the 3130 instrument 28 setup of the 3130xl instrument 28 3' A 85, 86 setup of the 3500 and 3500xL instruments 24 3130 instrument 28 extra peaks 77 3500 instrument 24 6-dye license activation 28 G spectral calibration 26, 30 GeneScan 600 LIZ Size Standard v2.0 48 600 LIZ Size Standard v2.0 48 GeneScan size standard, about 11 A I accuracy and reproducibility 63 import panels, bins, and marker stutter 36 alleles, off-ladder 65 instrument and software compatibility 15 allelic ladder, requirements for electrophoresis 23 artifacts 87 L limited product warranty 138 B LIZ size standard bins, import 36 about 11 biohazard safety 135 peak sizes 48 volume per reaction 27, 30 LIZ Size Standard v2.0 48 C characterization of loci, validation 88 control DNA M 007 11 marker stutter, import 36 profile 13 materials not supplied 127 D P developmental validation 57 panels direct amplification 22 check version 35 DNA control profile 13 import 36 documentation, related 136 PCR DS-37 matrix standard 26, 30 conditions 21 dye set for 6-dye samples 26, 30 perform 21 setup 131 work areas 131 E electrophoresis data collection software 24, 28 Q prepare samples 27, 30 quantification, DNA 17 142 NGM Detect™ PCR Amplification Kit User Guide Index R T required materials 127 terms and conditions 138 run module for electrophoresis thermal cyclers 3130 instrument 28 for use with kit 15 3130xl instrument 28 programming 21 3500 and 3500xL instruments 24 troubleshooting 124 S V safety, biohazard 135 validation, importance 56 sensitivity 92 size standard 48 spectral calibration 26, 30 W stutter, peaks 83 warranty 138 stutter file, import 36 work area, PCR setup 131 NGM Detect™ PCR Amplification Kit User Guide 143 thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 2 January 2019 QuantStudio™ 5 Real-Time PCR Instrument (for Human Identification) USER GUIDE Installation, maintenance, and administration for use with: HID Real‑Time PCR Analysis Software v1.3 and v1.4 Publication Number MAN0017162 Revision B.0 For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures. Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256 Revision history: MAN0017162 B.0 (English) Revision Date Description B.0 9 November 2022 • Updated to include HID Real‑Time PCR Analysis Software v1.4. • Updated to include instrument firmware v1.5.1. • For the instrument-to-computer connection, added firewall port 7443 (for instruments with firmware v1.5.1). A.0 15 June 2017 New document for Human Identification workflows using instrument firmware v1.3.1. The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Microsoft and Windows are trademarks of Microsoft Corporation. Pentium is a trademark of Intel Corporation. ©2022 Thermo Fisher Scientific Inc. All rights reserved. Contents ■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Instrument overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Installation, verification, and calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Instrument filters and supported dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 System dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Custom dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Parts of the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Parts of the home screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Comparison of features in desktop software and the instrument . . . . . . . . . . . . . . . . . . . . . 13 ■ CHAPTER 2 Install the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Workflow: Install the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Before you begin installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Unpack and install the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Power on and follow the startup wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Instrument and computer connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Computer-to-instrument configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Firewall ports that must be open . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ■ CHAPTER 3 General procedures to operate the instrument . . . . . . . . . . . . . . . . . . . 18 Precautions for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Power on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Power off the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Sign in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Sign out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Load and unload plate in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 View real-time data and plots on the instrument touchscreen . . . . . . . . . . . . . . . . . . . . . . . . 22 Adjust the display of real-time plots on the instrument touchscreen . . . . . . . . . . . . . . 22 Transfer EDS files from the instrument home screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 3 Contents ■ CHAPTER 4 Configure the instrument and manage instrument profiles . . . . . 23 Initial start up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Overview of instrument settings (Administrator) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Overview of instrument settings (Standard or Guest) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Manage instrument profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Instrument profiles and signed-in users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Create an administrator instrument profile during initial start-up . . . . . . . . . . . . . . . . . 28 Create a new instrument profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Edit an instrument profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Manage all instrument profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Link an instrument profile to the Thermo Fisher™ Connect Platform . . . . . . . . . . . . . . . . . . 30 Recommended order to set up profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Overview of local instrument profiles and Thermo Fisher™ Connect Platform profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Thermo Fisher™ Connect Platform instrument profile roles and functions . . . . . . . . . 32 Test the connection to the Thermo Fisher™ Connect Platform . . . . . . . . . . . . . . . . . . . 32 Link the instrument to your Thermo Fisher™ Connect Platform account . . . . . . . . . . . 33 Link a local profile to a Thermo Fisher™ Connect Platform profile . . . . . . . . . . . . . . . . 34 Unlink a Thermo Fisher™ Connect Platform account . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 If you link when you are signed in to the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 If you link when you are not signed in to the instrument . . . . . . . . . . . . . . . . . . . . . . . . . 37 Change the PIN for a Thermo Fisher™ Connect Platform profile . . . . . . . . . . . . . . . . . . 38 Require instrument profile sign-in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Manage the Sign Out Timer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Manage the instrument name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Enable Sleep Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Set the idling temperature for the heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Set the date and time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Restore factory defaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Enable Remote Monitoring Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Update instrument software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 ■ CHAPTER 5 Calibrate and verify instrument performance . . . . . . . . . . . . . . . . . . . . . 42 Calibration and verification schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Calibration descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 View calibration status and set reminders in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Perform ROI/uniformity, background, and dye calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Workflow: Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Prepare a calibration plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Perform calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 View calibration images and transfer results to USB . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Contents Identify contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Create a background plate (optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Calibrate dyes for HID‑validated workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Workflow: Calibrate dyes for HID‑validated workflows . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Prepare a calibration plate for HID‑validated workflows . . . . . . . . . . . . . . . . . . . . . . . . . 51 Add custom dyes to the instrument for HID‑validated workflows . . . . . . . . . . . . . . . . . 52 Perform a custom dye calibration for HID‑validated workflows . . . . . . . . . . . . . . . . . . . 53 Perform instrument verification using RNase P plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Instrument verification description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 RNase P instrument verification plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Performance specifications pass criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Prepare an RNase P plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Perform RNase P verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 ■ CHAPTER 6 Maintain the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Backup or restore the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Decontaminate the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Clean the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Detailed procedures for cleaning the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Replace the instrument fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Replace the fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Store, move, or ship the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Prepare the instrument to store, move, or ship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Move the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Return the instrument for service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 ■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Instrument troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Troubleshoot calibration failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Troubleshoot verification failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 ■ APPENDIX B Parts and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Kits, consumables, accessories, and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Consumables (96‑well, 0.2‑mL format) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 General-use materials and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 5 Contents ■ APPENDIX C Instrument specification and layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Configured system dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Instrument and computer connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Instrument clearances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Electrical requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Environmental requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 ■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Conformity symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Safety alerts on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Location of safety labels on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Safety information for instruments not manufactured by Thermo Fisher Scientific . . . . . . 82 Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Physical injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Electrical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Cleaning and decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Safety compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 EMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Environmental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 ■ APPENDIX E Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Obtain information from the Help system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 1 Product information ■ Instrument overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 ■ Installation, verification, and calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 ■ Instrument filters and supported dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 ■ Parts of the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 ■ Parts of the home screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 ■ Comparison of features in desktop software and the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Instrument overview The QuantStudio™ 5 Real-Time PCR Instrument uses fluorescence-based polymerase chain reaction (PCR) reagents to perform: • Quantitative detection of target nucleic acid sequences (targets). • Qualitative detection of targets (endpoint analysis, genotyping, and presence/absence). The instrument is configured with a 96‑well 0.2‑mL fixed block (6 color de‑coupled). To run experiments using HID assays, the instrument must be integrated with the HID Real‑Time PCR Analysis Software. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 7 Chapter 1 Product information 1 Installation, verification, and calibration Installation, verification, and calibration Your HID Support Representative will contact you to schedule the installation. During instrument installation, the service representative will perform the initial instrument verification using an RNase P plate and calibrate the instrument for the versions of ABY™ and JUN™ dye used by HID‑validated workflows. Instruments are factory calibrated, so ROI, uniformity, background, and system dye calibrations are not necessary at installation. You do have the option to install the instrument yourself. Before first use of the instrument, complete the following tasks: • Install the instrument (see page 14). • Verify instrument performance (see page 55). • Perform custom dye calibrations for ABY™ and JUN™ dyes (see page 51). After installation, perform regular calibration and verification according to the “Calibration and verification schedule” on page 43. 8 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 1 Product information Instrument filters and supported dyes 1 Instrument filters and supported dyes System dyes The instrument uses a de-coupled six-color optical filter set that supports the dyes shown in the following table and figure. For more information about the spectral dye calibration kits available for the instrument, contact your HID Support Representative. Peak Filter wavelength (nm)[1] Color Factory-calibrated dyes Example custom dyes filter Excitation Emission FAM™ dye, SYBR™ Green x1-m1 Blue 470 ± 15 nm 520 ± 15 nm SYT09 dye JOE™ dye, x2-m2 Green 520 ± 10 nm 558 ± 12 nm VIC™ dye HEX dye, TET™ dye[2] ™ NED™ dye, TAMRA™ dye, x3-m3 Yellow 550 ± 10 nm 587 ± 10 nm Cy®3 dye ABY™ dye [3] x4-m4 Orange 580 ± 10 nm 623 ± 14 nm ROX™ dye, JUN™ dye [3] Texas Red™ dye MUSTANG PURPLE™ dye, x5-m5 Red 640 ± 10 nm 682 ± 14 nm LIZ™ dye Cy™5 Deep- x6-m6 662 ± 10 nm 711 ± 12 nm None [4] Cy® 5.5 dye Red [1] The central wavelengths are the optimized wavelengths. [2] The HEX™ and TET™ dyes from Thermo Fisher Scientific fall within the emission wavelength range of the system, therefore they can be added and adapted for use on the instrument. [3] HID-validated workflows use versions of ABY™ and JUN™ dyes that are considered custom dyes. [4] This filter set currently does not support any dyes supplied by Thermo Fisher Scientific. Wavelength (nm) Emission Spectra x1-m1 x2-m2 x3-m3 x4-m4 x5-m5 x6-m6 Filters 1 2 3 4 5 1 x1-m1 — FAM™ dye, SYBR™ Green dye 2 x2-m2 — VIC™ dye 3 x3-m3 — ABY™ dye, NED™ dye, Cy®3 dye, TAMRA™ dye 4 x4-m4 — JUN™ dye, ROX™ dye, Texas Red™ dye 5 x5-m5 — Cy™5 dye, MUSTANG PURPLE™ dye QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 9 Chapter 1 Product information 1 Parts of the instrument Custom dyes The instrument can run assays designed with custom dyes. Custom dyes include: • Dyes that are not supplied by Thermo Fisher Scientific. • Dyes or formulations of dyes that are not system dyes for the instrument. Note: HID-validated workflows use versions of ABY™ and JUN™ dyes that are considered custom dyes for the instrument. To calibrate these ABY™ and JUN™ dyes, see page 51. To calibrate any other custom dye, contact your HID Support Representative. Parts of the instrument 1 Touchscreen – Controls the instrument. 2 USB port – For connection to an external network drive or external data storage device. 1 3 Instrument drawer – Contains sample plate. 2 3 The instrument includes three additional USB ports on the back of the instrument. Note: The instrument recognizes only one external storage device at a time for data transfer. 10 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 1 Product information Parts of the home screen 1 Parts of the home screen 1 2 3 4 5 9 6 8 7 1 Avatar and Instrument name 6 Settings button 2 Eject instrument drawer icon 7 Access templates buttons (not applicable for 3 Help system icon HID‑validated workflows) 4 Status dial 8 Connectivity icons 5 Instrument profile name; instrument block type 9 Sign In (or My Profile) button Table 1 Parts of the home screen Element of the home screen Function Avatar and Instrument name Set by the administrator to uniquely identify the instrument. Eject instrument drawer icon Touch to open or close the instrument drawer. Help icon Touch to launch the touchscreen Help system to access step-by-step instructions. Status dial When the instrument is not in use – Displays Set up run. Note: Instrument runs must be set up and started from the desktop software. When the instrument is in use – Displays the sample block temperature, the elapsed run time, and the run status. Note: Swipe the dial to the left or touch to access real-time views of the run. Instrument profile username Displays the username of a signed-in user and the instrument block type. and block type Note: If no user is signed in, the instrument defaults to the Guest profile. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 11 Chapter 1 Product information 1 Parts of the home screen Table 1 Parts of the home screen (continued) Element of the home screen Function Settings button Touch Settings to configure, calibrate, or learn about the instrument. Connectivity icons • – The instrument is connected via a wired configuration. • – A USB drive is plugged into the instrument. Sign In button • Touch Sign In to sign into an instrument profile. (My Profile button when a user • Touch My Profile to change instrument profile settings. is signed in) 12 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 1 Product information Comparison of features in desktop software and the instrument 1 Comparison of features in desktop software and the instrument To run experiments using HID assays, the instrument must be integrated with the HID Real‑Time PCR Analysis Software. Actions Desktop software Instrument Enter or edit properties [1] Edit experiment name; enter user name ✓ — Select instrument, experiment type, chemistry, run mode ✓ — Edit method [1] Edit the thermal protocol, reaction volume, optical filter selection ✓ — Set up plate and well details [1] Define samples ✓ — Assign samples to wells ✓ — Define targets ✓ — Assign targets to wells ✓ — Instrument run Start an instrument run [1] ✓ — Monitor a run in progress ✓ ✓ View time remaining ✓ ✓ View real-time plots — ✓ Run results Review run results (analyzed run data) ✓ — Configure analysis settings ✓ — Export Select export options for run data and run results (analyzed run data) ✓ — Export run data ✓ ✓ Export template settings ✓ — [1] Feature is available in the instrument touchscreen, but it is not validated for the HID assay workflow. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 13 2 Install the instrument ■ Workflow: Install the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 ■ Before you begin installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 ■ Unpack and install the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 ■ Power on and follow the startup wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 ■ Instrument and computer connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 ■ Computer-to-instrument configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Workflow: Install the instrument Perform all steps in “Before you begin installation” on page 14 ▼ “Unpack and install the instrument” on page 15 ▼ “Power on and follow the startup wizard” on page 15 ▼ “Perform instrument verification using RNase P plates” on page 55 ▼ “Calibrate dyes for HID‑validated workflows” on page 51 Before you begin installation A Thermo Fisher Scientific service representative will contact you to schedule an installation. However, you do have the option to install the instrument yourself. Before starting the installation: • Review the site requirements in the QuantStudio™ 5 Real-Time PCR Instrument Site Preparation Guide (for Human Identification) (Pub. No. MAN0016701). • Review the HID‑validated computer‑to‑instrument configuration (page 16). 14 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 2 Install the instrument Unpack and install the instrument 2 Unpack and install the instrument 1. Prepare the installation site as described in the QuantStudio™ 5 Real-Time PCR Instrument Site Preparation Guide (for Human Identification) (Pub. No. MAN0016701). 2. Follow the pre-printed instructions on the instrument box to unpack the instrument, accessories, and reference documentation. Save the packing material for future use or recycle it. The instrument box contains: • Reference documentation: Welcome note, unpacking and set up instructions card, system documentation insert • One instrument • Accessories: power cable, Ethernet cable, USB drive, reaction tube retainer • Shipping plate Note: Save the shipping plate but do not use it to operate the instrument. • Spectral calibration plates for HID‑validated workflows. Note: Store these plates at –20°C (–15°C to –25°C). 3. Place the instrument on the bench. 4. Plug the power cable into the power port on the back panel of the instrument, then plug the cable into an electrical receptacle. 5. Connect an Ethernet cable to the Ethernet port on the back panel of the instrument, then connect the cable to the computer. Power on and follow the startup wizard 1. Power on the instrument. 2. Follow the startup wizard through the following tasks: • Select the language for the instrument. • Accept the license agreement. • Configure the instrument date and time. • Create an administrator instrument profile. Note: You can perform any of the steps above at a later time if you do not have the information needed to complete the startup screens. See page 23. IMPORTANT! Before using the instrument for the first time, we recommend that you perform instrument verification using RNase P plates (see “Perform instrument verification using RNase P plates” on page 55). QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 15 Chapter 2 Install the instrument 2 Instrument and computer connections Instrument and computer connections 1 2 3 4 5 6 7 Figure 1 Instrument back panel 1 USB ports 4 RS232 Port—For service use only 2 WiFi USB port—Not applicable 5 Fuse Cover 3 Ethernet Port—RJ45 port for 100/1,000 Mbps Ethernet 6 Power Switch communication with the instrument 7 Power Port—100 to 240 VAC 3 2 1 1 4 Figure 2 Instrument‑to‑computer connections (barcode scanner connected to the computer) Minitower configuration 1 Detachable power supply cord compatible with local power supply receptacle. 2 Connection between the computer and the instrument. 3 Connection between the computer and the monitor, keyboard, and mouse. 4 Connection between the computer and the (optional) handheld barcode scanner. 16 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 2 Install the instrument Computer-to-instrument configuration 2 Computer-to-instrument configuration IMPORTANT! For HID use, the QuantStudio™ 5 Real-Time PCR System has been validated for a direct (computer-to-instrument) configuration. During installation, a Thermo Fisher Scientific service representative can set up only a direct configuration. Direct configuration • A computer provided by Thermo Fisher Scientific with the HID Real‑Time PCR Analysis Software • Computer‑to‑instrument connection: – Direct, wired connection between the computer and the instrument using an Ethernet cable Firewall ports that must be open Ports Condition Standard ports for instrument-to-Thermo Fisher™ Connect Platform and computer-to-Thermo 80/443 Fisher™ Connect Platform connections QuantStudio™ 5 Real-Time PCR System (with 0.2‑mL 96‑Well Sample Block) mDNS, 7000 with firmware v1.3.x Instrument-to-computer connection QuantStudio™ 5 Real-Time PCR System (with 0.2‑mL 96‑Well Sample Block) mDNS, 7443 with firmware v1.5.1 Instrument-to-computer connection mDNS, 5353 Instrument discovery QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 17 3 General procedures to operate the instrument ■ Precautions for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 ■ Power on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Power off the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 ■ Sign in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ■ Sign out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ■ Load and unload plate in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 ■ View real-time data and plots on the instrument touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 ■ Transfer EDS files from the instrument home screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Precautions for use CAUTION! PHYSICAL INJURY HAZARD. Do not remove the instrument cover. There are no components inside the instrument that you can safely service yourself. If you suspect a problem, contact technical support. CAUTION! FIRE HAZARD. For continued protection against the risk of fire, replace fuses only with listed and certified fuses of the same type and rating as those currently in the instrument. CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the sample block temperature can reach 100°C. Allow it to cool to room temperature before handling. CAUTION! Before using a cleaning or decontamination method other than those recommended by Thermo Fisher Scientific, confirm with Thermo Fisher Scientific that the proposed method will not damage the instrument. CAUTION! Use flat caps for tubes. Rounded caps can damage the heated cover. 18 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 3 General procedures to operate the instrument Power on the instrument 3 Power on the instrument 1. Touch anywhere on the touchscreen to determine if the instrument is in sleep mode. If the home screen is displayed, the instrument is already powered on. 2. If the home screen does not display, power on the instrument by pressing the switch on the rear panel. If left unattended, the instrument automatically enters sleep mode (enabled by default) to conserve power. Note: To customize the sleep mode setting, touch Settings4Instrument Settings4Sleep Mode. Power off the instrument The instrument operates in low-power mode when not in use. However, the instrument can be powered off completely so that the components use no power. Note: To power off the instrument for >1 week, see page 65 . 1. Power off the instrument using the power switch on the back of the instrument. 2. Power off the computer. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 19 Chapter 3 General procedures to operate the instrument 3 Sign in Sign in Create an instrument profile before signing into the instrument. See “Create a new instrument profile” on page 29. Note: An instrument profile is a user account specifically for the instrument. It is not related to any other user account for the system or software. 1. In the home screen, touch Sign In. 2. Touch Sign In, then select your username. 3. Enter your PIN, then touch Enter. Note: To enable access to Thermo Fisher™ Connect, see “Link an instrument profile to the Thermo Fisher™ Connect Platform” on page 30. Sign out 1. In the home screen, touch My Profile. 2. Touch Sign Out. 20 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 3 General procedures to operate the instrument Load and unload plate in the instrument 3 Load and unload plate in the instrument CAUTION! Use optical flat caps for tubes. Rounded caps can damage the heated cover. 1. Load the plate. a. Touch to eject the instrument drawer. b. Load the plate onto the plate adapter so that: • Well A1 of the plate is in the top-left corner of the plate adapter. • The barcode faces the front of the instrument. IMPORTANT! The instrument should be used by trained operators who have been warned of the moving parts hazard. Note: Do not remove the black plate adapter before loading a plate or tube strips. If used, tube strips may fit loosely in the adapter, but the heated cover will apply the appropriate pressure to seat the tube strips securely in the adapter. c. Touch to close the instrument drawer. 2. When the run ends, unload the plate. a. Touch to eject the instrument drawer. b. Remove the plate. c. Touch to close the instrument drawer. CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the plate temperature can reach 100°C. Allow it to cool to room temperature before handling. Note: If the instrument does not eject the plate, contact Support. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 21 Chapter 3 General procedures to operate the instrument 3 View real-time data and plots on the instrument touchscreen View real-time data and plots on the instrument touchscreen 1. In the instrument home screen, during an instrument run, touch or swipe left twice. 2. Touch Well details. 3. Touch Samples, Targets, or Tasks to select a graphical representation of each selection. 4. Touch Close to return to the home screen. Adjust the display of real-time plots on the instrument touchscreen 1. In the instrument home screen, during an instrument run, touch or swipe left twice to view real-time data and plots. 2. Touch Zoom. 3. Touch or to zoom in or out. 4. Touch the arrows to pan left, right, up, or down on the graph. 5. Touch Close to return to the default view. Transfer EDS files from the instrument home screen 1. In the home screen, when a run ends, touch Transfer File. 2. Select the data destination for the EDS file. 3. Navigate to and select a folder. 4. Touch OK. 5. Touch Transfer. Note: Touch Settings4Run History to transfer EDS files at any time. 22 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 4 Configure the instrument and manage instrument profiles ■ Initial start up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ■ Overview of instrument settings (Administrator) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 ■ Overview of instrument settings (Standard or Guest) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 ■ Manage instrument profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 ■ Link an instrument profile to the Thermo Fisher™ Connect Platform . . . . . . . . . . . . . . . . . . . . . . . . 30 ■ Require instrument profile sign-in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 ■ Manage the Sign Out Timer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 ■ Manage the instrument name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 ■ Enable Sleep Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 ■ Set the idling temperature for the heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 ■ Set the date and time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 ■ Restore factory defaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 ■ Enable Remote Monitoring Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 ■ Update instrument software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Initial start up Perform initial start up tasks: • After initially powering on the instrument (see page 19). • After restoring factory defaults (see “Restore factory defaults” on page 40). 1. Ensure that there is a direct, wired connection between the computer and the instrument using an Ethernet cable (see page 17). 2. Create an administrator instrument profile (see page 28). 3. (Optional) Manage the instrument name (see page 39). 4. Set the date and time (see page 39). QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 23 Chapter 4 Configure the instrument and manage instrument profiles 4 Overview of instrument settings (Administrator) Overview of instrument settings (Administrator) Touch Settings in the home screen to configure the instrument settings as needed. Access to most settings are restricted to administrator instrument profiles. Options Description Instrument Settings Instrument Name Enter a unique instrument name, and (optional) set an instrument avatar. Sleep Mode Enable the instrument to enter a sleep mode after a set length of inactivity. Heated Cover Set the idling temperature for the heated cover (before it enters sleep mode). Temperature Date/Time Set time zone and date and time formats. Restore Factory Restore the instrument to the factory settings. Defaults IMPORTANT! Back up the instrument before restoring factory defaults (see “Backup or restore the instrument” on page 60 ). When you restore an instrument to its factory defaults: · The following items are deleted: · All instrument profiles · All files stored on the instrument, including all EDS files · All custom dye calibrations, including calibrations for ABY™ and JUN™ dyes · RNase P verification · The following items are not deleted: · The most recent valid ROI/uniformity and background calibrations · The most recent system dye calibrations 24 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Overview of instrument settings (Administrator) 4 Options Description About Instrument About Instrument [1] Displays the Model Name, IP Address, Serial Number, and Firmware Version. License Agreement [1] Displays the End User Software License Agreement and the Limited Product Warranty. You can export the License Agreement to a USB drive. Notifications [1] — Enable home screen notifications of instrument errors. The number of new, unviewed notifications displays over Settings in the home screen. Maintenance and Service Monitoring Enable the Remote Monitoring Service to automatically notify Thermo Fisher Scientific support teams in real time of potential instrument issues. The service monitors and sends general instrument data, but the service does not monitor or send customer data. Instrument Statistics [1] Displays instrument usage information, including Disk Space Remaining, LED Life, and RNase P Status. Calibrations • Perform calibrations. – ROI and Uniformity – Dye – Custom (including calibrations for background and ABY™ and JUN™ dyes) • View calibration history and set calibration reminders in History and Reminders. RNase P Verification [1] Perform instrument performance verification using an RNase P plate. Self Verification Test [1] Check the instrument hardware functions. Log [1] View and export the Instrument Run Log. Backup / Restore • Backup the instrument [1] • Restore an instrument backup Ship Prep Mode [1] Place the instrument in a safe state for moving or long-term storage. Run History [1] — Displays the instrument runs and whether the EDS file for the run was transferred. Touch a run to view its details or to transfer or delete (Administrator only) its EDS file. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 25 Chapter 4 Configure the instrument and manage instrument profiles 4 Overview of instrument settings (Administrator) Options Description Manage Users Sign In Required Enable only signed-in users to access the instrument for any task, including accessing Settings. Enabling this feature disables the guest profile access to the instrument. Note: Instrument runs must be started from the desktop software, and actions during an instrument run are automatically logged to the guest instrument profile (even if a user was signed into the instrument). Sign Out Timer Set the time length of inactivity before a user is automatically signed out. Manage Profiles Access the profile information for the instrument. [1] Also available to standard and guest instrument profiles. 26 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Overview of instrument settings (Standard or Guest) 4 Overview of instrument settings (Standard or Guest) Touch Settings in the home screen to configure the instrument settings as needed. Access to most settings are restricted to administrator instrument profiles. Options Description About Instrument About Instrument Displays the Model Name, IP Address, Serial Number, and Firmware Version. License Agreement Displays the End User Software License Agreement and the Limited Product Warranty. You can export the License Agreement to a USB drive. Notifications — Enable home screen notifications of instrument errors. The number of new, unviewed notifications displays over Settings in the home screen. Maintenance and Service Instrument Statistics Displays instrument usage information, including Disk Space Remaining, LED Life, and RNase P Status. RNase P Verification Perform instrument performance verification using an RNase P plate. Self Verification Test Check the instrument hardware functions. Log View and export the Instrument Run Log. Backup / Restore Backup the instrument Ship Prep Mode Place the instrument in a safe state for moving or long-term storage. Run History — Displays the instrument runs and whether the EDS file for the run was transferred. Touch a run to view its details and to transfer its EDS file. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 27 Chapter 4 Configure the instrument and manage instrument profiles 4 Manage instrument profiles Manage instrument profiles Note: An instrument profile is a user account specifically for the instrument. It is not related to any other user account for the system or software. To See Create a profile • “Create an administrator instrument profile during initial start-up” on page 28 • “Create a new instrument profile” on page 29 Configure a profile • “Edit an instrument profile” on page 29 View or manage all profiles • “Manage all instrument profiles” on page 29 Instrument profiles and signed-in users An instrument profile is a user account specifically for the instrument. It is not related to any other user account for the system or software. Instrument profile Allowed actions type Administrator All maintenance and administrative tasks, including instrument configuration Standard View instrument status, transfer files, and many maintenance tasks (such as RNase P Verification and Self Verification Test) Guest View instrument status, transfer files, and many maintenance tasks (such as RNase P Verification and Self Verification Test) Note: All instrument runs and actions during a run are logged to the guest profile (even if a user was signed-in to the instrument when the run was started). Instrument runs must be started from the desktop software. The instrument will automatically sign-off a signed-in user when an instrument run begins. Note: To disable guest profile access: Settings4Manage Users4Sign In Required set to on. Create an administrator instrument profile during initial start-up During initial start-up, the user is automatically prompted to create an administrator instrument profile. See “Initial start up”. • The first instrument profile that is created during installation is given administrator privileges. • Administrators can grant administrative privileges to other users. See “Manage all instrument profiles” on page 29. 28 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Manage instrument profiles 4 1. Touch Name, enter a username, then touch Done. 2. Touch PIN, enter a four-digit numerical password, then touch Enter. Note: Touch the Show PIN checkbox to switch PIN display on or off. 3. Touch Confirm PIN, then repeat step 2. 4. Touch Create profile. Create a new instrument profile 1. In the home screen, touch Sign In, then touch Get Started. 2. Touch Name, enter a username, then touch Done. 3. Touch PIN Code, enter a four-digit numerical password, then touch Enter. Note: Touch the Show PIN checkbox to switch PIN display on or off. 4. Touch Confirm PIN, then repeat step 2. 5. Touch Create profile. 6. Sign in to the profile you just created. Note: To enable access to the Connect Platform, see “Link an instrument profile to the Thermo Fisher™ Connect Platform” on page 30 and “If you link when you are signed in to the instrument” on page 36. Edit an instrument profile 1. In the home screen, touch My Profile. Note: Administrators can also navigate to this screen by touching Settings4Manage Users4Manage Profiles. 2. Touch Edit. 3. Select the fields to edit, then make changes. 4. Touch Done. Manage all instrument profiles 1. In the home screen, access the All Profiles tab. • Touch My Profile4All Profiles. • Touch Settings4Manage Users4Manage Profiles4All Profiles. A list of users, the date the profile was created, and the user type displays. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 29 Chapter 4 Configure the instrument and manage instrument profiles 4 Link an instrument profile to the Thermo Fisher™ Connect Platform 2. Select the instrument profile to edit. 3. Edit the profile. • To delete the profile, touch Delete profile4Delete. • To reset the PIN, touch Reset PIN4Reset. Note: · The user will be directed to enter a new PIN at the next sign in. · The PIN can be reset on the instrument for a local instrument profile. It cannot be reset on the instrument for a Thermo Fisher™ Connect Platform profile. The PIN for a Connect Platform profile is reset on the Connect Platform. • To enable or disable administrative privileges, slide the control to Administrator or Standard, respectively. 4. Touch Done. Link an instrument profile to the Thermo Fisher™ Connect Platform Linking your instrument profile to the Thermo Fisher™ Connect Platform (cloud-based) allows you to access the following functions: • View an instrument status from the cloud. • Download templates from your cloud storage to the instrument. • Transfer run data from the instrument to your cloud storage. To link an instrument profile to the Connect Platform, see the following sections: • “Recommended order to set up profiles” on page 31 • “Overview of local instrument profiles and Thermo Fisher™ Connect Platform profiles” on page 31 • “Thermo Fisher™ Connect Platform instrument profile roles and functions” on page 32 • “Link a local profile to a Thermo Fisher™ Connect Platform profile” on page 34 • “Unlink a Thermo Fisher™ Connect Platform account” on page 36 • “If you link when you are signed in to the instrument” on page 36 • “If you link when you are not signed in to the instrument” on page 37 • “Change the PIN for a Thermo Fisher™ Connect Platform profile” on page 38 30 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Link an instrument profile to the Thermo Fisher™ Connect Platform 4 Recommended order to set up profiles Task Description Create a profile for the The first profile on the instrument becomes the instrument administrator. instrument administrator. See “Create an administrator instrument profile during initial start-up” on page 28. A Thermo Fisher™ Connect Platform administrator is distinct from an instrument administrator. Select a Thermo The first Connect Platform profile to be linked to the instrument is automatically Fisher™ Connect Platform assigned the role of a Connect Platform administrator. It is recommended that the administrator. first Connect Platform profile to be linked is the one that should be a Connect Platform administrator. The roles can be updated at a later time on the Connect Platform. Create a local instrument profile. Connect Platform-enabled features are not Each person creates a available. profile. (Optional) Link a local instrument profile to a Connect Platform profile. Assign additional instrument Any of the profiles can be assigned the role of instrument administrator. administrators. Overview of local instrument profiles and Thermo Fisher™ Connect Platform profiles • Local instrument profile – Experiment template files and data files are stored on the instrument. – Experiment template files and data files can be transferred to the desktop software. – Email notifications are not available. – Cannot view the instrument status from the Thermo Fisher™ Connect Platform. – Separate profiles are required for each instrument if multiple instruments are used. • Thermo Fisher™ Connect Platform profile – Experiment template files and data files are stored on the instrument. – Experiment template files and data files can be transferred to the Connect Platform or the desktop software. – Email notifications are available. – View the instrument status from the Connect Platform. – The same profile can be used for multiple instruments. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 31 Chapter 4 Configure the instrument and manage instrument profiles 4 Link an instrument profile to the Thermo Fisher™ Connect Platform Thermo Fisher™ Connect Platform instrument profile roles and functions The first user who links their local instrument profile to their Thermo Fisher™ Connect Platform account is assigned a Connect Platform profile with the administrator role. Instrument Location Functions allowed profile Standard Connect • Create, save, open, import, and run template files Platform • Create and modify run settings • View and export data files Administrator Connect All the permissions of a local administrator profile, plus the following functions Platform performed in the Connect Platform: • See a list of all the Connect Platform profiles that are linked to the instrument • Assign Connect Platform administrator roles to one or more users • Remove a user from an instrument • Disconnect the instrument from the Connect Platform • Change the instrument name Test the connection to the Thermo Fisher™ Connect Platform 1. In the home screen, tap (Settings)4Maintenance and service4Connect services. The Connect Services screen is displayed. 2. Tap Test connection. If the connection can be established, You are able to connect to the Connect platform will be displayed. 32 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Link an instrument profile to the Thermo Fisher™ Connect Platform 4 If the connection cannot be established, Unable to connect will be displayed. 3. Tap Close. Link the instrument to your Thermo Fisher™ Connect Platform account This section describes using your Thermo Fisher™ Connect Platform account when you use the instrument for the first time. The first time that you use your Connect Platform account on an instrument, you will be prompted to create a four-digit numerical PIN. This PIN is to use when signing in to the instrument with your Connect Platform account. It will apply to all other instruments when you use your Connect Platform account. This does not change the password when signing in to your Connect Platform account on a browser. After the PIN is set up on the instrument, it must be changed with your Connect Platform account on a browser, see “Create an administrator instrument profile during initial start-up” on page 28. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 33 Chapter 4 Configure the instrument and manage instrument profiles 4 Link an instrument profile to the Thermo Fisher™ Connect Platform The first time the instrument is used with a Connect Platform account, a region must be selected. 1. In the Sign In screen, tap Get started4Create a local instrument file4Connect to your Thermo Fisher™ Connect Platform account. 2. (Optional) Select the appropriate region. Option Description China For users in China U.S. For users in any country other than China 3. Tap a connection option. Option Action Mobile devices Note: Before selecting this option, install and sign in to the Connect Platform application on your mobile device. On the instrument: 1. Tap Mobile devices. 2. Hold the camera on your mobile device over the QR code that is displayed on the touchscreen. 3. Tap Close. PC A link code is displayed on the instrument. On a computer: 1. Access the Connect Platform. 2. Click Add instrument. 3. Select QuantStudio. 4. Enter the link code. 4. (Optional) In the Enter PIN screen, tap the PIN (4 digits required) field, enter a four-digit numerical PIN, then tap Enter. Tap the Show PIN checkbox to show or hide the PIN. 5. (Optional) Tap the Confirm PIN field, enter the four-digit numerical PIN again, then tap Enter. 6. Tap Done. Link a local profile to a Thermo Fisher™ Connect Platform profile This section describes linking a local instrument profile to a Thermo Fisher™ Connect Platform profile, if a local instrument profile was created first. The first time that you use your Connect Platform account on an instrument, you will be prompted to create a four-digit numerical PIN. This PIN is to use when signing in to the instrument with your Connect Platform account. It will apply to all other instruments when you use your Connect Platform account. This does not change the password when signing in to your Connect Platform account on a browser. 34 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Link an instrument profile to the Thermo Fisher™ Connect Platform 4 After the PIN is set up on the instrument, it must be changed with your Connect Platform account on a browser, see “Change the PIN for a Thermo Fisher™ Connect Platform profile” on page 38. The first time the instrument is used with a Connect Platform account, a region must be selected. For a detailed description of the profile, see “If you link when you are signed in to the instrument” on page 36. (Optional) Test the connection, see “Test the connection to the Thermo Fisher™ Connect Platform” on page 32. 1. In the home screen, tap (Profile). The My Profile screen is displayed. 2. Tap Connect4Link Account. The Connect to Connect Platform screen is displayed. 3. (Optional) Select the appropriate region. Option Description China For users in China U.S. For users in any country other than China 4. Tap a connection option. Option Action Mobile devices Note: Before selecting this option, install and sign in to the Connect Platform application on your mobile device. On the instrument: 1. Tap Mobile devices. 2. Hold the camera on your mobile device over the QR code that is displayed on the touchscreen. 3. Tap Close. PC A link code is displayed on the instrument. On a computer: 1. Access the Connect Platform. 2. Click Add instrument. 3. Select QuantStudio. 4. Enter the link code. 5. (Optional) In the Enter PIN screen, tap the PIN (4 digits required) field, enter a four-digit numerical PIN, then tap Enter. Tap the Show PIN checkbox to show or hide the PIN. 6. (Optional) Tap the Confirm PIN field, enter the four-digit numerical PIN again, then tap Enter. 7. Tap Done. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 35 Chapter 4 Configure the instrument and manage instrument profiles 4 Link an instrument profile to the Thermo Fisher™ Connect Platform Unlink a Thermo Fisher™ Connect Platform account Unlinking a Thermo Fisher™ Connect Platform account is done from the Connect Platform application on your computer. For more information about unlinking a Connect Platform account, see “If you link when you are signed in to the instrument” on page 36. 1. Sign in to your account on the desktop Connect Platform application. 2. In the left pane, click (Instrument). 3. Select the instrument, then click Disconnect. 4. Tap Confirm. If you link when you are signed in to the instrument In this scenario, your local instrument profile name is created manually on the instrument before you link. Your local instrument profile name differs from your Thermo Fisher™ Connect Platform instrument profile name. Phase Steps that occur Before you • You enter your local instrument profile name in the Sign In screen. link: • Your local instrument profile (UserABC) is displayed in the home screen of the instrument. • All plates and results that you create are accessible only when you are signed in with your local instrument profile. When you • You link your local instrument profile (see “Link a local profile to a Thermo Fisher™ Connect link: Platform profile” on page 34). • If this is the first time you link, a Connect Platform instrument profile is created using the FirstNameLastInitial of the user name from your thermofisher.com account. Example: [email protected] First name is User, Last name is Gray. The Connect Platform account username is User G. • Your local instrument profile (UserABC) is linked to your Connect Platform account ([email protected]). • Your Connect Platform instrument profile (User G.) replaces your local instrument profile. After you link: • Your Connect Platform instrument profile (User G.) and is displayed in the home screen of the instrument. • Plate files and data files from your local instrument profile can be transferred to the Connect Platform. • New plate files and data files are saved under your Connect Platform instrument profile. 36 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Link an instrument profile to the Thermo Fisher™ Connect Platform 4 (continued) Phase Steps that occur If your • Your local instrument profile (UserABC) is displayed in the home screen of the instrument. Connect • Plate files and data files that were saved locally to the instrument under your Connect Platform Platform instrument profile are accessible under your local instrument profile. account is • Plate files and data files that were saved to DataConnect on the Connect Platform are not unlinked: accessible from the instrument under your local instrument profile. • Plate files and data files are saved under local instrument profile and can be copied to the Connect Platform (see “Transfer EDS files from the instrument home screen” on page 22). • Your local instrument profile name (UserABC) is available for selection in the Sign In screen. If you link when you are not signed in to the instrument In this scenario, your local instrument profile name is created automatically at the time that you link. The same user name is used for your local instrument profile and your Thermo Fisher™ Connect Platform profile. Plates and results are accessible when you sign in with either profile. If you link to the Connect Platform when you are not signed in to the instrument: Phase Steps that occur Before you • In the Sign In screen, you tap Get Started4Connect. link: When you • You link your profile (see “Link the instrument to your Thermo Fisher™ Connect Platform link: account” on page 33). • If this is the first time you link, a Connect Platform instrument and a local instrument profile (with standard role) are created with the same name using the FirstNameLastInitial of the user name from your thermofisher.com account. Example: [email protected] First name is User, Last name is Gray. The Connect Platform account username is User G. • Your local instrument profile (User G.) is linked to your Connect Platform account ([email protected]). • Your Connect Platform instrument profile (User G.) replaces your local instrument profile. After you link: • Your Connect Platform instrument profile (User G.) and is displayed in the home screen of the instrument. • Plate files and data files from your local instrument profile can be transferred to the Connect Platform. • New plate files and data files are saved under your Connect Platform instrument profile. • Your Connect Platform instrument profile name (User G. ) is available for selection in the Sign In screen. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 37 Chapter 4 Configure the instrument and manage instrument profiles 4 Require instrument profile sign-in (continued) Phase Steps that occur If your • Your local instrument profile (User G.) is displayed in the home screen of the instrument. Connect • Plate files and data files that were saved under your Connect Platform instrument profile are Platform accessible under your local instrument profile. account is • New plates and results are saved under local instrument profile and can be copied to the unlinked: Connect Platform. • Your local instrument profile name (User G.) is available for selection in the Sign In screen. IMPORTANT! If you sign in with a local profile, without linking to the Connect Platform, sign out, then link to the Connect Platform (Get Started4Connect), you can potentially have two instrument profiles with different names. Plate files and data files from when you are signed in with one instrument profile are not accessible when you are signed in with the other instrument profile. Change the PIN for a Thermo Fisher™ Connect Platform profile This section describes changing a PIN for a Thermo Fisher™ Connect Platform profile. To change a PIN for a local instrument profile, see “Manage all instrument profiles” on page 29. 1. Sign in to the Connect Platform on a browser. 2. Click Update PIN number. 3. In the Update PIN number dialog box, enter a new PIN, then enter the new PIN a second time to confirm it. 4. Click Send. The 4-digit PIN to sign in to instruments with your Connect Platform profile is updated. This change applies to all of the instruments that you access with your Connect Platform profile. Require instrument profile sign-in 1. In the home screen, touch Settings4Manage Users4Sign In Required. 2. Slide the control On. Only signed-in users are allowed to access the instrument for any task, including access to Settings. 3. Touch Done. 38 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Manage the Sign Out Timer 4 Manage the Sign Out Timer 1. In the home screen, touch Settings4Manage Users4Sign Out Timer. 2. Touch the Edit Time field, then enter the desired duration of inactivity before automatic user sign out. 3. Touch Enter, then touch Done. Manage the instrument name 1. In the home screen, touch Settings4Instrument Settings4Instrument Name. 2. Touch the Instrument Name field, enter an instrument name, then touch Done. 3. Touch OK. Enable Sleep Mode 1. In the home screen, touch Settings4Instrument Settings4Sleep Mode. 2. Slide the control On to enable sleep mode. 3. Touch Edit Time, then enter the time length of inactivity before the instrument enters sleep mode. 4. Touch Enter, then touch OK. Set the idling temperature for the heated cover 1. In the home screen, touch Settings4Instrument Settings4Heated Cover Idle Temperature. 2. Slide the control On to set the idling temperature for the heated cover. 3. Touch the Edit Temperature field, then enter the desired idling temperature. 4. Touch Enter, then touch OK. Set the date and time 1. In the home screen, touch Settings4Instrument Settings4Date/Time. 2. Select a time zone from the dropdown list. 3. Select a date format. a. Touch Date Format, then select the preferred date format. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 39 Chapter 4 Configure the instrument and manage instrument profiles 4 Restore factory defaults b. Touch Next, touch the date field, then enter the date. c. Touch Enter, then touch Done. 4. Select a time format. a. Touch Time Format. b. Slide the control to select a 12‑hour or 24‑hour clock. c. Touch Next, touch the time field, then enter the time. d. Touch Enter, then touch Done. 5. Touch Done. Restore factory defaults This procedure can only be performed by an Administrator. IMPORTANT! Back up the instrument before restoring factory defaults (see “Backup or restore the instrument” on page 60 ). 1. In the home screen, touch Settings4Instrument Settings4Restore Factory Defaults. 2. Touch Restore Factory Defaults. 3. Power Off, then power On the instrument to apply the change. • Instrument profiles and files stored on the instrument are deleted, including all EDS files, RNase P verification, and any custom dye calibrations. • The most recent valid ROI/Uniformity, Background, and system dye calibrations are not deleted. After restoring factory defaults, perform initial start‑up tasks. See “Initial start up”. Enable Remote Monitoring Service Note: Enabling the Remote Monitoring Service allows the instrument to automatically notify Thermo Fisher Scientific support teams in real time of potential instrument issues. The service monitors and sends general instrument data, but the service does not monitor or send customer data. 1. In the home screen, touch Settings4Maintenance and Service4Monitoring. 2. Slide the control On to enable the Remote Monitoring Service. 3. Touch OK. 40 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 4 Configure the instrument and manage instrument profiles Update instrument software 4 Update instrument software IMPORTANT! Update the instrument software only under the specific guidance of your HID Support Representative. In the home screen: 1. Touch Settings4Maintenance and Service4Software Update. 2. Touch the location of the update files. 3. When prompted, confirm your request to update the software. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 41 5 Calibrate and verify instrument performance ■ Calibration and verification schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 ■ Calibration descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 ■ View calibration status and set reminders in the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 ■ Perform ROI/uniformity, background, and dye calibrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 ■ Calibrate dyes for HID‑validated workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 ■ Perform instrument verification using RNase P plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 42 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Calibration and verification schedule 5 Calibration and verification schedule The instrument is factory-calibrated and does not require calibration at installation. To ensure optimal performance, perform calibrations at the recommended frequency. Note: During instrument installation, the Thermo Fisher Scientific service representative will perform the initial instrument verification using an RNase P plate and calibrate the instrument for the versions of ABY™ and JUN™ dyes used by HID-validated workflows. IMPORTANT! Perform calibrations and instrument runs under the environmental conditions that are specified in “Environmental requirements” on page 77. Exposure to extreme temperatures can adversely affect the instrument performance and shorten the life span of the instrument components. To set the calibration frequency for the instrument, touch Settings4Maintenance and Service4Calibrations4History and Reminders4Edit4Exp interval field. Calibration Recommended frequency ROI/Uniformity • Every two years (recommended) • Always perform new Background and Dye calibrations after an ROI/Uniformity calibration. Note: Performing an ROI/Uniformity calibration invalidates all other calibrations. Background • Every two years (recommended) • Background calibration can also be performed, as needed: – To check for contamination (depends on usage and laboratory conditions). – To obtain the most accurate data for the removal of background fluorescence. Note: Performing a Background calibration does not invalidate any other calibration. Dye • Every two years (recommended) • During a Dye calibration, only the dyes on the given spectral calibration plate are calibrated. Note: Performing a Dye calibration for a given dye plate does not invalidate any other calibration. RNase P • After performing instrument calibrations (recommended) instrument • As needed to confirm instrument performance verification Note: To prepare custom dye plates and to perform custom calibrations, see “Calibrate dyes for HID‑validated workflows” on page 51. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 43 Chapter 5 Calibrate and verify instrument performance 5 Calibration descriptions Calibration descriptions Calibration description and purpose Pass Criteria ROI/Uniformity • The software captures images for each optical filter. The image for each filter • The software uses calibration data to map the increase in fluorescence distinguishes all wells of the to the plate wells during subsequent runs and to evaluate well-to-well plate. consistency of the signals. Each well in the image is distinct. Background • The software captures background images for each optical filter in the The plate images for all absence of sample and reagent, and it checks that the fluorescence from filters are free of abnormal each well is below a fluorescence threshold. fluorescence. • The software uses calibration data to remove background fluorescence during a run. Note: You can also run this calibration to determine if contamination is related to the sample block or the plate. Dye • The software extracts a spectral profile for each dye standard, then Dye spectra peak within the produces a set of spectral profiles plotted as fluorescence vs filter. same filter as their group. • The software uses calibration data to characterize and distinguish the individual contribution of each dye in the total fluorescence signals collected by the instrument. 44 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance View calibration status and set reminders in the instrument 5 View calibration status and set reminders in the instrument 1. In the home screen, touch Settings4Maintenance and Service4Calibrations4History and Reminders. 2. In the Calibration Reminders screen, view the status of each calibration type. 3. (Optional) Touch a calibration row to view the history of that specific calibration type, then touch Done. 4. Touch Edit to set the calibration reminder settings. For each calibration type: a. Slide the control On to enable the calibration reminder. b. Edit the Exp interval and Remind me fields. c. Touch Save. 5. (Optional) To transfer the calibration report, touch Export then follow the directions on the screen. 6. Touch Done. Perform ROI/uniformity, background, and dye calibrations Workflow: Calibration Note: The ROI and uniformity calibrations use the same calibration plate. Perform an ROI/uniformity calibration You are automatically prompted to perform background calibration. ▼ Perform a background calibration Perform any time that ROI/uniformity calibrations are current. ▼ Perform system dye calibrations Perform any time that ROI/uniformity and background calibrations are current. ▼ Perform dye calibrations for ABY™ and JUN™ dyes (page 51) Perform any time that ROI/uniformity and background calibrations are current. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 45 Chapter 5 Calibrate and verify instrument performance 5 Perform ROI/uniformity, background, and dye calibrations Prepare a calibration plate Materials required for calibration plate preparation • Plate(s) for the calibration that you are performing: – ROI plate (the same plate is used for ROI and uniformity calibrations) – Background calibration plate – Dye calibration plates Note: We recommend calibrating with all Spectral Dye Calibrations Plates even if you are not using all the dyes in the plates. Note: Do not discard the packaging for the calibration plates. Each calibration plate can be used up to 3 times if the following conditions are met: · The plate is stored in its packing sleeve at –15 to –25°C. · The plate is used within 6 months after opening. · The plate is used before the plate expiry date • Centrifuge with plate adapter; buckets cleaned before use • Powder‐free gloves • Safety glasses Thaw, vortex, and centrifuge a calibration plate 1. Remove the calibration plate from the freezer, then thaw the plate in its packaging. Keep plates protected from light until you perform the calibration. • Thaw each plate for 30 minutes. • Use each plate within 2 hours of thawing. IMPORTANT! Do not remove the plate from its packaging until you are ready to use it. The fluorescent dyes in the wells of calibration plates are photosensitive. Prolonged exposure to light can diminish the fluorescence of the dyes. 2. While wearing powder‐free gloves, remove the calibration plate from its packaging and retain the packaging. Do not remove the optical film. 3. Vortex the plate for 5 seconds, then centrifuge at 750–1,000 × g for 2 minutes. 4. Confirm that the liquid in each well is at the bottom of the well and free of bubbles. If it is not, centrifuge the plate again. 46 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Perform ROI/uniformity, background, and dye calibrations 5 IMPORTANT! Keep the bottom of the plate clean. Fluids and other contaminants on the bottom of the plate can contaminate the sample block and cause an abnormally high background signal. Perform calibrations 1. In the instrument home screen: Calibration Touch ROI/Uniformity[1] Settings4Maintenance and Service4Calibrations4ROI and Uniformity Background[2] Settings4Maintenance and Service4Calibrations4Custom4Background Dye Settings4Maintenance and Service4Calibrations4Dye [1] Automatically followed by Background calibration. [2] Initiate via this route if performing Background calibration only. 2. Follow the instructions on the screen to start the calibration. Note: Dye calibration only: Select the Dye Plate to run, then touch Next. 3. Load the plate. a. Touch to eject the instrument drawer. b. Load the plate onto the plate adapter so that: • Well A1 of the plate is in the top-left corner of the plate adapter. • The barcode faces the front of the instrument. IMPORTANT! The instrument should be used by trained operators who have been warned of the moving parts hazard. Note: Do not remove the black plate adapter before loading a plate or tube strips. If used, tube strips may fit loosely in the adapter, but the heated cover will apply the appropriate pressure to seat the tube strips securely in the adapter. c. Touch to close the instrument drawer. 4. Touch Start. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 47 Chapter 5 Calibrate and verify instrument performance 5 Perform ROI/uniformity, background, and dye calibrations 5. When the run is complete and the screen displays Calibration Complete, touch View Results to check the calibration status. Calibration status Action Passed Touch Next to proceed to the next required calibration. Failed See “Troubleshoot calibration failure” on page 68. Note: You can view the calibration images only after the ROI/Uniformity and Background calibrations pass. 6. When the run ends, unload the plate. a. Touch to eject the instrument drawer. b. Remove the plate. c. Touch to close the instrument drawer. CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the plate temperature can reach 100°C. Allow it to cool to room temperature before handling. Note: If the instrument does not eject the plate, contact Support. 48 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Perform ROI/uniformity, background, and dye calibrations 5 View calibration images and transfer results to USB The instrument performs the ROI, Uniformity, and Background calibrations in sequence. You can view the calibration images after the Background calibration is complete. 1. In the Calibration Status screen, touch Details. 2. In the Details screen, touch a calibration type to view its images and plots. Calibration Example results indicating successful calibration ROI Green circles around all wells and bright well centers. Note: Select the desired filter combination from the Filter Set dropdown list. Uniformity Signals from each well following a uniform trend. Background Few, if any, signals with abnormally high fluorescence. Dye Signals from each well following a uniform trend, and each dye peaks at the correct filter. 3. In the Calibration Status screen, touch Accept Results or Reject Results. Accepting the results saves the calibration data to the instrument and overwrites existing data. 4. (Optional) Touch Transfer EDS to transfer the calibration data to a USB. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 49 Chapter 5 Calibrate and verify instrument performance 5 Perform ROI/uniformity, background, and dye calibrations Identify contamination Signals that exceed the limit of normal fluorescence may indicate fluorescent contaminants on the calibration plate or the sample block. Common contaminants include ink residue from permanent pens, powder from disposable gloves, and dust. 1. View the calibration data and note the wells that failed the calibration. 2. Remove the plate from the instrument, rotate the plate 180°, then perform the calibration again. 3. Determine the location of the failed wells again as in step 1. Position of failed wells Action Identical The sample block is contaminated. Decontaminate the sample block (see page 61). Reversed The plate is contaminated. Discard the plate, then perform the calibration using a new calibration plate. 4. If the calibration fails after you decontaminate the sample block and replace the plate, contact Support. Create a background plate (optional) Whenever possible, use a background plate listed in Appendix B, “Parts and materials”. These plates contain a buffer that accurately simulates the reagents used for PCR, and, therefore, produces high- quality calibration data. If a background plate is not available, you can create one as described below. Required materials: • MicroAmp™ optical 96-well reaction plate • Optical adhesive cover or optical flat caps • Pipettor, 200‐µL (with pipette tips) • Powder‐free gloves • Safety glasses • Deionized water IMPORTANT! Wear powder-free gloves while creating the background plate. 1. Remove a reaction plate from its box and place it on a clean, dry surface. 2. Aliquot deionized water to each well of the reaction plate. The recommended volume for a 96‑well plate is 10–20 µL. 3. Seal the plate using an optical adhesive cover or optical flat caps. 4. Use the plate for background calibration. 50 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Calibrate dyes for HID‑validated workflows 5 Calibrate dyes for HID‑validated workflows HID‑validated workflows use versions of ABY™ and JUN™ dyes that are considered custom dyes for the instrument. These dyes require custom dye calibration. To calibrate any other custom dye, contact your HID Support Representative. IMPORTANT! To perform a custom dye calibration for HID‑validated workflows, use the following prepared spectral calibration plates: · ABY™ Spectral Calibration Plate, 96‑Well 0.2‑mL (Cat. No. 4461591) · JUN™ Spectral Calibration Plate, 96‑Well 0.2‑mL (Cat. No. 4461593) Workflow: Calibrate dyes for HID‑validated workflows Prepare a calibration plate (page 51) ▼ Add the custom dye to the instrument (page 52) ▼ Perform a custom dye calibration (page 53) Prepare a calibration plate for HID‑validated workflows Materials required for calibration plate preparation • Plate(s) for the calibration that you are performing: – ABY™ Spectral Calibration Plate, 96‑Well 0.2‑mL (Cat. No. 4461591) – JUN™ Spectral Calibration Plate, 96‑Well 0.2‑mL (Cat. No. 4461593) Note: Do not discard the packaging for the calibration plates. Each calibration plate can be used up to 3 times if the following conditions are met: · The plate is stored in its packing sleeve at –15 to –25°C. · The plate is used within 6 months after opening. · The plate is used before the plate expiry date • Centrifuge with plate adapter; buckets cleaned before use • Powder‐free gloves • Safety glasses Thaw, vortex, and centrifuge a calibration plate 1. Remove the calibration plate from the freezer, then thaw the plate in its packaging. Keep plates protected from light until you perform the calibration. • Thaw each plate for 30 minutes. • Use each plate within 2 hours of thawing. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 51 Chapter 5 Calibrate and verify instrument performance 5 Calibrate dyes for HID‑validated workflows IMPORTANT! Do not remove the plate from its packaging until you are ready to use it. The fluorescent dyes in the wells of calibration plates are photosensitive. Prolonged exposure to light can diminish the fluorescence of the dyes. 2. While wearing powder‐free gloves, remove the calibration plate from its packaging and retain the packaging. Do not remove the optical film. 3. Vortex the plate for 5 seconds, then centrifuge at 750–1,000 × g for 2 minutes. 4. Confirm that the liquid in each well is at the bottom of the well and free of bubbles. If it is not, centrifuge the plate again. IMPORTANT! Keep the bottom of the plate clean. Fluids and other contaminants on the bottom of the plate can contaminate the sample block and cause an abnormally high background signal. Add custom dyes to the instrument for HID‑validated workflows 1. In the instrument home screen, touch Settings4Maintenance and Service4Calibrations4Custom4Custom Dye. 2. Touch Add Custom Dye. 3. Enter the dye information: Field/option Action Custom Dye Name Enter the names for the custom dyes: • ABY-HID • JUN-HID IMPORTANT! Dye names are spacing sensitive. Type Select: • ABY-HID—Reporter • JUN-HID—Reporter 4. Touch Save. 52 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Calibrate dyes for HID‑validated workflows 5 Perform a custom dye calibration for HID‑validated workflows 1. Load the plate into the instrument. 2. In the instrument home screen, touch Settings4Maintenance and Service4Calibrations4Custom4Custom Dye. 3. Touch the custom dye to calibrate. 4. Review the custom dye information, (optional) make changes, then touch Update. 5. Enter the calibration temperature. 6. (Optional) Touch Reagents, then enter reagent information. 7. Touch Start. 8. When the run is complete and the screen displays Calibration Complete, touch View Results4Details. 9. Review the plot. Passing calibration results show uniform signals with peaks that are aligned with the dye wavelength. Filter wavelength (nm) Dye Peak filter Excitation Emission ABY-HID x3-m3 550 ± 10 587 ± 10 JUN-HID x4-m4 580 ± 10 623 ± 14 ABY‑HID dye calibration plot JUN‑HID dye calibration plot 10. Select an action depending on whether the custom dye calibration passed or failed. Calibration Action status Passed • Touch Accept Results or Reject Results. Note: Accepting the results saves the calibration data to the instrument and overwrites existing data. • (Optional) Touch Transfer EDS to transfer the calibration data to a USB. Failed • Perform the calibration again using a new custom dye plate. • See “Troubleshoot calibration failure” on page 68. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 53 Chapter 5 Calibrate and verify instrument performance 5 Calibrate dyes for HID‑validated workflows 11. Unload the plate from the instrument. CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the plate temperature can reach 100°C. Allow it to cool to room temperature before handling. 54 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Perform instrument verification using RNase P plates 5 Perform instrument verification using RNase P plates During instrument installation, your HID Support Representative will perform instrument verification. However, perform instrument verification: • After performing instrument calibrations. • As needed to confirm instrument performance. The instrument requires valid ROI/uniformity, background, and dye calibrations to perform instrument verification. Instrument verification description Purpose Description Pass criteria Confirms the Quantifies the number of The instrument passes performance specifications if the performance of copies of the human RNase P following inequality is true and the instrument successfully the instrument. gene in samples with distinguishes between unknown populations A and B. known concentrations of the [(CtA) – 3(σCtA)] > [(CtB) + 3(σCtB)] corresponding DNA template. where: • CtA = Average Ct of unknown population A • σCtA = Standard deviation of unknown population A • CtB = Average Ct of unknown population B • σCtB = Standard deviation of unknown population B QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 55 Chapter 5 Calibrate and verify instrument performance 5 Perform instrument verification using RNase P plates RNase P instrument verification plate The RNase P plate contains the reagents necessary for the detection and quantitation of genomic copies of the human RNase P gene (a single-copy gene encoding the RNase moiety of the RNase P enzyme). Each well contains: PCR master mix, RNase P primers, FAM™ dye-labeled probe, and a known concentration of human genomic DNA template. 1 2 3 4 5 6 7 8 Figure 3 96-well RNase P plate 1 Unknown A (5,000) 5 STD 5,000 copies 2 NTC (no template control) 6 STD 10,000 copies 3 STD 1,250 copies 7 STD 20,000 copies 4 STD 2,500 copies 8 Unknown B (10,000) Performance specifications pass criteria After the run, the software calculates average copy number values and standard deviation values. The instrument passes performance specifications if the following inequality is true and the instrument successfully distinguishes between unknown populations A and B. [(CtA) – 3(σCtA)] > [(CtB) + 3(σCtB)] • CtA = Average Ct of unknown population A • σCtA = Standard deviation of unknown population A • CtB = Average Ct of unknown population B • σCtB = Standard deviation of unknown population B The software automatically adjusts the threshold and omits a defined number of wells from the unknown populations to meet the performance specifications. To view any omitted wells, open the EDS file for the verification in the desktop software. 56 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Perform instrument verification using RNase P plates 5 Prepare an RNase P plate Materials required for RNase P plate preparation • RNase P instrument verification plate • Centrifuge with plate adapter; buckets cleaned before use • Powder‐free gloves • Safety glasses Thaw, vortex, and centrifuge an RNase P plate IMPORTANT! Expose the RNase P plate to room temperature for no more than 45 minutes, inclusive of thawing and preparation time. After thawing, the RNase P plate cannot be refrozen. 1. Remove the RNase P plate from the freezer, then thaw the plate in its packaging. • Thaw the plate for approximately 5 minutes. • Use the plate within 30 minutes of thawing. 2. Confirm that the bench, vortex, and centrifuge are clean. Before use, wipe the vortex and centrifuge using a lint‑free tissue. 3. While wearing powder‐free gloves, remove the plate from its packaging. 4. Vortex the plate for 5 seconds, then centrifuge at 750–1,000 × g for 2 minutes. 5. Confirm that the liquid in each well is at the bottom of the well and free of bubbles. If it is not, centrifuge the plate again. IMPORTANT! Keep the bottom of the plate clean. Fluids and other contaminants on the bottom of the plate can contaminate the sample block and cause an abnormally high background signal. Perform RNase P verification 1. In the home screen, touch Settings4Maintenance and Service4RNase P Verification. 2. Load the plate. a. Touch to eject the instrument drawer. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 57 Chapter 5 Calibrate and verify instrument performance 5 Perform instrument verification using RNase P plates b. Load the plate onto the plate adapter so that: • Well A1 of the plate is in the top-left corner of the plate adapter. • The barcode faces the front of the instrument. IMPORTANT! The instrument should be used by trained operators who have been warned of the moving parts hazard. Note: Do not remove the black plate adapter before loading a plate or tube strips. If used, tube strips may fit loosely in the adapter, but the heated cover will apply the appropriate pressure to seat the tube strips securely in the adapter. c. Touch to close the instrument drawer. 3. Touch Start. 4. When the run is complete and the screen displays Verification Complete, touch View Results to confirm the status of the run. Calibration status Action Passed Instrument is ready for use. Failed See “Troubleshoot verification failure” on page 69. 5. In the RNase P Verification Status screen, touch: • Accept Results to save the results to the instrument • Reject Results to delete the RNase P verification results • Export Results to export the calibration results to a USB 6. When the run ends, unload the plate. a. Touch to eject the instrument drawer. b. Remove the plate. c. Touch to close the instrument drawer. 58 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 5 Calibrate and verify instrument performance Perform instrument verification using RNase P plates 5 CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the plate temperature can reach 100°C. Allow it to cool to room temperature before handling. Note: If the instrument does not eject the plate, contact Support. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 59 6 Maintain the instrument ■ Backup or restore the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 ■ Decontaminate the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 ■ Replace the instrument fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 ■ Store, move, or ship the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 IMPORTANT! This chapter contains user maintenance procedures for the instrument. Procedures other than those described in this document must be performed by a qualified Thermo Fisher Scientific service representative. Backup or restore the instrument In the home screen: Touch Settings4Maintenance and Service4Backup/Restore. To Action Backup 1. Insert a USB into the front‑panel USB port. 2. Touch Backup Instrument. 3. Touch USB. 4. Enter a backup file name, then touch Done. 5. Select which elements to backup, or leave them all selected. 6. Touch Backup. Restore 1. Insert the USB that contains the backup file into the front‑panel USB port. 2. Touch Restore a Backup. 3. Touch USB. 4. Select the backup file, then touch Restore. 60 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 6 Maintain the instrument Decontaminate the sample block 6 Decontaminate the sample block Perform this procedure to eliminate fluorescent contaminants from the instrument sample block. Contamination is generally evident in failed background calibrations where one or more wells consistently exhibit abnormally high signals. CAUTION! PHYSICAL INJURY HAZARD. Do not remove the instrument cover. There are no components inside the instrument that you can safely service yourself. If you suspect a problem, contact Support. CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the sample block temperature can reach 100°C. Allow it to cool to room temperature before handling. CAUTION! Before using a cleaning or decontamination method other than those recommended by Thermo Fisher Scientific, confirm with Thermo Fisher Scientific that the proposed method will not damage the equipment. Materials required • Safety glasses • Powder-free gloves • Tissue, lint-free • Cotton or nylon swabs and lint-free cloths • Pipette (100-µL) with pipette tips • Deionized water • Ethanol, 95% solution • Na-hypochlorite, (0.1% v/v) solution Clean the sample block CAUTION! PHYSICAL INJURY HAZARD. During instrument operation, the sample block temperature can reach 100°C. Allow it to cool to room temperature before handling. IMPORTANT! Wear powder-free gloves when you perform this procedure. IMPORTANT! Always use deionized water to rinse wells after cleaning with bleach or ethanol solution. 1. Identify the contaminated wells of the sample block (see “Identify contamination” on page 50). 2. Prepare the instrument and access the sample block: a. Power off and unplug the instrument, then allow it to cool for 15 minutes. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 61 Chapter 6 Maintain the instrument 6 Decontaminate the sample block b. Touch to eject the instrument drawer. 3. Rinse the contaminated wells with deionized water (see “Detailed procedures for cleaning the sample block” on page 63). 4. Close the drawer and test the sample block for contamination: a. Push the instrument drawer back in to the instrument. b. Plug in, then power on the instrument. c. Perform a background calibration to confirm that you have eliminated the contamination. 5. If the contamination remains, clean the contaminated wells using a 95% ethanol solution: a. Repeat step 2 – step 3. b. Clean the contaminated wells using a 95% ethanol solution (see “Detailed procedures for cleaning the sample block” on page 63). c. Repeat step 3 – step 4 to rinse the sample block with deionized water and to confirm that you have eliminated the contamination. IMPORTANT! Always use deionized water to rinse wells after cleaning with bleach or ethanol solution. 6. If the contamination still remains, clean the contaminated wells using a Na-hypochlorite (0.1% v/v) solution: a. Repeat step 2 – step 3. b. Clean the contaminated wells using a Na-hypochlorite (0.1% v/v) solution (see “Detailed procedures for cleaning the sample block” on page 63). c. Repeat step 3 – step 4 to rinse the sample block with deionized water and to confirm that you have eliminated the contamination. IMPORTANT! Always use deionized water to rinse wells after cleaning with Na-hypochlorite or ethanol solution. 7. If the contamination continues to remain, contact Support. 62 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 6 Maintain the instrument Decontaminate the sample block 6 Detailed procedures for cleaning the sample block IMPORTANT! Use these cleaning procedures only in conjunction with the complete decontamination procedure (see “Decontaminate the sample block” on page 61). • Rinse the sample block with deionized water. a. Pipet a small volume of deionized water into each contaminated well. b. In each well, pipet the water up and down several times to rinse the well. c. Pipet the water to a waste beaker. d. Use a cotton swab to scrub inside of each contaminated well. e. Use a lint-free cloth to absorb the excess deionized water. • Clean the sample block with 95% ethanol. a. Pipet a small volume of 95% ethanol solution into each contaminated well. b. In each well, pipet the solution up and down several times to rinse the well. c. Pipet the ethanol solution to a waste beaker. IMPORTANT! Always use deionized water to rinse wells after cleaning with bleach or ethanol solution. • Clean the sample block with Na-hypochlorite (0.1% v/v) solution. a. Pipet a small volume of Na-hypochlorite (0.1% v/v) solution into each contaminated well. b. In each well, pipet the solution up and down several times to rinse the well. c. Pipet the bleach solution to a waste beaker. IMPORTANT! Always use deionized water to rinse wells after cleaning with bleach or ethanol solution. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 63 Chapter 6 Maintain the instrument 6 Replace the instrument fuses Replace the instrument fuses CAUTION! FIRE HAZARD. For continued protection against the risk of fire, replace fuses only with listed and certified fuses of the same type and rating as those currently in the instrument. Materials required • Fuses (2) – 10A, Time-Lag T, 250VAC, 5 × 20mm • Safety glasses • Powder-free gloves • Screwdriver, flathead Replace the fuses 1. Power off and unplug the instrument, then allow it to cool for 15 minutes. 2. Using a flat-head screwdriver, unscrew and remove the fuse holder. 3. Remove each fuse from its fuse holder and inspect it for damage. Carbon typically coats the inside of failed fuses. Good Failed 4. Replace each failed fuse. Note: The voltage and amperage ratings are on the fuse holder. 5. Install the fuse holder back into the instrument. 6. Plug in, then power on the instrument. The installation is successful if the instrument powers on. 64 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Chapter 6 Maintain the instrument Store, move, or ship the instrument 6 Note: Fuse failure can result from fluctuations in the supplied power to the system. To prevent further failures, consider installing an electrical protective device, such as a UPS or a surge protector. If issues with the fuse persist, contact Support. Store, move, or ship the instrument Prepare the instrument to store, move, or ship In the home screen: 1. Touch Settings4Maintenance and Service4Ship Prep Mode4Next. 2. Touch to eject the instrument drawer. 3. Load the packing plate or an empty plate, then touch to close the drawer. 4. Touch Lock Block. 5. Power off the instrument using the power switch on the back of the instrument. The instrument is now ready to store, move, or ship. Move the instrument CAUTION! PHYSICAL INJURY HAZARD. Do not attempt to lift the instrument or any other heavy objects unless you have received related training. Incorrect lifting can cause painful and sometimes permanent back injury. Use proper lifting techniques when lifting or moving the instrument. At least two people are required to lift it. IMPORTANT! Moving your instrument can create subtle changes in the alignment of the instrument optics. Recalibrate the instrument if necessary. • Ensure that the surface on which you place the instrument can support at least 35 kg (77 lbs). • Ensure that the path to transport the instrument is clear of obstructions. • At least two people are needed to lift and carry the instrument. • Keep your spine in a good neutral position. • Bend at the knees and lift with your legs. • Do not lift an object and twist your torso at the same time. • Coordinate your intentions with your assistant before lifting and carrying. IMPORTANT! After moving the instrument, perform an RNase P instrument verification run. If the run fails, perform ROI/ uniformity, background, and dye calibrations. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 65 Chapter 6 Maintain the instrument 6 Store, move, or ship the instrument Return the instrument for service The service process requires 2 to 3 weeks. Before returning the instrument for service, perform the following tasks. 1. Back up the instrument (see “Backup or restore the instrument” on page 60 ). 2. In the home screen, touch Settings4Instrument Settings4Reset Factory defaults. 3. Set the instrument to Ship Prep Mode (see “Prepare the instrument to store, move, or ship” on page 65). To return the instrument for service, perform the following tasks. 1. Contact your local customer care center or technical support group to obtain a copy of the Certificate of Instrument Decontamination, a service notification, a service call number, and packaging materials (if required). 2. Follow the instructions in the form to decontaminate the instrument. IMPORTANT! The instrument must be decontaminated before packing it for shipping. 3. Complete and sign a copy of the Certificate of Instrument Decontamination. 4. Fax the Certificate of Instrument Decontamination to the customer care center. 5. Pack the instrument in the provided packaging and follow the instructions in the table below. Prepare Include Exclude 1. Transfer any data files from the • Instrument Any accessories, including: instrument. • Completed and signed • Power cord 2. Load an empty plate in the Certificate of Instrument • Ethernet cable sample block. Decontamination • USB drive 3. Use the touchscreen to place Note: The instrument will not be • Wireless adapter the instrument in ship mode. accepted for service without a hard copy of the Certificate of Note: If included with Note: The empty plate and the instrument, these items Instrument Decontamination. ship mode protects the internal will be disposed of during components of the instrument during service and not returned. transport. 6. Attach the postage provided with the Certificate of Instrument Decontamination to the box, then ship the instrument to the designated facility. 66 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 A Troubleshooting Instrument troubleshooting Observation Possible cause Recommended action Insufficient disk space Insufficient disk space to save 1. In the home screen, touch message a run. Settings4Run History4Manage. 2. Delete or transfer experiments from the instrument. The touchscreen is black The instrument is in sleep Touch anywhere on the instrument mode. touchscreen. The instrument is not powered If you touch the instrument touchscreen and on. it remains black, check if the instrument is powered on. The power switch is located on the rear panel of the instrument. If the instrument does not power on, check that the instrument is properly plugged in. If the instrument does not power on and the instrument is properly plugged in, contact Support. Forgot PIN for instrument Non-administrator forgot See “Manage all instrument profiles” on profile instrument profile PIN. page 29. Administrator forgot instrument Have another administrator reset the PIN for profile PIN. the forgotten-PIN profile (see “Manage all instrument profiles” on page 29). If there is not another administrator profile on the instrument, you must restore factory defaults (see “Restore factory defaults” on page 40). QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 67 Appendix A Troubleshooting A Troubleshoot calibration failure Troubleshoot calibration failure Observation Possible cause Recommended action Calibration failed The plate was improperly Ensure the following: prepared. • The correct plate was used for the calibration performed. • The plate was properly thawed. • The plate was properly centrifuged. • The plate was properly sealed. The plate is damaged or Check for damage, improper plate seal, or contaminated. contamination. Order a replacement plate. If the replacement plate fails, contact Support. High fluorescence signal in Signals that exceed the See “Identify contamination” on page 50. individual wells limit of normal fluorescence may indicate fluorescent contaminants on the plate or the sample block. Calibration failed but plate is The incorrect plate was used Use the plate that matches the calibration undamaged for calibration performed. performed. The plate was improperly Repeat the calibration with the plate properly prepared. prepared. If the calibration fails again, order a replacement plate. If the replacement plate fails, contact Support. 68 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix A Troubleshooting Troubleshoot verification failure A Troubleshoot verification failure Observation Possible cause Recommended action Verification failed The plate was improperly Ensure the following: prepared. • The correct plate was used for the verification performed. • The plate was properly thawed. • The plate was properly centrifuged. • The plate was properly sealed. The plate is damaged or Check for damage, improper plate seal, or contaminated. contamination. Order a replacement plate. If the replacement plate fails, contact Support. High fluorescence signal The reaction volume is not Ensure that reaction volumes in the plate are correct. correct and match the volume that is entered in the Method tab. Signals that exceed the Examine the bottom of the reaction plate. If limit of normal fluorescence there is contamination, prepare a new plate may indicate fluorescent and run the experiment again. contaminants on the plate or Identify the location of contamination on the the sample block. plate or sample block. 1. Obtain a background plate. 2. Follow the procedures that are described in “Identify contamination” on page 50. Verification failed but plate is The incorrect plate was used Use the correct RNase P plate for verification. undamaged for verification. The plate was improperly Repeat the verification with a new properly prepared. prepared plate. Note: The verification procedure is an experiment run, so each RNase P plate can only be used once. If the verification fails again, order a replacement plate. If the replacement plate fails, contact Support. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 69 B Parts and materials ■ Kits, consumables, accessories, and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 ■ Consumables (96‑well, 0.2‑mL format) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 ■ Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 ■ General-use materials and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Kits, consumables, accessories, and reagents Unless otherwise indicated, all materials are available through thermofisher.com. Store all calibration and RNase P plates at –20°C. All other items can be stored at 15–30°C. Use all materials by the expiration date on the packaging. Catalog numbers that appear as links open the web pages for those products. 70 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix B Parts and materials Consumables (96‑well, 0.2‑mL format) B Consumables (96‑well, 0.2‑mL format) Consumable Amount Cat. No. MicroAmp™ Optical 8-Cap Strips 300 strips 4323032 MicroAmp™ Optical 8-Tube Strip, 0.2 mL 125 strips 4316567 MicroAmp™ Optical Tube without Cap, 0.2 mL 2,000 tubes N8010933 MicroAmp™ 96-Well Tray/Retainer Set for Veriti™ Systems 10 trays 4381850 10 plates N8010560 MicroAmp™ Optical 96-Well Reaction Plate 500 plates 4316813 20 plates 4306737 MicroAmp™ Optical 96-Well Reaction Plate with Barcode 500 plates 4326659 20 plates 4483354 MicroAmp™ EnduraPlate™ Optical 96-Well Clear Reaction Plates with Barcode 500 plates 4483352 MicroAmp™ EnduraPlate™ Optical 96-Well Clear GPLE Reaction Plates with 20 plates 4483348 Barcode 500 plates 4483351 MicroAmp™ EnduraPlate™ Optical 96-Well Fast Clear Reaction Plates with Barcode 20 plates 4483485 MicroAmp™ Optical Adhesive Film Kit 1 kit 4313663 MicroAmp™ Optical Film Compression Pad (Optional, used for some applications) 5 each 4312639 Instrument verification or calibration plate Cat. No. TaqMan™ RNase P Instrument Verification Plate, 96‑Well 0.2‑mL 4432382 Region of Interest (ROI) and Background Plates, 96‑Well 0.2‑mL (2 plates) 4432364 QuantStudio™ 3/5 10‑Dye Spectral Calibration Kit, 96‑Well 0.2‑mL A26343 (contains all 3 spectral calibration plates listed below) QuantStudio™ 3/5 Spectral Calibration Plate 1, 96‑Well 0.2‑mL A26331 (FAM™, VIC™, ROX™, SYBR™ dyes) QuantStudio™ 3/5 Spectral Calibration Plate 2, 96‑Well 0.2‑mL A26332 (ABY™, JUN™, MUSTANG PURPLE™ dyes) QuantStudio™ 3/5 Spectral Calibration Plate 3, 96‑Well 0.2‑mL A26333 (TAMRA™, NED™, Cy™5 dyes) Spectral calibration plates for dyes used with HID-validated workflows ABY™ Spectral Calibration Plate, 96‑Well 0.2‑mL 4461591 JUN™ Spectral Calibration Plate, 96‑Well 0.2‑mL 4461593 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 71 Appendix B Parts and materials B Accessories Accessories Item Amount Cat. No. MicroAmp™ Multi Removal Tool 1 tool 4313950 MicroAmp™ Cap Installing Tool (handle style) 1 tool 4330015 MicroAmp™ Optical Adhesive Film 25 films 4360954 100 films 4311971 MicroAmp™ Adhesive Film Applicator 5 applicators 4333183 General-use materials and consumables The following general-use materials and consumables are required to calibrate, maintain, and operate the instrument. Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier. Material/Consumable Source Centrifuge with 96‑well plate buckets MLS Cotton or nylon swabs and lint‑free cloths MLS Ethanol, 95% solution MLS Optical clear adhesive film for PCR MLS Na-hypochlorite, 0.1% v/v solution MLS Pipettors, 100‑µL and 200‑µL (with pipette tips) MLS Powder‑free gloves MLS Safety glasses MLS Screwdriver, flathead MLS Tissue, lint‑free MLS Deionized water MLS 72 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 C Instrument specification and layout ■ Configured system dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 ■ Instrument and computer connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 ■ Instrument clearances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 ■ Electrical requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 ■ Environmental requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 73 Appendix C Instrument specification and layout C Configured system dimensions Configured system dimensions Allow space for the configured instrument. A typical setup with a co-located minitower computer is shown below. Note: Dimensions are rounded to the nearest whole or half unit. 15 cm (6 in.) 50 cm (20 in.) 27 cm (10.5 in.) 30 cm (12 in.) 40 cm (16 in.) 207 cm (81.5 in.) 74 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix C Instrument specification and layout Instrument and computer connections C Instrument and computer connections 1 2 3 4 5 6 7 Figure 4 Instrument back panel 1 USB ports 4 RS232 Port—For service use only 2 WiFi USB port—Not applicable 5 Fuse Cover 3 Ethernet Port—RJ45 port for 100/1,000 Mbps Ethernet 6 Power Switch communication with the instrument 7 Power Port—100 to 240 VAC 3 2 1 1 4 Figure 5 Instrument‑to‑computer connections (barcode scanner connected to the computer) Minitower configuration 1 Detachable power supply cord compatible with local power supply receptacle. 2 Connection between the computer and the instrument. 3 Connection between the computer and the monitor, keyboard, and mouse. 4 Connection between the computer and the (optional) handheld barcode scanner. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 75 Appendix C Instrument specification and layout C Instrument clearances Instrument clearances During instrument installation and maintenance, it is necessary to access the back of the instrument. If the back of the instrument faces a wall, ensure that there is sufficient clearance on the bench to rotate the instrument for access. IMPORTANT! For safety, the power outlet for the instrument must be accessible. Component Top Front Sides Back Instrument 30 cm (12 in.) 30 cm (12 in.) 15 cm (6 in.) 15 cm (6 in.) Computer — 15 cm (6 in.) — 15 cm (6 in.) Electrical requirements WARNING! For safety, the power outlet used for powering the instrument must be accessible at all times. See “Instrument clearances” for information about the space needed between the wall and the instrument. In case of emergency, you must be able to immediately disconnect the main power supply to all the equipment. Allow adequate space between the wall and the equipment so that the power cords can be disconnected in case of emergency. WARNING! Use of accessories, transducers, and cables other than those specified or provided by the manufacturer of the equipment could result in increased electromagnetic emissions or decreased electromagnetic immunity of this equipment and result in improper operation. • Electric receptacle with grounding capability • Maximum power dissipation: ~960 W (not including computer and monitor) • Mains AC line voltage tolerances must be up to ±10 percent of nominal voltage Device Rated voltage Circuit required Rated frequency Rated power Instrument 100–240 ±10% VAC[1] 10 A 50/60 Hz 960 W Computer (laptop) 100–240 ±10% VAC 10 A 50/60 Hz 90 VA Computer (desktop) 125 VA 100–240 ±10% VAC 10 A 50/60 Hz Monitor 65 VA [1] If the supplied power fluctuates beyond the rated voltage, a power line regulator may be required. High or low voltages can adversely affect the electronic components of the instrument. 76 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix C Instrument specification and layout Environmental requirements C Environmental requirements Table 2 Environmental requirements Condition Acceptable range Installation site Indoor use only Electromagnetic Do not use this device in close proximity to sources of strong electromagnetic radiation interference (for example, unshielded intentional RF sources). Strong electromagnetic radiation may interfere with the proper operation of the device. Altitude Between sea level and 2000 m (6500 ft) above sea level Operating conditions • Humidity: 15–80% relative humidity (noncondensing) • Temperature: 15°C to 30°C (59°F to 86°F) Note: For optimal performance, avoid rapid or extreme fluctuations in room temperature. Storage and transport • Humidity: 20–80% relative humidity (noncondensing) conditions • Temperature: –30°C to 60°C (–22°F to 140°F) Thermal output During operation, the net thermal output, based on the actual current draw of the instrument, is expected to be approximately 960 W (3275 Btu/h). Vibration Ensure that the instrument is not adjacent to strong vibration sources, such as a centrifuge, pump, or compressor. Excessive vibration will affect instrument performance. Pollution degree The instrument has a Pollution Degree rating of II. The instrument may only be installed in an environment that has nonconductive pollutants such as dust particles or wood chips. Typical environments with a Pollution Degree II rating are laboratories and sales and commercial areas. The noise output of the instrument is £ 60 dB when running. Other conditions Ensure the instrument is located away from any vents that could expel particulate material onto the instrument components. Avoid placing the instrument and computer adjacent to heaters, cooling ducts, or in direct sunlight. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 77 D Safety ■ Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 ■ Safety alerts on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 ■ Safety information for instruments not manufactured by Thermo Fisher Scientific . . . . . . . . . . . . . 82 ■ Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 ■ Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 ■ Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 ■ Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. · Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. · Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain SDSs, visit thermofisher.com/support. Symbols on this instrument Symbols may be found on the instrument to warn against potential hazards or convey important safety information. In this document, the hazard symbol is used along with one of the following user attention words: • CAUTION!—Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. • WARNING!—Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. • DANGER!—Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. Symbol English Français Caution, risk of danger Attention, risque de danger Consult the manual for further safety Consulter le manuel pour d’autres information. renseignements de sécurité. 78 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix D Safety Symbols on this instrument D (continued) Symbol English Français Caution, risk of electrical shock Attention, risque de choc électrique Moving parts Parties mobiles Caution, hot surface Attention, surface chaude Potential biohazard Danger biologique potentiel Ultraviolet light Rayonnement ultraviolet Potential slipping hazard Danger de glisser potentiel On On (marche) Off Off (arrêt) On/Off On/Off (marche/arrêt) Standby En attente Earth (ground) terminal Borne de (mise à la) terre Protective conductor terminal (main Borne de conducteur de protection (mise à la ground) terre principale) Terminal that can receive or supply Borne pouvant recevoir ou envoyer une alternating current or voltage tension ou un courant de type alternatif Terminal that can receive or supply Borne pouvant recevoir ou envoyer une alternating or direct current or voltage tension ou un courant continu ou alternatif QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 79 Appendix D Safety D Symbols on this instrument (continued) Symbol English Français Do not dispose of this product in Ne pas éliminer ce produit avec les déchets unsorted municipal waste usuels non soumis au tri sélectif. CAUTION! To minimize MISE EN GARDE ! Pour minimiser negative environmental impact les conséquences négatives sur l’en‐ from disposal of electronic vironnement à la suite de l’élimination waste, do not dispose of de déchets électroniques, ne pas éli‐ electronic waste in unsorted miner ce déchet électronique avec les municipal waste. Follow local déchets usuels non soumis au tri sé‐ municipal waste ordinances for lectif. Se conformer aux ordonnances proper disposal provision and locales sur les déchets municipaux contact customer service for pour les dispositions d’élimination et information about responsible communiquer avec le service à la cli‐ disposal options. entèle pour des renseignements sur les options d’élimination responsable. Conformity symbols Conformity mark Description Indicates conformity with safety requirements for Canada and U.S.A. Indicates conformity with European Union requirements for safety and electromagnetic compatibility. Indicates conformity with Australian standards for electromagnetic compatibility. 80 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix D Safety Safety alerts on this instrument D Safety alerts on this instrument Additional text may be used with one of the symbols described above when more specific information is needed to avoid exposure to a hazard. See the following table for safety alerts found on the instrument. English Français CAUTION! Hazardous chemicals. MISE EN GARDE ! Produits chimiques dange‐ Read the Safety Data Sheets (SDSs) reux. Lire les fiches signalétiques (FS) avant de ma‐ before handling. nipuler les produits. CAUTION! Hazardous waste. Refer MISE EN GARDE ! Déchets dangereux. Lire les to SDS(s) and local regulations for fiches signalétiques (FS) et la réglementation locale handling and disposal. associées à la manipulation et à l’élimination des déchets. Location of safety labels on the instrument QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 81 Appendix D Safety D Safety information for instruments not manufactured by Thermo Fisher Scientific Safety information for instruments not manufactured by Thermo Fisher Scientific Some of the accessories provided as part of the instrument system are not designed or built by Thermo Fisher Scientific. Consult the manufacturer's documentation for the information needed for the safe use of these products. Instrument safety General CAUTION! Do not remove instrument protective covers. If you remove the protective instrument panels or disable interlock devices, you may be exposed to serious hazards including, but not limited to, severe electrical shock, laser exposure, crushing, or chemical exposure. CAUTION! Solvents and Pressurized fluids. Wear eye protection when working with any pressurized fluids. Use caution when working with any polymeric tubing that is under pressure: · Extinguish any nearby flames if you use flammable solvents. · Do not use polymeric tubing that has been severely stressed or kinked. · Do not use polymeric tubing with tetrahydrofuran or nitric and sulfuric acids. · Be aware that methylene chloride and dimethyl sulfoxide cause polymeric tubing to swell and greatly reduce the rupture pressure of the tubing. · Be aware that high solvent flow rates (~40mL/min) may cause a static charge to build up on the surface of the tubing and electrical sparks may result. 82 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix D Safety Instrument safety D Physical injury CAUTION! Moving and Lifting Injury. The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide. Improper lifting can cause painful and permanent back injury. Things to consider before lifting or moving the instrument or accessories: · Depending on the weight, moving or lifting may require two or more persons. · If you decide to lift or move the instrument after it has been installed, do not attempt to do so without the assistance of others, the use of appropriate moving equipment, and proper lifting techniques. · Ensure you have a secure, comfortable grip on the instrument or accessory. · Make sure that the path from where the object is to where it is being moved is clear of obstructions. · Do not lift an object and twist your torso at the same time. Keep your spine in a good neutral position while lifting with your legs. · Participants should coordinate lift and move intentions with each other before lifting and carrying. · For smaller packages, rather than lifting the object from the packing box, carefully tilt the box on its side and hold it stationary while someone else slides the contents out of the box. CAUTION! Moving Parts. Moving parts can crush, pinch and cut. Keep hands clear of moving parts while operating the instrument. Disconnect power before servicing. WARNING! Do not attempt to lift or move the instrument without the assistance of others. Use appropriate moving equipment and proper lifting technique, improper lifting may result in serious injury. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 83 Appendix D Safety D Safety and electromagnetic compatibility (EMC) standards Electrical safety WARNING! Fuse Installation. Before installing the instrument, verify that the fuses are properly installed and the fuse voltage matches the supply voltage. Replace fuses only with the type and rating specified for the unit. Improper fuses can damage the instrument wiring system and cause a fire. WARNING! Ensure appropriate electrical supply. For safe operation of the instrument: · Plug the system into a properly grounded receptacle with adequate current capacity. · Ensure the electrical supply is of suitable voltage. · Never operate the instrument with the ground disconnected. Grounding continuity is required for safe operation of the instrument. WARNING! Power Supply Line Cords. Use properly configured and approved line cords for the power supply in your facility. WARNING! Disconnecting Power. To fully disconnect power either detach or unplug the power cord, positioning the instrument such that the power cord is accessible. Cleaning and decontamination CAUTION! Cleaning and Decontamination. Use only the cleaning and decontamination methods specified in the manufacturer's user documentation. It is the responsibility of the operator (or other responsible person) to ensure the following requirements are met: · No decontamination or cleaning agents are used that could cause a HAZARD as a result of a reaction with parts of the equipment or with material contained in the equipment. · The instrument is properly decontaminated a) if hazardous material is spilled onto or into the equipment, and/or b) prior to having the instrument serviced at your facility or sending the instrument for repair, maintenance, trade-in, disposal, or termination of a loan (decontamination forms may be requested from customer service). · Before using any cleaning or decontamination methods (except those recommended by the manufacturer), users should confirm with the manufacturer that the proposed method will not damage the equipment. Safety and electromagnetic compatibility (EMC) standards The instrument design and manufacture complies with the following standards and requirements for safety and electromagnetic compatibility. 84 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix D Safety Safety and electromagnetic compatibility (EMC) standards D Safety compliance Reference Description EU Directive 2014/35/EU European Union “Low Voltage Directive” IEC 61010-1 Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1: General requirements EN 61010-1 UL 61010-1 CSA C22.2 No. 61010-1 IEC 61010-2-010 Safety requirements for electrical equipment for measurement, control and laboratory use – Part 2-010: Particular requirements for laboratory equipment for EN 61010-2-010 the heating of materials IEC 61010-2-081 Safety requirements for electrical equipment for measurement, control and laboratory use – Part 2-081: Particular requirements for automatic and semi- EN 61010-2-081 automatic laboratory equipment for analysis and other purposes EMC Reference Description Directive 2014/30/EU European Union “EMC Directive” EN 61326-1/ IEC 61326-1 Electrical Equipment for Measurement, Control and Laboratory Use – EMC Requirements – Part 1: General Requirements AS/NZS CISPR 11 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial, Scientific, and Medical (ISM) Radiofrequency Equipment ICES-001, Issue 4 Industrial, Scientific and Medical (ISM) Radio Frequency Generators FCC Part 15 Subpart B (47 CFR) U.S. Standard Radio Frequency Devices This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 85 Appendix D Safety D Safety and electromagnetic compatibility (EMC) standards Environmental design Reference Description Directive 2012/19/EU European Union “WEEE Directive” – Waste electrical and electronic equipment EU Directive 2011/65/EU & European Union “RoHS Directive” – Restriction of hazardous Commission Delegated Directive substances in electrical and electronic equipment (EU) 2015/863 Directive 2006/66/EC European Union “Battery Directive” GB/T 26572 Requirements of concentration limits for certain restricted substances in electrical and electronic products. China EEP Hazardous Substances Information Component Name QuantStudio™ 1 Real-Time PCR Instrument (96-Well 0.2-mL Block) Hazardous Substances (Pb) (Hg) (Cd) (Cr(VI)) (PBB) (PBDE) PCBA X O O O O O SJ/T11364. This table is compiled according to SJ/T 11364 standard. O: GB/T26572. Indicates that the concentration of the hazardous substance in all homogeneous materials for the part is below the relevant threshold of the GB/T 26572 standard. X: GB/T26572. Indicates that the concentration of the hazardous substance in at least one homogenous material of the part is above the relevant threshold of the GB/T 26572 standard. 86 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Appendix D Safety Chemical safety D Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions: · Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document. · Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). · Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). · Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. · Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) · After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. · Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. · IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling warning. WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 87 Appendix D Safety D Biological hazard safety Biological hazard safety WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards. WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment. · U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at: www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2020-P.pdf · World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/publications/i/item/9789240011311 88 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 E Documentation and support Related documentation Document Pub. No. QuantStudio™ 5 Real-Time PCR Instrument Site Preparation Guide (for Human Identification) MAN0016701 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) MAN0017162 HID Real‑Time PCR Analysis Software User Guide MAN0009819 Obtain information from the Help system The instrument has a Help system that describes how to use each feature of the touchscreen. Touch on the instrument touchscreen to access the Help system. Customer and technical support For support: • In North America—Send an email to [email protected], or call 888-821-4443 option 1. • Outside North America—Contact your local support office. For the latest services and support information for all locations, go to thermofisher.com/support to obtain the following information. • Worldwide contact telephone numbers • Product support • Order and web support • Safety Data Sheets (SDSs; also known as MSDSs) Additional product documentation, including user guides and Certificates of Analysis, are available by contacting Customer Support. QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 89 Appendix E Documentation and support E Limited product warranty Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/ global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support. 90 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Index A Connect profile, Overview 31 consumables, 96-well 0.2-mL 71 accessories 72 contamination, identify 50 administrator instrument profile 28 custom dye Administrator profile 32 add to the instrument 52 calibration, perform 53 B description 10 custom dye calibration, workflow, HID 51 background calibration custom dyes, HID‑validated workflow 51 create plate 50 description 44 plate, prepare 46, 51 D Background calibration date and time 39 run 47 disk space message when to perform 43 documentation, related 89 backup instrument 60 dye calibration biohazard safety 88 description 44 plate, prepare 46, 51 C Dye calibration run 47 calibration when to perform 43 Background 47 dyes custom dye. See custom dye custom 10 Dye 47 system 9 plate, prepare 46, 51 reminders 45 ROI/Uniformity 47 E schedule 43 Edit profile 38 status 45 EDS files, transfer 22 transfer results 49 view images 49 workflow 45 F calibration failure, troubleshoot 68 factory defaults 40 Change password 38 firewall ports 17 Change PIN 38 fluorescence high computer fuses, replace 64 connection to instrument or LAN 17 connections 16, 75 Connect G Link 37 guest profile, disable 38 Link to instrument 33 Roles Administrator 32 H Standard 32 heated cover temperature, set for instrument idling Unlink from instrument 36 39 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 91 Index Help system, access 89 Profile home screen, parts of 11 Connect Link 33 Unlink 36 I Edit 38 installation profiles before you begin 14 administrator 28 workflow 14 delete 29 instrument edit 29 profile, create 29 guest, disable 38 connections 16, 75 instrument 29 enable for remote monitoring 40 require sign-in 38 heated cover idle temperature 39 reset PIN 29 move 65 sign in 20 name 39 Profiles overview 7 Connect parts of 10 Administrator 32 power off 19 Standard 32 power on 19 Order to set up 31 prepare to store or move or ship 65 profile, overview 28 return for service 66 R touchscreen black real-time plots instrument filters 9 view on instrument touchscreen 22 instrument layout 73 zoom 22 instrument settings 24, 27 related documentation 89 instrument specification 73 Replace with index term instrument verification. See RNase P verification require sign in 38 restore instrument 60 RNase P verification L pass criteria 56 limited product warranty 90 plate description 56 load plate 21 run 57 Local instrument profile, Overview 31 RNase P verification, plate, prepare 57 ROI/uniformity calibration description 44 M plate, prepare 46, 51 maintenance 60 ROI/Uniformity calibration run 47 when to perform 43 N run data, transfer 22 network, configurations supported 17 S O safety, biohazard 88 online Help. See Help system sample block Order to set up profiles 31 clean 61 decontaminate 61 settings 24, 27 P sign in, require 38 Password, Change 38 sign out performance verification. See RNase P verification profile 20 PIN, Change 38 timer, set 39 PIN reset 29 sleep mode 39 92 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 Index software updates 41 plate, prepare 46, 51 Standard profile 32 unload plate 21 standby mode 39 unpack instrument 15 startup wizard 15 supported dyes 9 symbols, safety 78 V system dyes 9 verification failure, troubleshoot 69 T W terms and conditions 90 warranty 90 Thermo Fisher Connect profile, Edit 38 well details, view on instrument touchscreen 22 troubleshooting 67 workflow, custom dye calibration, HID 51 U Z uniformity calibration zoom 22 description 44 QuantStudio™ 5 Real-Time PCR Instrument User Guide (for Human Identification) | MAN0017162 93 QS 5HID_Instrument UG_MAN0017162-v3-GUID-633E64F5-3506-485C-B537-EA26655B5DD2-2022/10/04 00:17:18 en 19:13:23.447Z thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 9 November 2022

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