User Bulletin
Applied Biosystems 3500/3500xL Genetic Analyzer
June 2011
SUBJECT: Protocols for the analysis of AmpFlSTR® PCR Amplification
Kit PCR products and validation summary
This user bulletin describes protocols for processing AmpFlSTR® PCR Amplification
kit products on the Applied Biosystems 3500 Series Genetic Analyzers, using 3500
Data Collection software Version 1.0 and GeneMapper® ID-X Software Version 1.2 or
later. In addition, comprehensive information regarding the validation of the 3500
Series Genetic Analyzers for use with the AmpFlSTR® Kits is also included. This user
bulletin is intended to be used in conjunction with related 3500 documentation.
References to relevant supplemental documentation are provided where
appropriate.
In This User Section 1: Preparing the Instrument for Human Identification STR Analysis . . . . . . 4
Bulletin Section 2: 3500 Data Collection Software Components . . . . . . . . . . . . . . . . . . . . . . . . 23
Section 3: Sample Run Workflow and Run Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Section 4: Developmental Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Validated instrument protocol specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Documentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Overview The 3500 Series Genetic Analyzers for Human Identification Applications are the
first capillary electrophoresis (CE) instruments developed specifically to meet the
requirements of Human Identification (HID) laboratories. Features of these
analyzers include:
• 8-capillary 3500 System and 24-capillary 3500xL System provide higher
throughput.
• Advanced thermal system design improves temperature control for more
consistent data migration and contributes to reduced run times.
• Reduced signal variation from instrument to instrument, run to run, and
capillary to capillary.
• Innovative “snap-in-and-go” consumable design with Radio Frequency
Identification (RFID) technology helps to track and record key consumables
data.
• HID specific workflow, pre-configured for AmpFlSTR® kits, simplifies run
setup and software navigation.
• Integrated data collection and QC analysis software provides real-time
assessment of data quality and re-injection options.
• Solid State laser allows use with standard electrical power configurations and
requires no heat removal.
Supplementary For more detailed information on the 3500 Series Genetic Analyzers and related
references procedures, please refer to the following documents:
• Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661)
• 3500 and 3500xL Genetic Analyzers Quick Reference Card (Part no. 4401662)
• GeneMapper® ID-X Software Version 1.2 Quick Reference Card (Part no. 4426482)
• 3500 Data Collection v1.0 Software Release Notes (can be found on the 3500
Data Collection Software CD)
• User Bulletin: Applied Biosystems 3500/3500xL Genetic Analyzer (Part no.
4445098)
• GeneMapper® ID-X Software v1.2 User Bulletin (Part no. 4462639)
• Frequently Asked Questions (FAQs) and supplementary information is
contained on the Applied Biosystems website,
http://marketing.appliedbiosystems.com/mk/get/3500_HID_FAQS.
Note: The information and procedures contained in this user bulletin relate
specifically to the HID workflow and analysis of AmpFlSTR® PCR Amplification kit
products. Some of the recommendations may differ from information contained in
the related documentation. Users should follow the specific recommendations
contained in this guide unless directed specifically to related documentation.
2 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Document overview
Section 2
Section 1 Section 3 Section 4
Software
HID Calibrations Sample run Developmental validation
components
(see page 4) (see page 28) (see page 34)
(see page 23)
Spatial calibration Assays Assign plate Materials and methods
contents
(see page 4) (see page 23) (see page 34)
(see page 28)
Spectral Instrument Genotype concordance and
calibration protocols Load plate for reproducibility
the run (see page 41)
(see page 8) (see page 25)
(see page 29)
HID performance QC protocols Sizing precision and
check Evaluate data resolution
(see page 26)
real time (see page 43)
(see page 15)
(see page 30)
HID plate
Sensitivity and Normalization
templates
Specify (see page 46)
(see page 27)
reinjections
(see page 32) Sensitivity
(see page 46)
Baseline Noise Analysis
(see page 51)
Internal Standard
Normalization
(see page 55)
Mixture analysis
(see page 60)
Conclusions
(see page 64)
Ordering information (see page 65)
Validated instrument protocol specifications (see page 67)
Documentation (see page 69)
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 3
Section 1: Preparing the Instrument for Human Identification STR Analysis
Section 1: Preparing the Instrument for Human Identification
STR Analysis
This section covers:
Performing a spatial calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Performing a spectral calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Performing a HID Install Standard performance check . . . . . . . . . . . . . . . . . . . . . . . . 15
Note: To start the 3500 Genetic Analyzer properly, refer to the Applied Biosystems
3500/3500xL Genetic Analyzer User Guide (Part no. 4401661). Also, perform any
required maintenance tasks as indicated by the Instrument Gauges and Maintenance
Notifications on the Data Collection Dashboard Screen described in the Applied
Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Upon installation of a new 3500 series instrument for HID analysis, and prior to
performing HID analysis of AmpFlSTR® PCR Amplification kit products for the first
time, the field service engineer will complete the following procedures:
• Spatial calibration
• Spectral calibration
• HID Install Standard performance check
These procedures should be completed by individual laboratories or Life
Technologies Field Service Engineers as necessary after installation.
Note: In addition to the guidance in this section, refer to the Applied Biosystems
3500/3500xL Genetic Analyzer User Guide (Part no. 4401661) for additional guidance
pertaining to the instrument calibrations and performance check.
Save or print IMPORTANT! The software does not save historical calibration or performance
calibration reports! check results. Only the most recent information is maintained in the software;
therefore, after performing a calibration, the calibration reports should be saved
electronically for record keeping purposes. Follow the instructions for printing
calibration records or saving them electronically referenced in the Applied Biosystems
3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Performing a spatial calibration
The 3500 Series Data Collection 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 is detected by the CCD camera. A spatial
calibration is required to ensure correct alignment of the capillaries and optimal
detection of the signal in each capillary.
A new spatial calibration is recommended when:
• A capillary array is installed, replaced or temporarily removed from the
detection block
• The instrument is moved
• The detection cell door is opened
4 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Perform a spatial 1. Click Maintenance on the Dashboard. Click
calibration Spatial under the Calibrate heading on the left
of the navigation pane shown below.
2. Select No Fill, or select Fill to fill the array with
polymer before starting the calibration.
Step 2
Step 3
Step 4
3. (Optional) Select Perform QC Checks if you want the system to check each
capillary against the specified range for spacing and intensity. During the
calibration, the software calculates:
Attribute Calculation Threshold
Average peak height sum of all peak heights • 8-cap: 6400 RFU
number of peaks
• 24-cap: 3000 RFU
Uniformity (peak height standard deviation 0.2
similarity)
average peak height
Capillary spacing max spacing − min spacing 2 pixels
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 5
Performing a spatial calibration
4. Click Start Calibration. The display updates as the run progresses.
Note: If you engaged the optional QC
Check, and the average of any of the
QC values exceeds the threshold, a
Spatial QC Check error message is
displayed. Click OK and re-run the
Spatial Calibration.
Evaluate the When the run is complete, evaluate the spatial calibration profile to ensure that you
spatial calibration see the following:
profile 1. One sharp peak for each capillary. Small shoulders may be acceptable.
2. One marker (+) at the apex of every peak. No off-apex markers.
3. An even peak profile (all peaks about the same height).
4. Spacing should be between 13 and 16.
Note: If any peaks are lower than usual for the instrument or the heights
drastically slope up or down, repeat the calibration with a fill. See the following
figures for common acceptable Spatial Profiles.
Figure 1 Acceptable spatial profiles (8-capillary: top, 24-capillary: bottom)
5. If the results meet the criteria above, click Accept Results. If the results do not
meet the criteria listed above, click Reject Results, then refer to “Spatial
calibration troubleshooting” in the Applied Biosystems 3500/3500xL Genetic
Analyzer User Guide (Part no. 4401661) for troubleshooting information.
Export or print To export spatial calibration results:
spatial calibration
results
6 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
1. Click Export Results.
2. Enter an export file name.
3. Select the export file type.
4. Click Save.
View or print Note: Spatial and spectral calibration reports include the date on which a capillary
spatial calibration 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
report
instrument.
1. Click View Spatial Calibration 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.
4. Close the report.
IMPORTANT! After performing a calibration, you can save the calibration report
electronically for record keeping. The software does not save historical calibration results.
Only the most recent spatial calibration is maintained in the software. In the Printer dialog
box, select CutePDF Writer as the printer to print the report to a PDF file.
For additional information on performing and evaluating a spatial calibration, refer
to the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 7
Performing a spectral calibration
Performing a spectral calibration
A spectral calibration creates a matrix that corrects for the overlapping fluorescence
emission spectra of the dyes. Although each of these dyes emits its maximum
fluorescence at a different wavelength, there is some overlap in the emission spectra
between the dyes. The goal of multicomponent analysis is to effectively correct for
spectral overlap and minimize the presence of artifacts, such as spectral pull-up, in
the data.
A new spectral calibration is recommended when:
• The capillary array is changed or temporarily removed from the detection
block.
Note: Better dye separation is achieved when a new spectral calibration is
performed after removal or replacement of the capillary array and is therefore
highly recommended.
• The instrument is moved.
• The laser or CCD camera has been realigned/replaced by a service engineer.
• An increase in (pull-up and/or pull-down peaks) is observed.
• A new dye set is used on the instrument.
• The capillary array length or polymer type is changed.
• The detection cell door is opened (not required by the software, but
recommended).
Prepare the 1. If necessary, perform a spatial calibration as described in the previous section.
instrument 2. In the Dashboard, check consumable status and ensure that:
• Consumables are not expired
• Adequate injections remain for consumables
3. Ensure that the buffer levels are at the fill lines.
4. Set the oven temperature to 60°C, then click Start Pre-heat.
Note: Preheating for approximately 30 minutes helps mitigate subtle first-run
migration rate effects. The pre-heat function automatically turns off after 2
hours.
5. Check the pump assembly for bubbles and run the Remove Bubble wizard if
needed.
Prepare the For all chemicals, read the SDS, and follows the handling instructions. Wear
standard appropriate protective eyewear, clothing, and gloves.
calibration plate
8 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
1. Choose the appropriate Dye Set and Matrix Standards.
For AmpFlSTR® kits that use a... And use... AmpFlSTR® kit Examples
5-dye system, which includes the Matrix Standard Set DS-33 • Identifiler® Plus PCR Amplification Kit
following dyes: (Dye Set G5) • Identifiler® PCR Amplification Kit
• 6FAM™ • Identifiler® Direct PCR Amplification Kit
• VIC®
• NGM™ PCR Amplification Kit
• NED™
• NGM SElect™ PCR Amplification Kit
• PET®
• Yfiler® PCR Amplification Kit
• LIZ®
• SEfiler Plus™ PCR Amplification Kit
• Other five-dye AmpFlSTR® kits
4-dye system, which includes the Matrix Standard Set DS-32 • Profiler Plus® PCR Amplification Kit
following dyes: (Dye Set F) • COfiler® PCR Amplification Kit
• 5FAM™ • Profiler Plus® ID PCR Amplification Kit
• JOE™
• SGM Plus® PCR Amplification Kit
• NED™
• Other 4-dye AmpFlSTR® kits
• ROX™
2. Thaw and thoroughly vortex the Matrix Standard tube and spin the tube briefly
in a microcentrifuge.
3. Prepare the Matrix Standard by combining the following in a labeled 1.5-mL
colorless microcentrifuge tube:
Matrix Standard Set Matrix Standard Set
Reagent DS-33 DS-32
(Dye Set G5) (Dye Set F)
Matrix Standard (Part no. 4345833) 3 µl 4 µl
Hi-Di™ Formamide 297 µl 246 µl
(Part no. 4311320)
Final Volume (1.5mL tube) 300 µl 250 µl
4. Vortex thoroughly and spin the mixture briefly in a microcentrifuge.
5. Heat the tube at 95°C for 5 minutes to denature the sample and immediately
place the tube on ice for 3 minutes.
Note: In some instances, if the spectral calibration is of sub-optimal quality as
determined by inspection of the calibration data or the presence of excess pull-
up in the test sample data, an improved spectral may be obtained by
eliminating the chilling step after heating.
6. Load 10 µL of the spectral calibration mixture into each well.
Note: For a 96-well plate, use 10 µL of mixture per well. For a 384-well plate,
use 5 µL of mixture per well. Add the mixture to wells A1-H1 on a 3500
instrument or A1-H3 on a 3500xL instrument and load the plate assembly on
the instrument.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 9
Performing a spectral calibration
IMPORTANT! You are not required to create a plate for the calibration. The
software uses predetermined positions for the calibration.
8-capillary A1 through H1
96-well plate
24-capillary A1 through H1, A2 through H2, and A3
through H3
96-well plate
7. Centrifuge the plate to ensure that the samples are at the bottom of the wells.
IMPORTANT! 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.
Prepare the plate 1. Place the sample plate into the plate base.
assembly 2. Align the holes in the septa
strip with the wells of the
plate, then firmly press Plate retainer
downward onto the plate.
IMPORTANT! Make sure to
use the correct plate base for Plate with
standard plates versus 8-tube septa strip
strips and fast plates. Using
the wrong plate base may
affect performance. 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 strip are aligned. If not aligned, re-assemble and
then assemble the plate assembly.
IMPORTANT! The array tips will be damaged if the plate retainer and septa
strip holes do not align correctly.
Load the plate in IMPORTANT! Do not change electronic signature settings during a spectral
the instrument and calibration.
IMPORTANT! If you change polymer type, spectral calibrations for the original
initiate the
polymer type are not retained.
calibration
The following are the basic steps to initiate the spectral calibration. Refer to the
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661) for
more information.
10 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
1. Place the plate in the autosampler with the labels facing outward and the
notched corner of the plate in the notched corner of the autosampler.
2. Close the instrument door to re-initialize the instrument.
3. Click Maintenance on the dashboard.
4. Click on Spectral under Calibrate in the left navigation pane.
5. Select the number of wells and plate position on the instrument.
6. Select the chemistry standard and dye set.
(Optional) Select Allow Borrowing to have a failed capillary use a neighboring
capillary’s spectral data. Refer to the Applied Biosystems 3500/3500xL Genetic
Analyzer User Guide (Part no. 4401661) for more information.
5
6
7
7. Click Start Run. The following occurs:
• The system sets up three injections.
• 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 capillaries are shown in green and failing capillaries are shown in
red. Borrowed capillaries are shown in yellow with an arrow indicating the
adjacent capillary from which results were borrowed.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 11
Performing a spectral calibration
8. 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 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)
F 0.95 8.5
G5 0.95 13.5
9. If the data for all capillaries meet the acceptance criteria, click the Accept
Results button.
Note: If the data for all capillaries does not meet the acceptance criteria, click the
Reject Results button, repeat the analysis and begin the troubleshooting process. For
more information on troubleshooting spectral calibrations refer to the Applied
Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Evaluate the This section contains basic information regarding evaluation of the spectral
spectral calibration data. For more information regarding evaluation of spectral calibration
data, refer to the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no.
calibration data
4401661).
Excessive pull-up signal in the analyzed data is the most common manifestation of a
poor quality spectral calibration. The following characteristics will assist the user in
determining if the spectral calibration may be suitable:
• Order of the peaks in the profile from left to right:
F: Blue, green, yellow, red
G5: Blue, green, yellow, red, orange
• No extraneous peaks in the raw data profile.
• No gross dips, overlaps or other irregular morphology.
• Spectral profile peaks are separate and distinct.
12 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
• Ideally, spectral peak heights should be approximately similar to the peak
heights typically analyzed by the laboratory.
Figure 2 Order of the 4-dye (left) and 5-dye (right) spectral calibration peaks
A spectral calibration automatically sets up three injections. The number of
injections performed depends on the number of capillaries that pass or fail during an
injection and whether you select Allow Borrowing.
When borrowing is disabled, all capillaries must meet passing criteria. When
borrowing is enabled, all capillaries have to pass within the borrowing limits.
For a successful calibration, there must be at least one passing attempt for each
capillary within the three injection profile. A passing attempt is defined within the
parameters set regarding borrowing and are described in the following section and
in the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Spectral calibrations with borrowing disabled
When borrowing is disabled, any failure in the quality value or condition number
will prompt a reinjection of the matrix standards and new attempt at obtaining
acceptable values. If, after all three injections, no runs are acceptable, the calibration
will fail.
Spectral calibrations with borrowing enabled
When borrowing is enabled, the user can define the number of acceptable spectral
failures they will allow for a 3500xL (up to 3 maximum). For a 3500 series Genetic
Analyzer, only one failure is allowed.
Any failure in the quality value or condition number will prompt a reinjection of the
matrix standards and new attempt at obtaining acceptable values. If, after all three
injections, no runs are acceptable, the calibration will fail.
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.
Figure 3 Example of borrowing in a 3500 Series Genetic Analyzer
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 13
Performing a spectral calibration
Export or print View and print a spectral calibration report
spectral Note: Spatial and spectral calibration reports include the date on which a capillary
calibration results 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 Spectral Calibration 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.
4. Close the report.
IMPORTANT! After
performing a
calibration, save the calibration report electronically for record keeping. The
software does not save historical calibration results. Only the most recent spectral
calibration for each dye set is maintained in the software. To save the file
electronically as a PDF, in the Printer dialog box, select CutePDF Writer as the
printer.
Export spectral calibration results
To export spectral calibration results:
1. Click Export Spectral Calibration Results.
2. Specify an export file name and location.
3. Click Save.
14 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
View the spectral Select History View, then select a dye set to view the associated calibration history.
calibration history
Performing a HID Install Standard performance check
This section contains the basic information needed for performing the HID Install
Standard Performance Check. For more information on preparing the instrument
and running an instrument performance check, refer to the Applied Biosystems
3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Note: While the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no.
4401661) recommends users perform the HID Install Standard Performance Check
monthly, this is not a mandatory requirement for ongoing instrument operation in
an HID laboratory. The HID Install Standard Performance Check is intended to be
performed at installation by a Life Technologies Field Service Engineer. This
procedure may be repeated by the Service Engineer during maintenance or service
visits. It is not necessary to repeat this procedure after install and it is recommended
that each HID laboratory determine the appropriate Quality Control measures to
verify correct instrument operation. These measures may or may not include
repeated injections of allelic ladder to verify precision and correct genotyping
instead of performing the HID Install Standard Performance Check.
Prepare the 1. Vortex the AmpFlSTR® Identifiler®, Identifiler® Direct, or Identifiler® Plus
reagents allelic ladder, GeneScan™ 600 LIZ® Size Standard v2.0, and Hi-Di™ Formamide
(if stored in aliquoted tubes) and spin each tube briefly in a microcentrifuge.
2. Prepare performance-check chemistry by combining the following in a labeled
colorless 1.5-mL microcentrifuge tube:
Volume
Reagent
3500 3500xL
Allelic Ladder 8 µL 24 µL
GeneScan™ 600 Liz® Size Standard v2.0 4 µL 12 µL
Hi-Di™ Formamide 68 µL 204 µL
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 15
Performing a HID Install Standard performance check
3. Denature the sample at 95°C for 3 minutes, then store on ice until ready to use.
4. Aliquot 10 µL of the mixture into each appropriate well of the plate as shown
below.
5. Load the standards in injection position 1 in the plate as shown:
IMPORTANT! You do not create a plate for the performance check. The
software uses predetermined positions for the performance check run. You
cannot specify standard location on the plate. If you do not place standards in
the positions indicated, the calibration will fail.
8-capillary A1 through H1
96-well plate
24-capillary A1 through H1, A2 through H2, and A3
through H3
96-well plate
6. Briefly centrifuge the plate containing the standards.
7. Verify that each sample
is positioned correctly
in the bottom of its
well.
IMPORTANT! 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.
Prepare the plate 1. Place the sample plate into the plate base.
assembly 2. Align the holes in the septa
strip with the wells of the
plate, then firmly press Plate retainer
downward onto the plate.
IMPORTANT! Make sure to
use the correct plate base for Plate with
standard plates versus 8-tube septa strip
strips and fast plates. Using
the wrong plate base may
affect performance. Plate base
3. Snap the plate retainer (cover)
onto the plate, septa, and plate
base.
16 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
4. Verify that the holes of the plate retainer and the septa strip are aligned. If not
aligned, re-assemble and then assemble the plate assembly.
IMPORTANT! The array tips will be damaged if the plate retainer and septa
strip holes do not align correctly.
Load the plate in 1. Place the plate in the autosampler with the labels facing you (or the instrument
the instrument door) and the notched corner of the plate in the notched corner of the
autosampler.
2. Close the instrument door to re-initialize the instrument.
3. Access the HID Install Standard screen: Select
Maintenance, then select HID Install Standard
in the navigation pane.
4. Select the plate type and plate position in the
instrument.
Note: You do not create a plate for the
performance check. The software uses
predetermined positions for the run. You
cannot specify the standard locations on the
plate.
5. Click Start Run.
Step4
Step 5
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 17
Performing a HID Install Standard performance check
What you see The system performs one run and indicates the number of observed allele and size
during a run standard peaks.
The Capillary Run Data display updates 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.
Note: The following example is for the HID install standard.
Number of peaks
per capillary
Plot and allele
size/height
information for the
selected capillary
Allele results for all
capillaries
How the software The software evaluates peaks in the data for each capillary. To be identified as a
determines possible allele, peaks must be within the following ranges (nominal allele size, or
reference bin size, is hard-coded):
passing and failing
capillaries for the • All markers except TH01: ± 0.7 bp of nominal size for the allele
fragment/HID • TH01: ± 0.5 bp of nominal size for the allele
performance check 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.
18 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Result Description Threshold
Avg Peak Height Average of peak heights for observed allele peaks of the included Fragment: >175 RFU
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 average peak height that do not meet thresholds fail.
Note: Review the data for failed alleles as described below.
Result Description
For information only
Nominal Size Expected allele fragment peak size (bp).
Mean Average fragment size for the observed allele peaks.
Peak Height % Percentage of observed allele peaks with a peak height above the minimum threshold.
>Min
Sizing Accuracy Difference between the allele size and the mean allele size.
Evaluate 1. Examine the number of size standard and allele peaks found for each capillary.
fragment/HID Note: The number of expected peaks shown below is for the HID install
install standard standard.
data
Expected
Observed
2. If the expected number of alleles and size standard peaks are found, click
Accept Results.
If the expected number of alleles and size standard peaks are not found,
troubleshoot as described below.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 19
Performing a HID Install Standard performance check
Troubleshoot 1. Click a capillary with fewer
than the expected number of
peaks to display detailed
information for each allele in
the table below the plot.
2. Double-click the Size
column to sort results and
identify the alleles that were
not found.
A “0” Size value indicates
that an allele falls outside
the expected size window
(Nominal Size ± 0.7 bp or ±
0.5 for TH01).
3. Troubleshoot failing data:
a. Analyze the install
standard data files in your secondary analysis software (GeneMapper®
ID-X Software v1.2 or later using Identifiler® kit panels and bins).
b. Evaluate the failed data and examine the alleles not found by the 3500
Series Data Collection Software.
c. If the alleles are properly called in the secondary analysis software, you
can:
• Deselect the Include checkmark for a capillary.
• Click Recalculate.
• Accept the install standard results.
Note: The GeneMapper® ID-X Software may identify alleles not identified
by the 3500 Series Data Collection Software because of the bin-offsetting
feature (which uses the observed alleles in the allelic ladder samples to
adjust the reference bin locations for samples).
d. If the alleles are not properly called:
• (Optional) Click View Detail Report to save a record of the failed
run. To save the report electronically (.pdf), print the report and select
CutePDF Writer as the printer. For more information, see “Save
historical performance check reports (.pdf) for record keeping” on
page 21.
• Click Reject Results.
• Rerun the install standard to determine if the problem may be caused
by sample preparation, a poor injection, a capillary issue, or a system
problem (which may require instrument service). For more
information, refer to the Applied Biosystems 3500/3500xL Genetic
Analyzer User Guide (Part no. 4401661) for fragment/HID install
standard troubleshooting information.
20 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Example HID install standard results
View previously run Select History View, then select an install standard to view the associated calibration
install standards information.
View and print a IMPORTANT! Ensure that all dyes are selected before viewing the report. The report
fragment or HID will contain incomplete data if all dyes are not selected.
install standard Note the following:
detail report • Install standard 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.
• To generate a report for a failed installation standard run, you must do so
before you click Reject Results.
1. Click View Detail 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.
4. Close the report.
Save historical IMPORTANT! After performing a performance check, save the performance check
performance check report electronically for record keeping. The software does not save historical
calibration results. Only the most recent spectral calibration for each dye set is
reports (.pdf) for
maintained in the software.
record keeping
1. Click View Detail Report.
2. Click Print.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 21
Performing a HID Install Standard performance check
3. In the Printer dialog box, select CutePDF Writer as the printer.
4. Specify a name and location for the report.
Note: While the number of expected allele peaks is 205, the actual number observed
may be lower than 205 and may still be acceptable. The reference file for these peaks
in the software is a static reference, meaning that no bin offsetting has been applied.
Therefore, it is advisable that any questionable data or runs be analyzed in
Genemapper® ID-X software (v1.2 or later) to determine if the instrument is
performing as expected for AmpFlSTR® kits. These files are located at X:\Applied
Biosystems\3500\Data\[Run Date] and stored according to the date the run was
performed. The run date can be identified by examining the History View tab in the
HID Install Standard portion of the Maintenance menu.
22 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Section 2: 3500 Data Collection Software Components
This section highlights the features of the 3500 data collection software that are
designed to enhance the human identification laboratory workflow:
Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Instrument protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
QC protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
HID plate templates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
For more information regarding basic run set up for the AmpFlSTR® kits, refer to the
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
The 3500 Data Collection software is designed to minimize the number of steps
required to initiate a sample run while maximizing flexibility. The software has been
pre-configured to streamline the set up and analysis of the AmpFlSTR® kits. Assays
and Plate Templates are the primary information that the data collection software
uses when performing HID analyses on the 3500 Series Genetic Analyzers. Users can
only enter (or import) sample names and assign an Assay to begin a run. This section
contains more information on creating and using these files to optimize workflow
efficiency.
Assays
An assay is a configurable component of the software that contains all the
information the instrument requires to perform a run.
Each assay contains:
• One or more instrument protocols which define the chemistry or kit being
analyzed and the run parameters
• A primary QC analysis protocol
The 3500 data collection software contains a library of assay templates pre-
configured to support the AmpFlSTR® kits. New assays can be created and added to
the library by the user. For more information on creating assays, refer to the Applied
Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661). The following
is an example of the validated configuration for the HID Assay for the Identifiler®
kit:
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 23
Assays
The default HID Assay settings contain one instrument protocol, which determines
the instrument run parameters, and one QC protocol which assists the user in
evaluating the data for reinjections or reanalysis. When the user imports an assay for
use with a particular sample run, the option is available to assign multiple
instrument protocols to enable subsequent injections of the same sample set to be
performed under different conditions. The user is also able to change the QC
protocol selected for the sample run.
24 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Instrument protocols
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. While defined Instrument Protocol parameters are established for
validated protocols, users may create their own protocols to fit laboratory
procedures. For more information on creating instrument protocols, refer to the
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661). The
following is an example of a validated HID instrument protocol for the Identifiler®
kit:
IMPORTANT! The software allows users to define instrument protocol parameters.
Any adjustments made to the validated parameters defined by Applied Biosystems,
should be supported by appropriate validation studies. The Normalization Target
value should be adjusted according to the individual laboratory’s results.
Note: “Validated instrument protocol specifications” on page 67 contains a
complete list of all validated preloaded Assays and Instrument Protocols.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 25
QC protocols
QC protocols
A QC protocol is a required component of an assay and determines how primary
analysis of the data will occur in the Data Collection software. A QC protocol defines
peak detection, threshold, and sizing values applied during primary analysis. This
primary analysis allows the user to view sized data in the Data Collection software
for completed injections while additional run data is still being collected.
Note: The QC analysis protocol is intended to assist users in performing a basic
quality assessment of the data during a run. A more complete assessment of the final
data can be performed using GeneMapper® ID-X Software v1.2 or later. To simplify
data analysis, it may be advisable for users to align the parameters defined in the QC
protocols in the 3500 Data Collection software with those used in GeneMapper®
ID-X Software.
For more information on creating QC protocols, please refer to Applied Biosystems
3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
The following is an example of a QC protocol for the Identifiler® kit as validated at
Applied Biosystems utilizing the Internal Standard Normalization function:
IMPORTANT! Parameters within the QC protocol may be adjusted following
appropriate validation by the laboratory. The Peak Amplitude Threshold settings
shown above are defined based on developmental validation studies and noise
analysis performed by Applied Biosystems. Evaluations in each individual HID
laboratory will assist the user in setting appropriate Peak Amplitude Thresholds for
their specific workflow.
26 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
HID plate templates
The instrument workflow may be further streamlined through the use of plate
templates. Users may create their own plate templates, modify user created
templates, or customize the factory-provided defaults to fit laboratory specific
procedures. For more information on creating a plate from a template, refer to the
Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Templates allow users to easily manage the options available and restrict the choices
to simplify the set up of a plate. In the following example, a new template was
created to allow the user to select from multiple Identifiler® kit assays, two File
Name Conventions, and five Results Groups. This example shows sample names
(numbers) pre-filled, but the user may choose to type each name individually or
utilize the sample import feature.
Example of a new plate template configured by the user.
Users are provided with multiple options for visualizing the plate layout, as well as
assigning sample names, Assays, File Name Conventions, and Results Groups. It is
suggested that users name custom Library objects with titles that reflect the
important parameters defined within the template.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 27
Section 3: Sample Run Workflow and Run Setup
Section 3: Sample Run Workflow and Run Setup
This section covers:
Common operations in the dashboard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Assign plate contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Load plates for the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Evaluate data in real time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Specify re-injections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
For a simplified list of steps required for setting up for a run, refer to the 3500 and
3500xL Genetic Analyzers Quick Reference Card (Part no. 4401662). While many options
are available for creating a plate, in this section we illustrate a run set up using the
“Create Plate from Template” menu option. For illustration purposes, specific
conditions for running the validated parameters for the Identifiler® Plus kit on a
3500xL will be detailed. For more information on creating a plate from a template,
refer to the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no.
4401661).
Common operations in the dashboard
1. Define plate properties:
a. In the Dashboard, click Create Plate from Template, Choose “HID” from
the Filter menu, then select the appropriate Plate Name
Note: For the Identifiler® Plus kit, choose “5dye_36_POP4_xl”.
b. Click Open to define the plate details. Enter the Plate Name in the “Name”
field.
Note: A checkbox for Perform Auto-Analysis is present here. To perform
autoanalysis, a full version of the GeneMapper® ID-X Software v1.2 or
later needs to be co-installed with the 3500 Data Collection software which
limits the number of clients that can connect to the full install. Therefore
auto-analysis is not supported for human identification applications on the
3500 series Genetic Analyzers and is not recommended for HID
laboratories.
c. Click the Assign Plate Contents button at the bottom of the screen.
Assign plate contents
1. Enter sample names:
a. Click the Plate Map or Table View tab, then in the plate view, click a well
to select it.
28 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
b. Type a sample name directly into the field, then press Enter.
c. (Optional) You can right-click and use the Fill Series options to populate all
sample names on the plate.
Note: Users may import sample names by using the “Import” option at
the top of the window. Users are advised to manually create a desired plate
first and select “Export” to define a standardized and compatible Import
file template. This file may then be used as the plate import template,
streamlining the import of plates in future runs.
IMPORTANT! Use of Microsoft Excel templates, or .txt files generated
from Microsoft Excel worksheets, may contain hidden characters that
render the format of the file unsuitable for sample import. Users may
choose to use .csv files for sample import to allow editing in Excel while
preserving the proper format. Some versions of Microsoft Excel or
laboratory generated worksheets may be incompatible for use for any file
import template editing.
d. To select the Assay, File Name Conventions and Results Groups, select the
wells you want to change, then select the check box(es) of interest. The
assay is the only mandatory selection.
Note: For the Identifiler® Plus kit, choose “IFP+Norm_POP4_xl”.
IMPORTANT! To utilize internal standard normalization, select an assay
that contains a sizecalling protocol or a QC protocol that specifies an
internal standard normalization size standard (e.g. GeneScan™ 600 LIZ®
Size Standard v2.0).
e. (Optional) In the Customize Sample Info pane, select the sample type
(Sample, Positive Control, Negative Control, Allelic Ladder, Hi-Di™, or No
Data).
f. Save the changes made by selecting Save Plate, then Save, Save As, or
Save as Template.
g. Click Link Plate for Run in the Assign Plates for Run screen or Load Plates
for Run in the navigation pane to assign the plate and specify the position
of the plate in the autosampler.
Load plates for the run
1. Inspect the information presented on the screen. Confirm that the linked plate is
in the correct position of the autosampler.
2. Click Start Run to begin the run immediately, or review the injection list by
clicking Create Injection List in the Load Plates for Run screen.
Note: In the Preview Run screen, users may rearrange the injection list or
assign duplicate injections. The re-inject option is available upon completion of
the first injection and prior to cessation of all runs in the injection list.
3. Click Start Run if the Create Injection List option was chosen.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 29
Evaluate data in real time
Evaluate data in real time
Review results for After each injection, click View Fragment/HID Results in Review Results. If you
the currently access the View Fragment/HID Results screen while an instrument run is in
progress, the samples table lists results for completed injections in the current run.
running plate
Select one or more samples in the samples table to display their data in the plot view
and sizing table view.
Review previously If you access the View Fragment/HID Results screen when no run is in progress and
run samples 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.
Note: By default, the Fragment
Samples view is selected. If you
are importing HID files, click HID
Samples.
30 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Review sample 1. In the samples view, click the Table Settings button,
quality 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.
Flag/Symbols Description
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 (fragment
analysis only) At least one peak contains a pull-up peak.
A pull-up peak is identified when the peak height of the minor peak is ≤ X% of and
within ±Y data point of the major peak, where X and Y are values you specify.
Broad Peak (HID analysis
only) At least one peak exceeds the Broad Peak threshold.
Broad peaks affect Sizing Quality.
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.
Normalization Limit • – Sample was collected with a normalization size standard, sample
Normalization Factor is within range.
• – Sample was collected with a normalization size standard, sample
Normalization 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 Dx Series Genetic Analyzer CS2
instrument.
Note: If the Sizing Quality is , normalization is not applied, even if the
Normalization Factor is within the normalization range.
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.
Note: If the Sizing Quality is
, normalization is not
applied, even if the
Normalization Factor is within
the normalization range.
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.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 31
Specify re-injections
For more information, refer to the Applied Biosystems 3500/3500xL Genetic Analyzer
User Guide (Part no. 4401661).
Specify re-injections
You can specify a re-injection before the run completes. 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.
1. Select the injections or wells to re-inject:
Note: Re-inject is grayed 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.
IMPORTANT! Re-injection wells cannot be selected by clicking on the plate map.
The user should select samples for reinjection by clicking on the samples in the
Instrument Run Views and Flags section. Samples or flags can be used to specify
reinjections.
Goal Action
To collect data for all wells in an injection 1. Select the injection in the injection
list.
2. Click Re-inject.
To collect data for only specific wells 1. Select the injection.
(Samples with assays that specify more 2. Select in the array view the capillary
than one instrument protocol are listed that corresponds to the well or
one time in the injection list for each sample of interest.
instrument protocol.)
3. Click Re-inject.
Note: You can also specify re-injections
for specific samples in Review Results.
To collect data for only samples that 1. Select the samples in the flag table.
contain flags
2. Click Re-inject.
2. In the Re-injection dialog box, make selections for the following options, then
click OK:
• The protocol to use for the re-injection: original, modified, new, or one
from the library
• When to make the re-injection
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.
32 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
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).
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.
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.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 33
Section 4: Developmental Validation
Section 4: Developmental Validation
This section covers:
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Genotype concordance and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Sizing precision and resolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Sensitivity and Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Baseline Noise Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Internal Standard Normalization (ISN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Mixture Analysis with GeneMapper® ID-X Software Version 1.2. . . . . . . . . . . . . . . . 60
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Overview
The Applied Biosystems Human Identification Product Assurance Group validated
the use of the 3500 series Genetic Analyzers (3500 and 3500xL) with the AmpFlSTR®
PCR Amplification Kits.
The validation experiments were performed according to the DNA Advisory Board
(DAB) Quality Assurance Standards, effective October 1, 1998 (DNA Advisory
Board, 1998), and the revised guidelines from the Scientific Working Group on DNA
Analysis Methods [Revised Validation Guidelines-Scientific Working Group on DNA
Analysis Methods (SWGDAM). Forensic Sci. Communications (July 2004) Volume 6 (3)].
The studies performed include:
• Genotype concordance and reproducibility (page 41)
• Sizing precision and resolution (page 43)
• Sensitivity and Normalization (page 46)
–Sensitivity (page 46)
–Baseline Noise Analysis (page 51)
–Internal Standard Normalization (ISN) (page 55)
• Mixture Analysis with GeneMapper® ID-X Software Version 1.2 (page 60)
IMPORTANT! Each laboratory should perform internal validation studies to
establish appropriate guidelines, protocols, and procedures prior to implementing a
new technology.
Materials and methods
Kits The following AmpFlSTR® kits were tested during this validation study:
• MiniFiler™ PCR Amplification Kit
• Yfiler® PCR Amplification Kit Yfiler
34 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
• Profiler Plus® PCR Amplification Kit
• Cofiler® PCR Amplification Kit
• Identifiler® PCR Amplification Kit
• Identifiler® Direct PCR Amplification Kit
• Identifiler® Plus PCR Amplification Kit
• NGM™ PCR Amplification Kit
• SGM Plus® PCR Amplification Kit
• SEfiler Plus™ PCR Amplification Kit
• Sinofiler™ PCR Amplification Kit
Not every AmpFlSTR® kit was utilized in every study. Representative kits were used
in each study to demonstrate the ability of the 3500 series instruments to achieve
accurate results within the parameters of each test. Selection of representative kits
for each study was based on factors including dye chemistry, use of a particular size
standard, and other similarities among the AmpFlSTR® kits.
Instrumentation Three 24-capillary instruments (3500xL) and three 8-capillary instruments (3500)
were used in the validation experiments. Each instrument was installed and
calibrated by field service engineers prior to the experiments. The spectral
calibrations described in “Performing a spectral calibration” on page 8 were
performed on all six instruments with both DS33 (G5) and DS32 (F) matrix
standards. The HID performance check was performed on all six instruments with
the HID Install Standard and GeneScan™ 600 LIZ® v2 size standard. All of the
instruments met all specifications for sizing precision and all spectral calibration
specifications including peak height (see “Evaluate fragment/HID install standard
data” on page 19).
Quantification of the DNA samples was performed using the Quantifiler® or
Quantifiler® Duo DNA Quantification kits on a 7500 Real-Time PCR System
(Applied Biosystems) to determine input DNA concentrations, and using GeneAmp
PCR System 9700 thermal cyclers equipped with gold-plated blocks for PCR
amplification. Where required, 3130xl instruments were used with the same sample
set for genotype concordance and overall STR profile comparison.
Factory-provided AmpFlSTR® assays containing the recommended instrument
protocol and run information were utilized for all studies. For information on each
assay and specific run conditions see “Validated instrument protocol specifications”
on page 67.
The AmpFlSTR® kits were validated on instruments using the consumables listed in
the following table. Anode buffer, cathode buffer and polymer were changed
according to the recommended maintenance schedule described in the Applied
Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 35
Materials and methods
Consumable Part Number
POP-4® Polymer 960 Samples 4393710
Cathode Buffer Container 4408256
Anode Buffer Container 4393927
GeneScan™ 600 LIZ® v2 Size Standard 4408399 (kit)
4408293 (tube)
GeneScan™ 500 ROX™ Size Standard 4310361 (9600 reactions)
401734 (800 reactions)
Hi-Di™ Formamide, 5 mL bottle 4401457
3500 36cm Capillary Array, 8 capillary 4404683
3500xL Genetic Analyzer Capillary Array, 24 4404687
capillary, 36 cm
36 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Samples The DNA samples tested in each experiment are listed in the following table.
GeneScan™ 600 LIZ® v2 size standard was used with the 5-dye chemistries and
GeneScan™ 500 ROX™ size standard was used with the 4-dye chemistries.
Table 1 Studies performed, kits tested, and DNA inputs
Replicates
PCR amplification kits
Study Samples DNA input per reaction per
tested
instrument
Genotype • 40 male and 42 female Identifiler®, Identifiler® 0.5-2 ng total DNA 1 replicate
Concordance extracted gDNA Direct, Identifiler® Plus, per sample
and samples‡. 1 positive MiniFiler™ , NGM™, SGM
2 injections
Reproducibility control, 1 NTC, and Plus®, Yfiler®, SEfiler
allelic ladder Plus™, Profiler Plus®,
• Identifiler® Direct: 84 Cofiler®, and Sinofiler™
(1.2 mm) punches of
blood stained FTA
paper
Sizing Allelic ladder§ Identifiler®, Identifiler® 1 µl per well 24 replicates
Precision and Direct, Identifiler® Plus,
4 injections
Accuracy MiniFiler™, NGM™,
Yfiler®, SEfiler Plus™,
Sinofiler™, SGM Plus®,
Profiler Plus®, and
COfiler®
Sensitivity 007 control DNA, 3 gDNA Identifiler®, Identifiler Identifiler® (3 lots GS600 5 replicate
samples, NTC and allelic Plus, NGM™, SGM v2) and SGM Plus® (GS DNA samples
ladder Plus®, MiniFiler™ 500 ROX): 0.125 ng,
3 replicate
0.5ng, 1 ng, 3 ng;
NTC
MiniFiler™ (2 lots GS600):
0.125 ng, 0.25 ng, 0.5 ng, 4 injections
0.75 ng
Internal Identifiler® Plus, NGM™:
Standard 0.062 ng, 0.125 ng,
Normalization 0.5 ng, 1 ng, and 1.5 ng
(ISN)
Mixture 4 pairs of male and Identifiler®, SGM Plus®, Identifiler®, SGM Plus®: 1 replicate
Analysis female gDNA, each with Profiler Plus®, total input 1 ng per sample
mixture ratio of 1:0, 1:1, COfiler®
Profiler Plus®, COfiler®:
1:3, 1:5, 1:9, and 0:1;
2 ng
1 injection
1 positive control
1 NTC and allelic ladder
Resolution SEfiler Plus™ allelic SEfiler Plus™ 1 µl per well 24 replicates
ladder
1 injection
‡ Includes African American, Caucasian, Hispanic, and Asian donors.
§ Identifiler® Direct, Identifiler® Plus, Identifiler®, NGM™, MiniFiler™, SGM Plus®, Yfiler®, Sinofiler™, and SEfiler Plus™
Kits with GeneScan™ 600 LIZ® Size Standard v2.0 and Profiler Plus®, COfiler® Kits using GeneScan™ 500 ROX™ Size
Standard®.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 37
Materials and methods
Table 2 Sample sources and kits and instruments used for preparation and amplification
Study Sample source Extraction Quantitation Amplification ‡ CE §
Genotype Commercially sourced BloodPrep™ Chemistry Quantifiler® 9700 gold block 3130xl
Concordance material and kit- on the ABI PRISM™ 6100 Human on and
and specific positive Nucleic Acid PrepStation 7500 3500
Reproducibility control
Sizing Precision Identifiler® allelic N/A N/A N/A 3500
and Accuracy ladder
Sensitivity Commercially sourced BloodPrep™ Chemistry Quantifiler® 9700 gold 3500
material and 007 on the ABI PRISM™ 6100 Duo on 7500 developmental
Internal Nucleic Acid PrepStation validation block
Standard
Normalization
(ISN)
Mixture Analysis SERACARE DNA Quant Duo on 9700 gold block 3500
7500
Resolution SEfiler Plus™, N/A N/A N/A 3500
Identifiler®, and
research methods
‡ On a GeneAmp® PCR System 9700 with gold block in 9600 emulation mode. Amplifications used 25 µl reaction volumes
for the 5 dye chemistries and 50µl reaction volumes for the 4 dye chemistries.
§ A mixture of Hi-Di™ Formamide: Size Standard:PCR product was prepared for each sample, control or allelic ladder
according to a 8.5:0.5: 1 ratio, respectively.
Data Analysis
All data were analyzed using GeneMapper® ID-X v1.2 software with internal standard
normalization enabled or disabled in the Analysis Method. The following kit standard
panels, bins, and stutter files were used for data analysis at the settings shown in the
table:
• MiniFiler™ PCR Amplification Kit
• Yfiler® PCR Amplification Kit
• Profiler Plus® PCR Amplification Kit
• Cofiler® PCR Amplification Kit
• Identifiler® PCR Amplification Kit
• SGM Plus® PCR Amplification Kit
• SEfiler Plus™ PCR Amplification Kit
• Identifiler® Direct PCR Amplification Kit (v1X)
• Identifiler® Plus PCR Amplification Kit (v1X)
• NGM™ PCR Amplification Kit (v1X)
• Sinofiler™ PCR Amplification Kit (v2X)
38 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Table 3 Analysis settings
Size Standard GeneScan™ 500 ROX™ ‡or
GeneScan™ 600 LIZ® v2§
Peak Detection Mode Advanced
Smoothing Light
Size Calling Method Local Southern, 3rd Order Least Squares
(MiniFiler™ only)
Baseline Window Size 51
Minimum Peak Half Width 2
Polynomial Degree 3
Peak Window Size 15
‡ CE_G5_HID_GS500 size standard peaks defined: 75.0, 100.0, 139.0, 150.0, 160.0, 200.0, 300.0,
340.0, 350.0, 400.0, 450.0
§ GS600_LIZ size standard peaks defined: 80.0, 100.0,114.0, 120.0, 140.0, 160.0, 180.0, 200.0,
214.0, 220.0, 240.0, 250.0, 260.0, 280.0, 300.0, 314.0, 320.0, 340.0, 360.0, 380.0, 400.0, 414.0, 420.0,
440.0, 460.0
Additional parameters were examined according to set criteria. The following table
describes the type of study and the associated criteria.
Table 4 Study criteria matrix
Study type Test cases Test method Passing criteria
Genotyping Concordance Confirm that the STR All 3500/3500xL allele calls
profiles are complete and are 100% concordant with
accurate by comparison to the 31xx genotype results
known STR profiles and for all the kits tested.
data generated from 31xx
runs using GeneMapper®
ID-X v1.2.
Reproducibility Confirm that the STR STR profiles from the same
profiles generated on samples or controls run on
different 3500 series different instrument and
instruments and different across injections are 100%
injections are complete concordant.
and accurate using
GeneMapper® ID-X v1.2.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 39
Materials and methods
Study type (continued) Test cases Test method Passing criteria
Sizing Precision Calculate the standard The sizing precision does
deviation of the mean base not exceed a standard
pair size for each allele in deviation of 0.15 bp within
the allelic ladder within an an injection (8- or 24-cap)
injection and across a or across a plate (96 wells)
plate. for all alleles in all kits
tested.
Accuracy Calculate the size range The size range does not
(max-min) for each allele exceed 0.5 bp per injection
in the allelic ladder (88 or 24-cap) for all alleles
samples across all in all kits tested.
samples per injection and
per plate.
Sensitivity Minimum DNA input Determine the lowest DNA Kit dependent.
input amount per reaction
that can generate a full
STR profile above the
GeneMapper® ID-X peak
amplitude threshold‡.
Maximum recommended Determine the highest No allele dropout or off-
DNA input DNA input amount per scale data for kit
reaction that can generate recommended range.
a full STR profile without
camera saturation
(offscale peaks).
Artifacts Identify reproducible No new reproducible
artifacts. artifacts within the read
region.
Pull-up Calculate the maximum Mean pull-up is ≤ 5%.
and mean pull-up for each
peak in each color for on-
scale data.
Internal Standard Internal Size Standard Determine the Normalization Factors are
Normalization (ISN) average peak height and Normalization Factor by between 0.3 and 3.
Normalization Factor dividing the average peak
height of the ISN peaks by
the internal standard
normalization threshold
setting.
ISN thresholds setting§:
• 3200 RFU for
Identifiler® Direct,
Identifiler® Plus,
MiniFiler™, Yfiler®,
NGM™
• 3800 for Identifiler® and
Sinofiler
40 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Study type (continued) Test cases Test method Passing criteria
Mixture Analysis Full profile for minor The highest mixture ratio Detection of minor
contributor that can generate contributor peaks at 1:9
detectable alleles for the ratio with total DNA input
minor contributor. of 1ng.
Concordance between
mixture analysis values
between different versions
of GeneMapper® ID-X
software.
‡ Values determined for instruments used during the developmental validation. Each laboratory should perform their
own studies to determine the appropriate settings for their instruments.
§ Variation among instruments, among injections, and among capillaries was also examined.
Genotype concordance and reproducibility
The genotype concordance and reproducibility study was conducted to demonstrate
that the STR profiles of the kits are complete and accurate, by comparing known STR
profiles to kit data generated by runs on the 3500 and 3500xL instruments using
GeneMapper® ID-X v1.2 and the 3130xl instrument.
Experimental For details of study parameters, see Table 1, “Studies performed, kits tested, and
design DNA inputs,” on page 37, Table 2, “Sample sources and kits and instruments used
for preparation and amplification,” on page 38, and Table 4, “Study criteria matrix,”
on page 39.
The data were analyzed using GeneMapper® ID-X v1.2 software with a peak
detection threshold of 175 RFU (see “Baseline noise and threshold determination for
the G5 and F Dye Sets” on page 51 for more information on the 175 RFU threshold
setting). Samples were analyzed with the ISN feature enabled and disabled in
GeneMapper® ID-X v1.2 software and with a kit-specific ISN threshold as defined in
the validated instrument protocols in “Internal Standard Normalization (ISN)” on
page 55. STR profiles generated on the 3500 and 3500xL instruments were compared
to data previously generated on the 3130xl Genetic Analyzer to confirm genotype
concordance.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 41
Genotype concordance and reproducibility
Results For all the kits tested, the results demonstrated that the genotype profiles from six
3500 or 3500xL instruments were 100% concordant with each other and with the
3130xl results. Also, the genotype profiles showed 100% concordance between the
analysis methods with ISN activated and ISN deactivated.
F
Table 5 Concordance results (82 samples per run)
Internal Standard
Kit Concordance
Normalization
Identifiler® 100% Enabled
100% Disabled
Identifiler® Direct 100% Enabled
100% Disabled
Identifiler® Plus 100% Enabled
100% Disabled
MiniFiler™ 100% Enabled
100% Disabled
NGM™ 100% Enabled
100% Disabled
SGM Plus® 100% Enabled
100% Disabled
Yfiler® 100% Enabled
100% Disabled
SEfiler Plus™ 100% Enabled
100% Disabled
Profiler Plus® 100% Enabled
100% Disabled
Cofiler® 100% Enabled
100% Disabled
Sinofiler™ 100% Enabled
100% Disabled
42 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Sizing precision and resolution
To assess the ability of the 3500 instrument to reproducibly measure the size of an
amplified DNA fragment, allelic ladder aliquots were injected multiple times on
three 3500 and three 3500xL instruments.
Experimental For details of study parameters, see Table 1, “Studies performed, kits tested, and
design DNA inputs,” on page 37, Table 2, “Sample sources and kits and instruments used
for preparation and amplification,” on page 38, and Table 4, “Study criteria matrix,”
on page 39.
Precision was expressed as a standard deviation (SD) of the mean base pair size
calculated for each allele within each locus across one injection and across multiple
injections. The SD of the mean size of each allele within a run was calculated and
plotted by marker. The graph below demonstrates how the two different types of
analyses were performed on a 3500 and 3500xL; however, both single and multiple
injections were performed on both instrument types.
One injection (3500 instrument) Multiple injections (3500xL instrument)
• 8 data points per mean • 96 data points per mean
• 12 injections per plate • 4 injections per plate
• 12 columns • 3 Instruments
• 3 Instruments
Results The SD in sizing within an injection and across multiple injections was less than
0.15 bp on both instruments for all kits tested using a full plate of 96 samples. The
median standard deviation is similar regardless of STR fragment size and capillary
array type. Sample allele sizing error rarely exceeds ±0.5 nt when the SD is ≤ 0.15 bp
(see Smith, et al. 1995, Biotechniques 18(1):122-8.)
Figures 4 and 5 show the data distribution of all alleles from wells containing
Identifiler® Allelic Ladder. Comparable results were observed for all kits. The box
portion of the box plots represent the data that fell within the 75th percentile, with
the median indicated by a line in the center of the box plot. The whisker lines
protruding from the boxes extend to the 90th percentile, and the individual outliers
beyond the whiskers represent the small percentage of data that were not in the 90th
percentile.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 43
Sizing precision and resolution
Figure 4 Standard Deviation of Mean Size for Identifiler® Allelic Ladder: within one injection
on 3500 (8 capillaries) and 3500xL (24 capillaries)
Figure 5 Standard Deviation of Mean Size for Identifiler® Allelic Ladder: across multiple
injections (entire 96-well plate) on 3500 (8 capillaries) and 3500xL (24 capillaries)
Resolution
The study tested the ability of the system to reliably detect and resolve alleles that
differ in length by a single base pair (bp). In addition, research methods were
developed to evaluate larger fragments (>300 bp) differing in size by 1 bp (data not
shown).
44 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Experimental For details of study parameters, see Table 1, “Studies performed, kits tested, and
design DNA inputs,” on page 37, Table 2, “Sample sources and kits and instruments used
for preparation and amplification,” on page 38, and Table 4, “Study criteria matrix,”
on page 39.
A minimum of 160 injections of the SEfiler Plus™ Kit Allelic Ladder (containing SE-
33 alleles 21, 21.1, 21.2 separated by a single base pair) and the Identifiler® Kit Allelic
Ladder (containing TH01 alleles 9.3,10) were performed on each of three 3500 and
three 3500xL instruments.
Results The SEfiler Plus™ SE-33-marker allele triplet (21, 21.1, and 21.2) and the Identifiler®
TH01 marker alleles (9.3 and 10), were resolved for all samples tested. Research
methods were used to confirm single base-pair resolution up to 400 bp.
Figure 6 Representative data for the SE-33 marker on a 3500xL instrument
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 45
Sensitivity and Normalization
Figure 7 Representative data for the TH01 marker on a 3500xL instrument
Sensitivity and Normalization
Sensitivity
The study tested the ability of the systems to generate a full and accurate STR profile
with given amounts of DNA input.
Each laboratory should perform their own evaluation to determine appropriate peak
amplitude settings for their instruments, laboratory protocols, and chemistry.
Experimental For details of study parameters, see Table 1, “Studies performed, kits tested, and
design DNA inputs,” on page 37, Table 2, “Sample sources and kits and instruments used
for preparation and amplification,” on page 38, and Table 4, “Study criteria matrix,”
on page 39. POP-4® polymer was used for all instrument runs. Sensitivity study of
the Identifiler® Direct kit was not applicable because it uses FTA paper punches.
Data were analyzed with the ISN targets set at 3800 RFU for Identifiler® kit and
Sinofiler® kit data and 3200 RFU for all other 5-dye kits. The ISN target, which is the
expected average signal of the ISN peaks from the GeneScan™ 600 LIZ® Size
Standard v2.0, was defined based on analysis of data across multiple 3500
instruments. A peak amplitude threshold setting of 175 RFU was used.
Each replicate was injected twice on each instrument at sample well locations
selected such that each sample would be injected in different capillaries across the
array. A total of 10 data points were collected for each sample and concentration on
each instrument. For the kits that used multiple lots of GeneScan™ 600 LIZ® Size
Standard v2.0 size standard, the CE runs were repeated for each lot. Three injections
on each instrument for each sample were performed with the Identifiler® Plus and
NGM™ kits, resulting in a total of 12 replicates for each sample at each concentration.
Six CE plates (one for each instrument) were prepared according to the following
procedure:
46 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
1. Prepare a master mix of the size standard and Hi-Di™ Formamide for all plates
using 0.5 µL of size standard and 8.5 µL of Hi-Di™ Formamide for each well
2. Pipet 9 µL of the master mix into each well. Pipet 1 µL of each sample from the
PCR plates or the allelic ladder to the CE plates. Denature the plates at 95°C for
3 minutes and cool on ice until use.
3. Run the CE plates on each instrument with 2 or 3 injections (kit dependent).
The following table shows an example of the plate layouts used for 007 Control DNA
(columns 1– 3) and genomic DNA samples H1126 (columns 4– 6), H1152 (columns
7– 9), and H1161 (columns 10– 12) amplified at input amounts of 125 – 750 pg across
the plate.
Table 6 Plate layout for MiniFiler™ kit sensitivity study
1 2 3 4 5 6 7 8 9 10 11 12
007 007 007 H1126 H1126 H1126 H1152 H1152 H1152 H1161 H1161 H1161
A 0.125 0.25 ng 0.5 ng 0.125 0.25 ng 0.5 ng 0.125 0.25 ng 0.5 ng 0.125 0.25 ng 0.5 ng
ng ng ng ng
007 007 007 H1126 H1126 H1126 H1152 H1152 H1152 H1161 H1161 H1161
B 0.75 ng 0.125 0.25 ng 0.75 ng 0.125 0.25 ng 0.75 ng 0.125 0.25 ng 0.75 ng 0.125 0.25 ng
ng ng ng ng
007 007 007 H1126 H1126 H1126 H1152 H1152 H1152 H1161 H1161 H1161
C 0.5 ng 0.75 ng 0.125 0.5 ng 0.75 ng 0.125 0.5 ng 0.75 ng 0.125 0.5 ng 0.75 ng 0.125
ng ng ng ng
D 007 007 007 H1126 H1126 H1126 H1152 H1152 H1152 H1161 H1161 H1161
0.25 ng 0.5 ng 0.75 ng 0.25 ng 0.5 ng 0.75 ng 0.25 ng 0.5 ng 0.75 ng 0.25 ng 0.5 ng 0.75 ng
E 007 007 007 H1126 H1126 H1126 H1152 H1152 H1152 H1161 H1161 H1161
0.125 0.25 ng 0.5 ng 0.125 0.25 ng 0.5 ng 0.125 0.25 ng 0.5 ng 0.125 0.25 ng 0.5 ng
ng ng ng ng
F 007 007 007 H1126 H1126 H1126 H1152 H1152 H1152 H1161 H1161 H1161
0.75 ng 0.125 0.25 ng 0.75 ng 0.125 0.25 ng 0.75 ng 0.125 0.25 ng 0.75 ng 0.125 0.25 ng
ng ng ng ng
G 007 007 NTC H1126 H1126 NTC H1152 H1152 NTC H1161 H1161 NTC
0.5 ng 0.75 ng 0.5 ng 0.75 ng 0.5 ng 0.75 ng 0.5 ng 0.75 ng
H AL AL AL AL AL AL AL AL AL AL AL AL
Results The sensitivity study evaluated allelic dropout, absolute peak height, peak height
versus concentration, inter-color and intra-locus balance, and heterozygote peak
height ratio. The data were also used to evaluate noise and spectral pull-up.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 47
Sensitivity
Allelic dropout
With few exceptions, the majority of samples tested for all kits produced a full
profile at 0.125 ng of input DNA using a 175 RFU peak detection threshold. The rate
of allelic dropout was sample-dependent and varied based on the input DNA
concentration, amplification kit chemistry, sensitivity of individual instruments and
whether ISN was enabled or disabled.
The allele call rate was highest for the SGM Plus® (99.97%), Identifiler® Plus
(99.95%), NGM™ (98.65%), and Identifiler® (97.24%) kits. In this study, the allele call
rate was 100% for all three kits that utilize ISN.
Figure 8 shows the allele call rate (the ratio of number of alleles observed:number of
alleles in a full profile). The data incorporates all four different DNA sources and all
six instruments. The SGM Plus® kit does not use ISN.
Figure 8 Allele call rate by kit with 0.125 ng DNA input. (ISN enabled: green; disabled: red)
Peak Height vs. Concentration
Figure 9 shows the average peak height with standard error bars for five
AmpFlSTR® kits. The graph includes data for two of the four concentrations used for
each kit, 0.125 ng and the optimal concentration, which is 0.5 ng for MiniFiler™ and
1.0 ng for all other kits tested. Each bar represents the average of all dye colors, all
replicates, and 6 instruments for the 007 Control DNA. The other DNA samples
produced similar results.
48 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
The graph includes data analyzed with ISN enabled and disabled. The SGM Plus®
kit does not use ISN.
Figure 9 Average Peak Heights of Five AmpFlSTR® Kits for 007 Control DNA at two
concentrations (ISN enabled: green; disabled: red)
The results show the expected peak heights for the relative DNA concentrations.
Peak heights increased slightly with ISN enabled, demonstrating that the Internal
Standard Normalization Targets used were appropriate for this set of six
instruments. Each laboratory should perform a similar analysis on their individual
instruments when evaluating Normalization Targets.
Intra-color balance and inter-color balance
Intra-color balance for a dye is calculated by:
1. Dividing the peak heights of homozygous alleles by 2 and averaging the peak
heights of heterozygous alleles
2. For the resulting peak heights at all loci within a color, dividing the lowest peak
height for any allele by the highest peak height for any allele
Inter-color balance is the ratio of average peak heights between dyes.
The bars in the graph below represent the intra-color peak balance ratio for five
AmpFlSTR® kits separated by dye color to also show inter-color balance. All alleles
from markers of the same dye color were averaged for each replicate of 007 Control
DNA and all instruments. Other DNAs that were tested produced similar results.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 49
Sensitivity
Note: The data below represents commonly observed intra-color ratios for pristine
samples. Degraded, inhibited, compromised samples, or samples using modified
amplification procedures may produce different results.
Figure 10 Intra-color ratio for 007 Control DNA at recommended concentration
Intra-locus balance/Heterozygote Peak Height Ratio (PHR)
Intra-locus balance is the peak height of the lower of two alleles in a heterozygous
pair divided by the peak height of its partner allele. This balance is generally
impacted more by factors related to amplification, such as DNA input amount, than
by factors relating to capillary electrophoresis.
The 3500 systems exhibited the intra-locus balance behavior that was expected from
validations of this kit with Applied Biosystems 310 and 31xx genetic analyzers.
These data represent 007 Control DNA at nominal concentration, although the other
DNAs tested performed similarly. All six instruments were utilized for this testing.
Note: The data that follows represents commonly observed intra-locus ratios for
pristine samples. Degraded, inhibited, compromised samples, or samples using
modified amplification procedures may produce different results.
50 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Figure 11 Average intra-locus peak balance (PHR) for 007 Control DNA at recommended
concentration
Baseline Noise Analysis
Background noise can be defined as any signal which does not represent the signal
of interest. All biochemical detection systems have a level of inherent noise that
should be evaluated to distinguish true signal from background noise. Peak heights
of artifacts, anomalies, and other sources of baseline noise vary depending on the
sensitivity of each instrument, the type of chemistry used, and in most cases, the
sample injection parameters and the amount of amplicon loaded onto the
instrument.
Refer to the appropriate kit User Manual or User Bulletins for information on known
chemistry-related artifacts.
Baseline noise and threshold determination for the G5 and F Dye Sets
Noise in a CE system can be generated inherently from the hardware components or
as a result of the introduction of chemicals or dye-labeled fragments into the system.
Background capillary electrophoresis instrument signal may be attributed to
electrical noise from the instrument electronics or background Raman signal from
the water in the polymer. Because of these factors, it is impossible to eliminate all the
baseline noise in a detection system. To analyze the baseline noise coming from the
dye sets used in the multiplex STR chemistries (G5 and F), experiments were
conducted to estimate signal to noise levels of each and to identify optimal peak
amplitude threshold settings for data analysis.
Non-normalized data from five instruments and normalized data from four
instruments were used to examine the noise in each color of the G5 dye set. Four
instrument runs from two instruments were used to investigate the noise level in dye
set F.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 51
Baseline Noise Analysis
The DS33 Installation Standard was used to evaluate default noise levels for the G5
dye set. The noise level for each dye was analyzed individually. The default 175 RFU
peak amplitude threshold was established by taking 10X the average standard
deviation (SD) of the noise in the G5 dye set for each color. The DS33 Installation
Standard represents the G5 dye set but contains few fragments, allowing noise
measurements to be made between peaks.
Note: Calculating 10X the SD of the baseline noise translates to a 99.9999999999%
confidence that true peaks can be distinguished from peaks due to instrument noise
as shown in the table below.
Table 7 Confidence interval distribution by Standard Deviation (SD)
SD Percentage
1 68.27%
2 95.45%
3 99.73%
Figure 12 Noise from each dye in the G5 dye set (Y = 10✕average SD)
52 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Figure 13 Noise from each dye in the F dye set (Y = 10✕average SD)
As shown, for the G5 dye set, the highest values were observed in the NED and PET
dye channels. Figures 12 and 13 demonstrate that for a 10 ✕ SD analysis, a threshold
of 175 RFU should be sufficient to capture the noise generated in the system. Each
lab should evaluate background noise for each instrument based on internal
validation studies.
Additionally, these data demonstrate the difference among dyes within a dye set.
Laboratories may wish to use this information to establish variable RFU thresholds
based on dye color.
The 3500 series instrument software operates on a RFU scale similar to the 37xx
series instruments, which is approximately three to four times the scale of the 31xx
or 310 instruments. While each instrument and laboratory will perform their own
evaluation, taking into account the difference in scale between platforms, the
baseline noise level of the 3500 data in this study is comparable to the relative
baseline noise seen with the 31xx platform.
Additionally, no reproducible dye artifacts specific to analysis performed on the 3500
instruments were observed in the read region of any marker, as compared to
analysis on the 31xx instruments.
Using SGM Plus® samples for the F dye set, we also confirmed that a 175 RFU
threshold is sufficient to capture noise levels in the F dye set, and that the overall
noise signal in the F dye set is lower than in the G5 dye set.
Note: Some methods for customer analysis of Analytical Thresholds may vary from
the engineering-based methods used here. Please contact your Life Technologies
HID Support representative for more information.
Spectral pull-up Spectral pull-up occurs when spectral calibration does not completely eliminate
signal in one dye that is due to signal in a second dye from the same sample. Spectral
pull-up was examined by visual inspection of the Identifiler®, MiniFiler™, and SGM
Plus® kit gDNA samples used in the sensitivity study. In addition, extensive analysis
was performed on the Identifiler® Plus kit data and is presented here.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 53
Baseline Noise Analysis
The study analyzed data from runs on three 3500 instruments and three 3500xL
instruments using the Identifiler® Plus kit with 1.5 ng input DNA. The tabular data
was exported and pull-up peaks were screened using spreadsheet analysis. A 50
RFU threshold was used for capturing a full complement of observed pull-up peaks,
including low level pull-up in addition to higher signal pull-up.
The percentage of pull-up was determined by dividing the peak height of the pull-
up peak (daughter peak) by the peak height of the parent peak. This study was
performed with Internal Standard Normalization off.
The data below represent 338 incidences of pull-up on the three 3500 series
instruments and 1067 observations of pull-up on the 3500xL instruments.
Figure 14 Average, minimum, and maximum pull-up observed for Identifiler® Plus at 1.5 ng
template DNA on six instruments (3500: blue, 3500xL: red)
Platform Mean SD Median Min Max N
3500 0.83 0.38 0.77 0.27 4.58 338
3500xL 1.17 0.65 1.01 0.18 4.02 1067
The average observed pull-up was 0.83% (SD 0.38%) for the 3500 instruments and
1.17% (SD 0.65%) for the 3500xL instruments.
In total, 3.2% of pull-up peaks observed with the 3500xL and 2.7% of those observed
with the 3500 instrument exceeded a baseline threshold of 175 RFU. In addition,
0.93% of 3500xL and 0.09% of 3500 data indicated pull-up percentages of >3%.
The highest pull-up peak observed on the 3500 instruments had a peak height 4.58%
of its parent peak, which was 7743 RFU. For the 3500xL instruments, the highest
pull-up peak was 4.02% of a parent that was 18,073 RFU.
These data align with the expected performance of the 3500 series genetic analyzers
and indicate pull-up percentages comparable to other capillary electrophoresis
platforms from Applied Biosystems.
54 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Pull-up percentage levels vary from injection to injection, dye to dye, and capillary
to capillary. Different kits and dye sets also affect the level of pull-up observed.
Users should perform a full analysis of the pull-up on their system, as described in
this document. A complete understanding of the pull-up characteristics of the
instrument assists the user in setting proper thresholds for analysis. Users are
cautioned against relying on individual data points to make assessments of pull-up.
Instead, a comprehensive analysis such as the one performed here provides a
complete assessment of the pull-up in the system.
Note: Off-scale data (as determined in the Raw Data or Off Scale Flags) does not
produce reliable peak heights and therefore is not suitable for the evaluation of
pull-up percentages.
Internal Standard Normalization (ISN)
Size Standard Normalization is a feature on 3500 systems that is designed to reduce
peak height variation across instruments. This feature attenuates signal variations
across instruments that may be due to variations in instrument hardware, capillary
array, sample salt load, and injection. For fragment analysis and HID applications,
the 3500 Series Data Collection Software includes an Internal Standard
Normalization (ISN) feature for use with the GeneScan™ 600 LIZ® Size Standard v2.0
(GS600 LIZ v2). For more information on internal standard normalization, refer to
the Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661).
Experimental For details of study parameters, see Table 1, “Studies performed, kits tested, and
design DNA inputs,” on page 37, Table 2, “Sample sources and kits and instruments used
for preparation and amplification,” on page 38, and Table 4, “Study criteria matrix,”
on page 39.
The sensitivity sample runs for the Identifiler®, MiniFiler™, Identifiler® Plus, and
NGM™ kits were used to test the ISN feature. All samples were run with ISN
activated in the 3500 data collection software. The data were analyzed with the ISN
option enabled and separately with the ISN option disabled in the GeneMapper®
ID-X v1.2 software.
See Table 6, “Plate layout for MiniFiler™ kit sensitivity study,” on page 47 for the
plate layout for the four samples and four DNA input amounts used for the
MiniFiler™ ISN test. Two lots of size standard were used and samples were run
separately with ISN enabled and disabled.
The ISN performance was evaluated by comparing the amount of peak height
variation observed between injections and between instruments with the ISN feature
enabled and disabled. In addition, lot-to-lot variation in GeneScan™ 600 LIZ® Size
Standard v2.0 peak heights was evaluated with Identifiler® and MiniFiler™ kit data.
After the peak height analysis was complete, ISN Targets were determined based on
the median peak heights obtained across all six instruments for each AmpFlSTR® kit
chemistry, using the median of the average peak heights of the internal standard
normalization peaks in the GeneScan™ 600 LIZ® Size Standard v2.0 size standard
(200, 220, 240,260, 280, 300,314, 320, 340, 360, 400) with both PCR reaction mix and
amplified DNA. Results showed similar average peak heights for the size standard.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 55
Internal Standard Normalization (ISN)
Results The results of this study are expressed as Coefficients of Variation (CVs). The CV of
peak heights for an injection is 100 ✕ [SD of peak heights / Mean peak height]. The
study measured injection-to-injection, capillary-to-capillary, and run-to-run
variation.
The injection-to-injection variation results are shown in the following figure. Each
data point in the graph represents the average height of all the peaks in an injection
profile of a sample at a particular concentration on a particular instrument. Each box
plot contains 96 data points (4 samples ✕ 4 concentrations ✕ 6 instruments). Five
capillaries at locations distributed across the array were each injected two times, for
a total of ten injections.
Figure 15 Injection-to-injection Coefficient of Variation of the peak heights for MiniFiler™ Kit
samples vs. two lots of size standard
These data demonstrate that the performance for ISN on the 3500 instruments was as
expected. When the ISN feature was disabled, the average injection-to-injection CV,
which indicates the approximate inherent variation, was approximately 12% for the
MiniFiler™ kit. When ISN was enabled, the average injection-to-injection CV was
reduced significantly to less than 5%.
The following figure shows instrument-to-instrument CV results for MiniFiler™ kit
data plotted against two lots of the GeneScan™ 600 LIZ® Size Standard v2.0, with
ISN enabled or disabled. Each box plot contains 16 data points (4 samples x 4
concentrations). Each point represents the CV across 6 instruments using the average
height of peaks from the same sample at the same concentration analyzed on the
same instrument.
56 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Figure 16 GeneScan™ 600 LIZ® Size Standard v2.0 instrument-to-instrument variation
When the ISN was disabled, the average instrument-to-instrument CV was
approximately 14% for MiniFiler™ kit data in this study. With ISN enabled, the
average instrument-to-instrument CV was reduced significantly to approximately
8%.
The data also demonstrate that different lots of the GeneScan™ 600 LIZ® Size
Standard v2.0 gave similar results for the instrument-to-instrument CV, indicating
that the performance of the two lots of size standard were consistent. This is also
supported by the following analysis showing the average peak height of the size
standard over multiple manufacturing lots with Identifiler® kit data.
The following figure shows median peak heights for GeneScan™ 600 LIZ® Size
Standard v2.0 analyzed in a background of Identifiler® Sensitivity Samples and three
lots of size standard. Each box contains 192 data points from all instruments, all
samples, and all concentrations run with each lot of the standard.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 57
Internal Standard Normalization (ISN)
Figure 17 GeneScan™ 600 LIZ® Size Standard v2.0 median peak heights: Identifiler®
samples
This data also indicates that the GeneScan™ 600 LIZ® Size Standard v2.0 peak
heights were not impacted by the PCR input DNA concentration.
58 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Normalization The following figure shows median peak heights for GeneScan™ 600 LIZ® Size
Factor Standard v2.0 analyzed using MiniFiler™ sensitivity samples. This data represents
two lots of size standard. The peak heights of the size standard were as expected,
and resulted in NF values of approximately 1.0.
Figure 18 GeneScan™ 600 LIZ® Size Standard v2.0 median peak heights: MiniFiler™
samples
If the Internal Standard Normalization Targets (NT) are set appropriately, the
Normalization Factor (NF) should not vary greatly from 1.0 (that is, the average
observed value from the sample size standard equals the NT). Therefore, users
should set the NT at approximately the median of the peak heights observed during
INS validation.
If the NF is consistently closer to 0.3 or 3.0 than to 1.0, users should review the
sample setup conditions and begin troubleshooting the source of the variation in
peak heights of the size standard or re-evaluate the internal standard normalization
target.
Normalization The Normalization Targets were established by reviewing the GeneScan™ 600 LIZ®
Targets Size Standard v2.0 peak height data for all kits tested and determining the median
peak height for the 11 peaks used during ISN. The following graph represents the
median peak heights of the GeneScan™ 600 LIZ® Size Standard v2.0 size standard
when analyzed in the presence of PCR product from five different AmpFlSTR®
chemistries. The Internal Standard Normalization Target was chosen such that the
intended Normalization Factor on subsequent data would be approximately 1.0.
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 59
Mixture Analysis with GeneMapper® ID-X Software Version 1.2
Figure 19 GS600 size standard peak heights injected with Identifiler® (IF), Identifiler® Direct
(IFD), Identifiler® Plus (IFP), MiniFiler™ (MF), and NGM™ kit samples
This exemplar data demonstrate that the average peak height for GS600 when
coinjected with Identifiler® kit samples is higher than the size standard peak heights
observed after coinjection with other chemistries. From this data and other analyses,
ISN Target values of 3800 RFU were established for Identifiler® and Sinofiler kit data,
and 3200 for Identifiler® Plus, Identifiler® Direct, MiniFiler™, Yfiler®, and NGM™ kit
data.
Mixture Analysis with GeneMapper® ID-X Software Version 1.2
To evaluate the ability of the 3500 systems to analyze DNA samples with more than
one contributor, a range of mixed DNA samples were analyzed. The default peak
amplitude threshold of 175 RFU was used to analyze the samples using
GeneMapper® ID-X Software v1.2.
For details of study parameters, see Table 1, “Studies performed, kits tested, and
DNA inputs,” on page 37, Table 2, “Sample sources and kits and instruments used
for preparation and amplification,” on page 38, and Table 4, “Study criteria matrix,”
on page 39.
Experimental For all samples in a project, the GeneMapper ID-X Mixture Analysis Tool categorizes
design and segregates eligible samples according to the minimum number of contributors
contained in each sample (1, 2, or 3 or more contributors), separates contributor
profiles into individual major and minor genotype combinations, and calculates
population statistics. For more information on the Mixture Analysis tool, see the
GeneMapper® ID-X Software Version 1.1 (Mixture Analysis Tool) Getting Started Guide
(Part no. 4396773).
60 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
For this study, four pairs of genomic DNA samples were created with seven mixture
ratios of 1:0, 1:1, 1:2, 1:3, 1:5, 1:9, and 0:1 (28 total samples). The total DNA input
amount in each reaction was the suggested DNA input amount for each kit: 1 ng for
Identifiler® and 2 ng for SGM Plus®, Profiler Plus®, and Cofiler®. The percentage of
DNA from each contributor therefore varied accordingly depending on the dilution.
For example, 1 ng DNA input for a 1:9 mixture results in 0.9 ng of one sample and
0.1 ng of the second target DNA for each sample in the mixture.
The allele dropout rates were measured for each mixture ratio in each kit. The
genotyping data were then imported into the Mixture Analysis tool in the software.
Dilution series The number of observed alleles in each mixture profile was tabulated and compared
to the expected number of alleles from the known profile.
The percent allele call rates are shown in Table 8 for Identifiler®, SGM Plus®, Profiler
Plus®, and Cofiler® respectively. A high percentage of the expected alleles were
detected through the 1:9 dilution series utilizing a 175 RFU threshold.
Table 8 Allele call rates for all dilutions using recommended template DNA input
amounts, 5 AmpFlSTR® kits
Allele call rate
Mix ratio
Identifiler® Identifiler® Profiler
SGM Plus® Cofiler®
Norm-On Norm-Off Plus®
1:1 100.0% 100.0% 100.0% 100.0% 100.0%
1:2 100.0% 100.0% 100.0% 100.0% 100.0%
1:3 100.0% 100.0% 100.0% 100.0% 100.0%
1:5 99.3% 98.4% 99.2% 99.9% 98.6%
1:9 98.4% 96.5% 89.0% 98.6% 98.2%
.hid file processing Samples were imported into the Mixture Analysis tool in the GeneMapper® ID-X
v1.2 software to demonstrate successful processing of .hid files. The GeneMapper®
ID-X v1.2 software validation was described previously in the GeneMapper® ID-X
Software Version 1.2 User Bulletin (Part no. 4462639). Five analysis categories were
used for mixture analysis depending on the kit used and the Mixture Analysis
method employed.
•MIT_175
–175 RFU for the mixture interpretation threshold (MIT).
–Mixture Analysis software default peak height ratio (PHR) thresholds.
Note: RFU values less than 175 are included for illustrative purposes only.
Data for RFU values less than 175 was not analyzed.
•PHR_175
–175 RFU for the mixture interpretation threshold (MIT).
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 61
Mixture Analysis with GeneMapper® ID-X Software Version 1.2
–Customized PHR thresholds and peak height ranges set according to the
intra-locus peak height ratios of the single source samples (1:0 and 0:1
mixtures) in the data set of each kit tested.
Table 9 Mixture analysis methods used with defined MIT and PHR thresholds
MA MIT
Kits PHR Threshold (rfu)
method (rfu)
MIT_175 175 50-150 151-300 301-1000 1001-99999
MA Default
0.4 0.54 0.63 0.75
PHR_175 175 175-5500 5501-99999
Norm Off
0.67 0.77
Identifiler® PHR_175 175 175-8500 8501-99999
Norm On
0.67 0.77
SGM Plus® PHR_175 175 175-99999
0.75
Profiler PHR_175 175 175-3540 3541-99999
Plus®
Cofiler® PHR_175 175 0.685 0.82
For the Identifiler® samples, the data were analyzed with the ISN option both
enabled and disabled. Since ISN changes the absolute peak heights, the peak height
range for each PHR threshold was set accordingly.
During mixture analysis, the samples were segregated into two categories,
1-contributor and 2-contributor, according to the MIT and PHR settings in each MA
method. The samples under the 2-contributor category were then deconvoluted to
give the potential genotype profiles of both contributors according to the PHR and
residual thresholds.
No significant difference in sample segregation and deconvolution was observed
between the ISN options
Mixture Ratio The MA tool in GeneMapper® ID-X v1.2 software calculates the Average Mx value as
prediction an estimation of the minor contributor proportion of the sample. Samples used in
this test were prepared for the study with known estimated ratios, and were used to
evaluate how well the MA calculations fit the expected values.
62 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
The Average Mx values were plotted against the expected minor contributor
proportions of all samples analyzed using different MA methods and ISN options.
The expected minor contributor proportions were calculated as the ratio of the minor
contributor DNA concentration to the total DNA concentration for the sample sets.
A linear regression fit line was plotted for each group of data.
Figure 20 Example: Regression of average vs. expected minor contributor proportions for
Identifiler® samples
The regression lines indicated a good fit of the curves to the data and low variation
among the individual data points. The regression line slopes were very similar to
results from 3130xl testing with the same DNA inputs and mixture ratios, indicating
that the two platforms demonstrated similar performance with mixed samples. The
data also demonstrate no statistical difference between the default and customized
MA methods, and between the ISN options.
Table 10 Slopes of regression lines for average minus expected Mx ratios
Internal Standard Slope with Slope with
Kits
Normalization MIT_175 PHR_175
Identifiler® On 0.844 0.8419
Off 0.8549 0.8473
SGM Plus® N/A 0.8292 0.87
Profiler Plus® N/A 0.9278 0.9003
Cofiler® N/A 0.8978 0.8807
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 63
Conclusions
Conclusions
• High-quality data were obtained on the 3500 and 3500xL Genetic Analyzers
with the AmpFlSTR® PCR Amplification Kits.
• Accurate genotyping results were obtained using GeneMapper® ID-X v1.2
software.
• Resolution estimations confirm single base pair analysis up to 400 base pairs.
• Sizing precision, measured by calculating the standard deviation in the size
values obtained for an allele within an injection was <0.15bp for all injections on
both the 3500 and 3500xL instruments.
• Noise levels and pull up percentages, when adjusted for the relative RFU
scaling of the 3500 instrument, are similar to existing platforms such as 31xx.
64 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Ordering information
Consumables Note: For reagent or consumable shelf-life expiration date, see the package label.
Description Part Number
Matrix Standard Set DS-33 3500 Series (Dye Set G5) 4345833
Matrix Standard Set DS-32 (Dye Set F) 4345831
3500 Capillary Array (36 cm) 4404683
3500xL Capillary Array (36 cm) 4404687
Anode Buffer Container (ABC) 3500 Series 4393927
Cathode Buffer Container (CBC) 3500 Series 4408256
Hi-Di™ Formamide 4401457
GeneScan™ 600 LIZ® Size Standard v2.0 Size Standard 3500 4408399
Series
3500 POP-4® Polymer, 384 samples 4393715
3500 POP-4® Polymer, 960 samples 4393710
Conditioning Reagent 3500 Series 4393718
8-Strip Septa for 3500/3500xL Genetic Analyzers 4410701
96-Well Septa for 3500/3500xL Genetic Analyzers 4412614
Septa Cathode Buffer Container, 3500 series 4410715
96-well retainer & base set (Standard) 3500/3500xL Genetic 4410228
Analyzers
GeneScan™ 500 ROX™ Size Standard 401734
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 65
Ordering information
HID analysis dye
sets
The following dye sets are supported with the HID Fragment Analysis 36_POP4 run
module:
Dye set F (4-dye):
•COfiler®
•Profiler Plus®
•Profiler Plus® ID
•SGM Plus®
•Other 4-dye kits
Dye set G5 (5-dye)
•Identifiler®
•MiniFiler™
•SEfiler™ Plus
•SinoFiler™
•Yfiler®
•Other 5-dye kits
66 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Validated instrument protocol specifications
The kits were validated by the assays shown in the following table, using POP-4®
polymer in a 36-cm capillary.
Primary analysis
Kit Assay Instrument protocol Size standard
protocol
AmpFlSTR® CO_POP4 HID36_POP4_F F_LS(75-400) CE_F_HID_GS500(75-
COfiler® 400)
PCR
Amplification CO_POP4_xl HID36_POP4xl_F F_LS(75-400) CE_F_HID_GS500(75-
Kit 400)
AmpFlSTR® IF_POP4 HID36_POP4_G5_NT3800 G5_LS(80-400) GS600_LIZ_(80-400)
Identifiler®
PCR IF_POP4_xl HID36_POP4xl_G5_NT3800 G5_LS(80-400) GS600_LIZ_(80-400)
Amplification
IF+Norm_POP4 HID36_POP4_G5_NT3800 G5_LS(80- GS600_LIZ+Normaliza
Kit
400)+Normalization tion_(80-400)
IF+Norm_POP4_xl HID36_POP4xl_G5_NT3800 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
AmpFlSTR® IFD_POP4 HID36_POP4_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Identifiler®
Direct PCR IFD_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Amplification
IFD+Norm_POP4 HID36_POP4_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
Kit
400)+Normalization tion_(80-400)
IFD+Norm_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
AmpFlSTR® IFP_+Norm_POP4 HID36_POP4_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
Identifiler® 400)+Normalization tion_(80-400)
Plus PCR
Amplification IFP_POP4 HID36_POP4_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Kit
IFP_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
IFP+Norm_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
AmpFlSTR® MF_POP4 HID36_POP4_G5_NT3200 G5_3rd(80-400) GS600_LIZ_(80-400)
MiniFiler™
PCR MF_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Amplification
MF+Norm_POP4 HID36_POP4_G5_NT3200 G5_3rd(80- GS600_LIZ+Normaliza
Kit
400)+Normalization tion_(80-400)
MF+Norm_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin 67
Validated instrument protocol specifications
Primary analysis
Kit Assay Instrument protocol Size standard
protocol
AmpFlSTR® NGM_POP4 HID36_POP4_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
NGM™ PCR
Amplification NGM_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Kit
NGM+Norm_POP4 HID36_POP4_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
NGM+Norm_POP4_ HID36_POP4xl_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
xl 400)+Normalization tion_(80-400)
AmpFlSTR® PP_POP4 HID36_POP4_F F_LS(75-450) CE_F_HID_GS500(75-
Profiler 450)
Plus® ID
PCR PP_POP4_xl HID36_POP4xl_F F_LS(75-450) CE_F_HID_GS500(75-
Amplification 450)
Kit
AmpFlSTR® SEfP_POP4 HID36_POP4_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
SEfiler Plus™
PCR SEfP_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Amplification
SEfP+Norm_POP4 HID36_POP4_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
Kit
400)+Normalization tion_(80-400)
SEfP+Norm_POP4_ HID36_POP4xl_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
xl 400)+Normalization tion_(80-400)
AmpFlSTR® SF_POP4 HID36_POP4_G5_NT3800 G5_LS(80-400) GS600_LIZ_(80-400)
Sinofiler™
PCR SF_POP4_xl HID36_POP4xl_G5_NT3800 G5_LS(80-400) GS600_LIZ_(80-400)
Amplification
SF+Norm_POP4 HID36_POP4_G5_NT3800 G5_LS(80- GS600_LIZ+Normaliza
Kit
400)+Normalization tion_(80-400)
SF+Norm_POP4_xl HID36_POP4xl_G5_NT3800 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
AmpFlSTR® SGM_POP4 HID36_POP4_F F_LS(75-450) CE_F_HID_GS500(75-
SGM Plus® 450)
PCR SGM_POP4_xl HID36_POP4xl_F F_LS(75-450) CE_F_HID_GS500(75-
Amplification 450)
Kit
AmpFlSTR® YF_POP4 HID36_POP4_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
Yfiler®
YF_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80-400) GS600_LIZ_(80-400)
PCR
Amplification YF+Norm_POP4 HID36_POP4_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
Kit 400)+Normalization tion_(80-400)
YF+Norm_POP4_xl HID36_POP4xl_G5_NT3200 G5_LS(80- GS600_LIZ+Normaliza
400)+Normalization tion_(80-400)
68 Applied Biosystems 3500/3500xL Genetic Analyzer User Bulletin
Documentation
For more detailed information on the 3500 Series Genetic Analyzers and related
procedures, refer to:
• Applied Biosystems 3500/3500xL Genetic Analyzer User Guide (Part no. 4401661)
• 3500 and 3500xL Genetic Analyzers Quick Reference Card (Part no. 4401662)
• GeneMapper® ID-X Software Version 1.2 Quick Reference Card (Part no. 4426482)
• 3500 Data Collection V1.0 Software Release Notes (on the 3500 Data Collection
Software CD)
• User Bulletin: Applied Biosystems 3500/3500xL Genetic Analyzer (Part no. 4445098)
• GeneMapper® ID-X Software Version 1.2 User Bulletin (Part no. 4462639)
Frequently Asked Questions (FAQs) and supplementary information are available at
the Applied Biosystems website, http://www.appliedbiosystems.com/3500hid.
For Research, Forensic, or Paternity Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Information in this document is subject to change without notice.
APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED
TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL
APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED
OF THE POSSIBILITY OF SUCH DAMAGES.
Limited Use Label License: Research Use Only
The purchase of this product conveys to the purchaser the limited, non-transferable right to use the product only to perform internal research for the sole
benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for
internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial
services such as reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please
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The trademarks mentioned herein are the property of Life Technologies or their respective owners. © 2011 Life Technologies. All rights reserved.
www.appliedbiosystems.com Part Number 4469192 Rev. A
Kliendilepingu nr 2021-230 lisa 3
VOLIKIRI
13. märts 2024. a
Lanlab OÜ, registrikood 10437034, asukoht Ravila tn 63, 50411 Tartu, mida esindab
seaduse ja põhikirja alusel juhatuse liige Ain Langel, isikukood 35805032720, e-post
[email protected], (edaspidi „Klient“)
annaab käesolevaga Advokaadibüroo Kõrgesaar ja Laasik OÜ vandeadvokaat Tambet
Laasik´ule volituse esindada ennast kõigi seadusest tulenevate õigustega alljärgneva
õigusteenuse osutamisel:
Kliendi õigusalane nõustamine ja esindamine Riigi Tugiteenuste Keskuse
riigihanke „Kapillaarsekvenaatorite ja qPCR seadmete kuluvahendid (EKEI)“
(viitenumber 272907) vaidlustamisel.
Ülalnäidatud eesmärkide saavutamiseks on advokaadil õigus võtta tarvitusele ükskõik
milliseid meetmeid ja teha toiminguid, mida ta peab vajalikuks volikirjas toodud
eesmärkide saavutamiseks.
Käesolev volikiri on antud edasivolitamisõigusega Advokaadibüroo Kõrgesaar ja Laasik
OÜ advokaatidele.
Käesolev volikiri kehtib tähtajatult.
___________________________
Ain Langel
Lp Angelika Timusk Riigihangete vaidlustuskomisjon
[email protected] 13. märts 2024 . a , nr 1 - 32 Vaid lustaja : OÜ Lanlab registrikood: 10437034 Vaid lustaja lepinguline esindaja: vandeadvokaat Tambet Laasik Advokaadibüroo Kõrgesaar & Laasik Rüütli 23, 51006 Tartu tel: +372 666 0077 e-post:
[email protected] SEISUKOHT Riigi Tugiteenuste Keskuse riigihankes „ Kapillaarsekvenaatorite ja qPCR seadmete kuluvahendid (EKEI)“ (viitenumber 272907) LanLab OÜ esitatud vaidlustuse kirjalikus menetluses SENINE MENETLUS OÜ LanLab esitab käesolevaga enda seisukoha hankija Riigi Tugiteenuste Keskuse 8. märtsi 2024. a vastusele. Menetluses kogutud tõendid ei kinnita hankija järelduste õigsust, vaid vastupidi tõendavad seda, et kolmas isik ei vastanud kvalifitseerimistingimustele, mistõttu vaidlustus tuleb rahuldada. Seda järgmistel põhjendustel. ÕIGUSLIKUD PÕHJENDUSED Hankija selgitas p 2.3, et tema hinnangul „ kolmas isik vastab kvalifitseerimise tingimustele. Juhime tähelepanu, et hankija ei ole kvalifitseerimistingimuses sätestanud, et pakkuja peaks olema 50 000 € eest müünud kuluvahendeid nii kapillaarsekvenaatoritele kui eraldi qPCR seadmetele ega piiritlenud, et kuluvahendeid peaks olema müüdud Eesti Kohtuekspertiisi Instituudil kasutusel olevatele masinatele. Hankija ei ole tingimuses täpsustanud, millises proportsioonis peab olema müüdud ühele või teisele seadmele “. Selline seisukoht ei ole õige ega täpne. Kvalifitseerimistingimuse sõnastus oli järgmine: „ Pakkuja peab olema hanketeate avaldamise kuupäevale eelneva 36 kuu jooksul teostanud vähemalt ühe samalaadse lepingu, mille objektiks on kuluvahendite tarne kohtuekspertiisi valdkonnas kasutatavatele kapillaarsekvenaatoritele ja qPCR seadmetele maksumusega vähemalt 50 000 aastas (käibemaksuta) “. See sõnastus ei toeta hankija väidet. Selleks, et hankija väide oleks õige, peaks kvalifitseerimistingimus olema sõnastatud teisiti: „ Pakkuja peab olema 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) “. Olemasolev tingimus, kus on sõna „ ja “ tähendab seda, et pakkuja peab olema müünud mõlema masina kuluvahendeid mitte ainult ühe nendest. Tõsi, on vaieldav, kas mõlemale seadmele eraldi pidi müüma kuluvahendeid 50 000 euro eest, või piisab, et kokku oli müük 50 000 euro eest. Küll on aga selge, et toodete müügi proportsioon ei ole täielikult irrelevantne. Iseenesest ei ole muidugi oluline, kas müüdud kuluvahenditest on nt 32% kapillaarsekvenaatoritele ja 68% qPCR seadmetele või on see proportsioon 57/43, 20/80 vms, kuid tähtis on see, et see proportsioon ei saa olla 0/100, vaid vähemalt mingis ulatuses peab olema müüdud kuluvahendeid mõlema seadme ta r beks. Hankija vastuse kohaselt on kolmas isik varem müünud aga vaid järgmist kaupa: „ Thermo Scientific ™ 339650, Nunc ™ 15mL, Conical Sterile Polypropylene Centrifuge Tubes “ . Erinevalt hankija vastuses väidetust, ei ole need kuluvahendid, mida saab kasutada nii kapillaarsekvenaatoritele kui ka qPCR seadmetele . Juhime siinkohal tähelepanu tootja kasutusjuhendile ( Lisa 1 ) , mille lk 65 on välja toodud kõik Eesti Kohtuekspertiisi Instituudis kasutusel oleva kapillaarelektroforeesi instrumendi kuluvahendid: Matrix Standard Set DS-33 3500 Series ( Dye Set G5) cat # 4345833 Matrix Standard Set DS-32 ( Dye Set F) cat # 4345831 3500 Capillary Array (36 cm) cat # 4404683 3500xL Capillary Array (36 cm) cat # 4404687 Anode Buffer Container (ABC) 3500 Series cat # 4393927 Cathode Buffer Container (CBC) 3500 Series cat # 4408256 Hi-Di ™ Formamide cat # 4401457 GeneScan ™ 600 LIZ® Size Standard v2.0 Size Standard 3500 Series cat # 4408399 3500 POP-4® Polymer , 384 samples cat # 4393715 3500 POP-4® Polymer , 960 samples cat # 4393710 Conditioning Reagent 3500 Series cat # 4393718 8-Strip Septa for 3500/3500xL Genetic Analyzers cat # 4410701 96-Well Septa for 3500/3500xL Genetic Analyzers cat # 4412614 Septa Cathode Buffer Container , 3500 series cat # 4410715 96-well retainer & base set (Standard) 3500/3500xL Genetic Analyzers cat # 4410228 GeneScan ™ 500 ROX™ Size Standard cat # 401734 Iseenesest on loomulikult ka teisi tootjaid, kes toodavad kapillaarelektroforeesi seadmeid, ning EKEI kasutuses olev instrument ei ole ainus võimalik. Samas on nende kõigi kuluvahendid oma olemuselt siiski samad (kapillaarid, jooksutamise- ja pesupuhvrid ning suurusmarkerid) ning mitte ükski neist ei kasuta kuluvahendina OÜ Lanmer poolt nimetatud ning nende poolt EPJ- le tarnitud toodet. EKEI vastuses välja toodud näite toode, mis on üldine laboriplastik ( Thermo Scientific ™ 339650, Nunc ™ 15mL, Conical Sterile Polypropylene Centrifuge Tubes ) , ei ole klassifitseeritav kui kapillaarelektroforeesi masina kuluvahend ning ei ole sel põhjusel ka välja toodud punktis 2.7 olevas tabelis. Kui täpne olla, siis see ei sobi tegelikult kummalegi masinale Seetõttu p alume uuesti üle kontrollida, kas Lanmer OÜ poolt tõenduseks toodud lepingus ja väljastatud arvetes viimase 36 kuu jooksul enne hanke väljastamise kuupäeva on välja toodud mõni ülaltoodu d nimekirjas nimetatud toode või sellele samaväärne . Siiani esile toodud toode seda ei ole, mistõttu palume ka teised üle kontrollida. KOKKUVÕTE Eeltoodut kokku võttes tuleb nentida, et menetluse käigus kogutud andmed kinnitavad seda, et kolmas isik OÜ Lanmer ei ole täitnud eelviidatud kvalifitseerimistingimust, ta ei ole müünud k apillaarsekvenaatorite kuluvahendeid, ta on alusetult kvalifitseeritud ning OÜ LanLab vaidlustus tuleb rahuldada. Lugupidamisega /allkirjastatud digitaalselt/ Tambet Laasik Vandeadvokaat Lisad: Applied Biosystems 3500/3500xL Genetic Analyzer kasutusjuhend volikiri