User BulletinApplied Biosystems 3130 Series Genetic Analyzers
July 2006
SUBJECT: Performance Verification After Upgrade from ABI PRISM® 3100 Series Genetic Analyzers
In This UserBulletin
This user bulletin includes the following topics:
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Before Upgrading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43.2 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
4 Experimental Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7.1 Results of a 3100-Avant-to-3130 Instrument Upgrade . . . . . . . . .127.2 Results of a 3100-to-3130xl Instrument Upgrade. . . . . . . . . . . . .247.3 Results of a 3100-Avant-to-3130xl Instrument Upgrade . . . . . . .367.4 Results of a 3130-to-3130xl Instrument Upgrade. . . . . . . . . . . . .48
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
1 Introduction
InstrumentUpgrade Paths
The following paths are available to upgrade an ABI PRISM® 3100 or 3100-Avant™ Genetic Analyzer to an Applied Biosystems 3130 or 3130xl Analyzer:
InstrumentComponents
Modified
The instrument components modified during the upgrade include:
• Polymer delivery system• Oven door gasket• CCD camera shutter• Detection cell heater• Data Collection Software upgrade to 3130 Series version
3.0 software
For details on the upgraded components, refer to the Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0, Protocols for Processing AmpFlSTR PCR Amplification Kit PCR Products User Bulletin (PN 4363787).
PerformanceVerification
StudiesPerformed
Although components of the optical system are not changed during the upgrade, DAB/SWGDAM guidelines require a performance verification after an upgrade. The following performance verification studies ensure that key performance parameters have not changed:
• Sensitivity• Mixture• Precision• Resolution• Concordance
IMPORTANT! Human identification laboratories should perform their own performance verification studies.
Instrument Can be upgraded to
3100 3130xl
3100-Avant 3130
3130xl
3130 3130xl
2 Before Upgrading
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 3
2 Before UpgradingIMPORTANT! Perform the following steps before upgrading an instrument:
• Review the Applied Biosystems 3130 and 3130xl Genetic Analyzer Site Preparation Guide to ensure that the laboratory environment meets specifications for a 3130 or 3130xl instrument.
• Back up all sample data. During the upgrade, the instrument computer is reimaged (all data are erased) before the Data Collection software is installed. It is not necessary to back up spatial calibration and spectral calibration files because these calibrations are performed at the end of the upgrade.
Reagents
4 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
3 Materials and Methods
3.1 ReagentsTo minimize variability between studies, the following setup procedures were used throughout the study:
• For the sensitivity studies, serial dilutions were prepared from the quantified stock control DNA (9947A, 007, and 9948) based on the average concentration determined using the Quantifiler® Human DNA Quantification Kit and the ABI PRISM® 7000 Sequence Detection System with SDS Software v1.0.
• For all studies, multiple PCR product reactions were pooled to provide sufficient quantity and minimize variability.
• For all studies, one manufactured lot of each of the following kits was used:
3.2 InstrumentsThree instruments of each type were used for each study. Figure 1 shows the instrument upgrade paths, instrument numbers, and the performance assessment test numbers performed. Table 1 on page 5 explains the performance assessment tests.
Figure 1 Tests performed on each instrument before and after upgrades
Kit AB Part Number
Quantifiler® Human DNA Quantification Kit 4343895
AmpFlSTR® Identifiler® PCR Amplification Kit 4322288
AmpFlSTR® Profiler Plus® PCR Amplification Kit 4303326
3100-Avant-003
Test 1
Upgrade to3100-Avant-040
3100-Avant-019
3130-003
Test 2
3130-040
3130-019
3130xl-003
Test 3
3130xl-040
3130xl-019
3100-006
Test 4
3100-015
3100-028
3130xl-006
Test 5
3130xl-015
3130xl-028
Upgrade to
Upgrade to
3 Materials and Methods
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 5
Table 1 Performance assessment tests (studies performed in each test: sensitivity, mixture, precision, resolution, and concordance)
Test Instrument Instrument ID Numbers Purpose
1 3100-Avant™ before upgrade
003, 040, and 019 To generate baseline data on the3100-Avant Genetic Analyzer before upgrade.
2 3130:
• After upgrade from 3100-Avantand
• Before upgrade to 3130xl
003, 040, and 019 3100-Avant-to- 3130 upgrade: To generate data using the same studies as Test 1, then to compare the results from Test 1 to the results from Test 2.
3130-to-3130xl upgrade: To generate baseline data on the 3130 Genetic Analyzer before upgrade.
3 3130xl after upgrade from 3130
003, 040, and 019 To generate data using the same studies as Test 1, then to compare the results from Test 1 to the results from Test 3.
4 3100 before upgrade 006, 015, and 028 To generate baseline data on the3100 Genetic Analyzers before upgrade.
5 3130xl after upgrade from 3100
006, 015, and 028 To generate data using the same studies as Test 4, then to compare the results from Test 4 to the results from Test 5.
Instruments
6 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
4 Experimental Setup
Quantitation All DNA samples were quantitated using the Quantifiler® Human DNA Quantification Kit according to the protocols defined in the Quantifiler® Kits User's Guide.
Amplification DNA was amplified:
• By PCR on a GeneAmp® PCR System 9700.
Note: The Aluminum 96-Well GeneAmp® PCR System 9700 Sample Block Module is not supported for use with the AmpFlSTR® chemistries.
• Using the following kits and reaction volumes according to the recommended protocols in the AmpFlSTR® user's manuals:
Sensitivity Study Sensitivity studies were run using three different DNA samples: 007, 9947A, and 9948.
When DNA input quantity is low, stochastic PCR effects may result in heterozygote peak imbalance, possible allele dropout, and low overall relative fluorescence signal.
Before and after upgrade, sensitivity studies were conducted to determine the detection capabilities of the instruments. As expected and reported in Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection v3.0 (PN 4363787), variation in sensitivity from instrument to instrument was observed.
Kit Reaction Volume (µL)
AB Part Number
AmpFlSTR® Identifiler® PCR Amplification Kit
25 4322288
AmpFlSTR® Profiler Plus®
PCR Amplification Kit50 4303326
4 Experimental Setup
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 7
Serial dilutions were made with 0.1 mM Na2EDTA (T10E0.1 buffer). The following DNA input amounts were amplified:
All samples were replicated twice on the plate, and each replicate was injected three times (for a total of six injections per sample).
The sensitivity study evaluated the following before and after upgrade of each instrument:
• The ability to detect a full genotype profile in a range of DNA input amounts, determined by evaluating the number of alleles that fell below the detection threshold
• Average peak heights• Average heterozygote peak height ratios
The GeneMapper® ID Software v3.2 analysis threshold for the sensitivity study was set at 50 RFU (relative fluorescence units).
Peak Height Calculations
Peak heights for all alleles from all samples in a test were used to calculate a single average peak height value. This average peak height value was then compared between tests.
To compare peak heights by chemistry, peak heights for all alleles from all samples in a test run using one chemistry were used to calculate a single average peak height value. This average peak height value was then compared between tests performed using the same chemistry.
Kit DNA Input Amounts Amplified
AmpFlSTR Identifiler PCR Amplification Kit
• 1.5 ng• 1.0 ng• 500 pg
• 250 pg• 125 pg
AmpFlSTR Profiler Plus PCR Amplification Kit
• 3.0 ng• 2.0 ng• 1.0 ng
• 500 pg• 250 pg
Instruments
8 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Mixture Study DNA samples 9947A and 9948 were used to prepare a set of DNA mixtures at the following 9947A:9948 ratios: 0:1, 1:1, 2:1, 4:1, 9:1, and 1:0 (Table 2).
All amplified samples were replicated twice on the plate, and each replicate was injected three times (for a total of six injections per sample). The samples were evaluated for the ability to detect the minor contributor at each ratio.
Total DNA input was 1.5 ng per reaction for the Identifiler® Kit and 3.0 ng per reaction for the Profiler Plus® Kit kit.
The GeneMapper ID Software v3.2 analysis threshold for this study was set at 50 RFU (relative fluorescence units).
Number of Expected Alleles
The number of expected alleles for mixtures was determined from the 1:1 mixture ratio. When the major and minor contributor shared an allele, the shared allele was counted as a single allele.
If an allele from the minor contributor fell in the stutter position of an allele from the major contributor, the allele from the minor contributor was identified only if its peak height exceeded the stutter filter threshold. Typically, minor contributor alleles in the stutter position of a major contributor allele are filtered as stutter in the 1:10 mixture ratio.
Table 2 Mixture composition
Kit Mixture Ratio
Total Female DNA Input (ng)
Total Male DNA Input (ng)
AmpFlSTR Identifiler PCR Amplification Kit
1:0 1.50 0.001:1 0.75 0.754:1 1.20 0.309:1 1.35 0.150:1 0.00 1.50
AmpFlSTR Profiler Plus PCR Amplification Kit
1:0 3.00 0.001:1 1.50 1.504:1 2.40 0.609:1 2.70 0.300:1 0.00 3.00
5 Data Collection
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 9
Precision Study Allelic ladders from the Identifiler® Kit and Profiler Plus® Kit were used to calculate the standard deviation of the size (in bp) for each allele in each allelic ladder. Sixteen replicates of each ladder were loaded on a 96-well plate, and each replicate was injected three times (for a total of 48 injections per sample).
Sizing averages were calculated within a run (that is, 16 samples for the 3100 and 3130xl 16-capillary instruments and four samples for the 3100-Avant and 3130 4-capillary instruments).
Sizing precision between runs of allelic ladder was not calculated during this study.
Resolution Study The TH01 locus of the Identifiler® Kit allelic ladder was examined to evaluate the resolution and detection of alleles differing in size by 1 base pair.
ConcordanceStudy
The genotype results from the sensitivity study were evaluated for concordance.
5 Data CollectionData was collected using the following versions of software:
All instruments used the default Run Module with the appropriate dye set.
Instrument Instrument ID Numbers
Data Collection Version
3100-Avant™ 003 2.0040 1.0019 1.0
3100 006 1.1015 1.1028 2.0
3130 003, 040, and 019 3.03130xl 003, 040, 019,
006, 015, and 0283.0
Instruments
10 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
For more information, see:
• User Bulletin ABI PRISM® 3100/3100-Avant™ Genetic Analyzers Using Data Collection v2.0: Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products (PN 4350218, Rev. A)
• User Bulletin Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0 (PN 4363787)
• ABI PRISM® 3100 Genetic Analyzer User's Manual (PN 4315834B)
• Applied Biosystems 3130/3130xl Genetic Analyzers: Getting Started Guide (PN 4352715)
6 Data Analysis
Initial DataCompiling and
Analysis
All data were analyzed using GeneMapper® ID Software v3.2 with:
• HID_Advanced Analysis Method • Peak Detection Threshold of 50 RFU
For information on setting up the analysis method, see the GeneMapper® ID Software Version 3.1 and 3.2 Human Identification Analysis Tutorial (P/N 4357520 Rev. A).
Final DataAnalysis
Minitab® Statistical Software was used to:
• Calculate averages • Generate the box and whisker plots
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 11
7 Results and DiscussionElectropherograms in this section were generated using GeneMapper® ID Software Version 3.2.
Data plots in this section were generated using Minitab® Statistical Software and reported in Box-and-Whisker-Plot format (Figure 2).
Figure 2 Box-and-whisker plot elements
Maximum value
Minimum value
75th percentile
Median value
25th percentile
Outliers
where:• Whiskers extend to the minimum and maximum data values.• Median = The middle of the data when it is ordered from smallest to largest value. • 25th percentile (lower quartile) = The median of the lower half of the data. • 75th percentile (upper quartile) = The median of the upper half of the data. • Minimum value = The smallest observed value. • Maximum value = The largest observed value.• Values beyond the whiskers are outliers. Outliers are unusually large or small
values and are represented as dots on the plot.
Results of a 3100-Avant™-to-3130 Instrument Upgrade
12 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.1 Results of a 3100-Avant™-to-3130 Instrument Upgrade
7.1.1 Sensitivity Study
The sensitivity study evaluated the following before and after upgrade of each instrument:
• The ability to detect a full genotype profile in a range of DNA input amounts, determined by evaluating the number of alleles that fell below the detection threshold
• Average peak heights• Average heterozygote peak height ratios
Number of AllelesBelow Detection
Threshold
Table 3 shows the number of alleles that fell below the detection threshold before and after upgrading the 3100-Avant instrument to a 3130 instrument:
• Before upgrade – Sixty four (64) alleles (out of a total of 8725) alleles fell below the detection threshold (Figure 3).
• After upgrade – Sixty four (64) alleles (out of a total of 9915) alleles fell below the detection threshold (Figure 4).
As expected, instrument-to-instrument variation was observed between instruments of the same type before and after upgrade.
Table 3 Sensitivity study – Number of alleles below detection threshold – 3100-Avant™-to-3130 instrument upgrade
Test 1 (before upgrade) Test 2 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3100-Avant-003 22 3678 3130-003 2 3259
3100-Avant-040 5 3082 3130-040 9 3273
3100-Avant-019 37 1965 3130-019 53 3383
Total 64 8725 Total 64 9915
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 13
Full genotypes were consistently obtained with 250 pg DNA input amounts for all samples amplified with the Identifiler® and Profiler Plus® kits before and after upgrading the 3100-Avant instrument to a 3130 instrument. All alleles that fell below the detection limit were obtained from the 125 pg amplification.
Alleles that fall below the threshold can be caused by:
• Low DNA input, which results in low overall relative fluorescence
• Stochastic PCR effects, which result in:– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3100-Avant™-to-3130 Instrument Upgrade
14 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 3 Sensitivity study – Identifiler® Kit electropherograms for a 3100-Avant™ instrument before upgrade (Y-scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
Serial dilutions of 9947A DNA
0.125 ng
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 15
Figure 4 Sensitivity study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 3) after 3100-Avant™-to-3130 instrument upgrade (Y scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
Serial dilutions of 9947A DNA
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
0.125 ng
Results of a 3100-Avant™-to-3130 Instrument Upgrade
16 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Peak Height A comparison of peak heights for each DNA input amount before and after instrument upgrade showed little variation in sensitivity (for a description of how peak heights were evaluated, see page 7):
• Overall, a 1.2% increase in peak heights was observed after upgrading the 3100-Avant instruments to 3130 instruments.
• Identifiler® Kit data showed a 4.1% decrease in overall peak height after upgrade (Figure 5).
• Profiler Plus® Kit data showed an 11.2% increase in overall peak height after upgrade (Figure 6).
Figure 5 Sensitivity study – Comparison of Identifiler® Kit peak heights by DNA input amount for all samples before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 17
Figure 6 Sensitivity study – Comparison of Profiler Plus® Kit peak heights by DNA input amount for all samples before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
HeterozygotePeak Height
Ratio
Heterozygote peak height ratios were calculated and plotted for each DNA sample and concentration in the sensitivity study (for a description of how peak heights were evaluated, see page 7).
Heterozygote peak height ratios for all loci in the Identifiler® Kit (Figure 7) and Profiler Plus® Kit (Figure 8) averaged 80% or greater before and after upgrading the 3100-Avant instruments to 3130 instruments.
Heterozygote peak height ratios below the 70% cutoff before and after upgrade occurred mainly with lower DNA input amounts (Figures 9 and 10).
Low heterozygote peak height ratios can be caused by:
• Low DNA input, which results in low overall relative fluorescence
• Stochastic PCR effects, which result in:– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3100-Avant™-to-3130 Instrument Upgrade
18 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 7 Sensitivity study – Comparison of Identifiler® Kit
heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
Figure 8 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 19
Figure 9 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by input amount for all samples before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
Figure 10 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by input amount for all samples before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
Results of a 3100-Avant™-to-3130 Instrument Upgrade
20 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.1.2 Mixture Study
Mixed DNA profiles were evaluated, and the number of alleles falling below 50 RFU was recorded before and after instrument upgrade.
Number of AllelesBelow Detection
Threshold
For a description of how the number of expected alleles was determined, see page 8.
Table 4 shows the number of alleles that fell below the detection threshold before and after upgrading the 3100-Avant instrument to a 3130 instrument:
• Before upgrade – Fifty seven (57) alleles (out of a total of 5832) fell below the detection threshold (Figure 11).
• After upgrade – Thirty eight (38) alleles (out of a total of 5643) fell below the detection threshold (Figure 12).
The limit of detection of the minor contributor was approximately 9:1 before and after upgrading the 3100-Avant instrument to a 3130 instrument
Table 4 Mixture study – Number of alleles below detection threshold – 3100-Avant™-to-3130 instrument upgrade (data for 1:1 mixture not included)
Test 1 (before upgrade) Test 2 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3100-Avant-003 12 1944 3130-003 12 1944
3100-Avant-040 33 1944 3130-040 13 1755
3100-Avant-019 12 1944 3130-019 13 1944
Total 57 5832 Total 38 5643
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 21
Figure 11 Mixture study – Identifiler® Kit electropherograms for a 3100-Avant™ instrument before upgrade (Y-scale is constant in each of the upper panels)
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9 FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
Results of a 3100-Avant™-to-3130 Instrument Upgrade
22 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 12 Mixture study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 11) after 3100-Avant™-to-3130 instrument upgrade
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 23
7.1.3 Precision Study
The standard deviation in the size value for each allele of the allelic ladder was calculated within a run (four samples per run) before and after upgrading each of the three instruments:
• All Identifiler® Kit and Profiler Plus® Kit runs before and after upgrade yielded standard deviation values <0.15 bp (Figure 13).
• A small overall increase in the standard deviation was observed after upgrade in the Identifiler® Kit data (0.005 bp) and Profiler Plus® Kit data (0.007 bp) (data not shown).
Figure 13 Precision study – Sizing data for Identifiler® Kit and Profiler Plus® Kit allelic ladders before (Test 1) and after (Test 2) 3100-Avant™-to-3130 instrument upgrade
7.1.4 Resolution Study
The TH01 locus of the Identifiler® Kit allelic ladder was examined to evaluate the resolution and detection of alleles differing in size by 1 base pair. The 9.3 and 10 alleles of TH01 were resolved and individually detected for all injections before and after upgrading all instruments.
7.1.5 Concordance Study
Amplification of the 9947A, 9948, and 007 DNA samples produced correct genotypes for all loci for both the Identifiler® Kit and the Profiler Plus® Kit before and after upgrading all instruments.
Results of a 3100-to-3130xl Instrument Upgrade
24 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.2 Results of a 3100-to-3130xl Instrument Upgrade
7.2.1 Sensitivity Study
The sensitivity study evaluated the following before and after upgrade of each instrument:
• The ability to detect a full genotype profile in a range of DNA input amounts, determined by evaluating the number of alleles that fell below the detection threshold
• Average peak heights• Average heterozygote peak height ratios
Number of AllelesBelow Detection
Threshold
Table 5 shows the number of alleles that fell below the detection threshold before and after upgrading the 3100 instrument to a 3130xl instrument:
• Before upgrade – Fifty six (56) alleles (out of a total of 7218) fell below the detection threshold (Figure 14).
• After upgrade – Four (4) alleles (out of a total of 10330) fell below the detection threshold (Figure 15).
As expected, instrument-to-instrument variation was observed between instruments of the same type before and after upgrade.
Table 5 Sensitivity study – Number of alleles below detection threshold – 3100-to-3130xl instrument upgrade
Test 4 (before upgrade) Test 5 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3100-006 Not included§
3183 3130xl-006 2 3396
3100-015 55 3540 3130xl-015 2 3432
3100-028 1 3678 3130xl-028 0 3502
Total 56 7218 Total 4 10330
‡. Data from failed injections not included§. Instrument 3100-006 injections failed
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 25
Full genotypes were consistently obtained with 250 pg DNA input for all samples amplified with the Identifiler® Kit and Profiler Plus® Kit before and after upgrading the 3100 instrument to a 3130xl instrument. All alleles that fell below the detection limit were obtained from the 125 pg amplification.
Alleles that fall below the threshold can be caused by:• Low DNA input, which results in low overall relative
fluorescence • Stochastic PCR effects, which result in:
– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3100-to-3130xl Instrument Upgrade
26 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 14 Sensitivity study – Identifiler® Kit electropherograms for a 3100 instrument before upgrade (Y-scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
Serial dilutions of 9947A DNA
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
0.125 ng
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 27
Figure 15 Sensitivity study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 14) after 3100-to-3130xl instrument upgrade (Y scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
Serial dilutions of 9947A DNA
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
0.125 ng
Results of a 3100-to-3130xl Instrument Upgrade
28 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Peak Height A comparison of peak heights for each DNA input amount before and after instrument upgrade showed some variation in sensitivity (for a description of how peak heights were evaluated, see page 7):
• Overall, a 8.9% increase in peak heights was observed after upgrading the 3100 instruments to 3130xl instruments.
• Identifiler® Kit data showed a 4.5% increase in overall peak height after upgrade (Figure 16).
• Profiler Plus® Kit data showed a 13.7% increase in overall peak height after upgrade (Figure 17).
Figure 16 Sensitivity study – Comparison of Identifiler® Kit peak heights by input amount for all samples before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 29
Figure 17 Sensitivity study – Comparison of Profiler Plus® Kit peak heights by input amount for all samples before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
HeterozygotePeak Height
Ratio
Heterozygote peak height ratios were calculated and plotted for each DNA sample and concentration in the sensitivity study (for a description of how peak heights were evaluated, see page 7).
Heterozygote peak height ratios for all loci in Identifiler® Kit (Figure 18) and Profiler Plus® Kit (Figure 19) kits averaged 80% or greater before and after upgrading the 3100 to 3130xl instruments.
Heterozygote peak height ratios below the 70% cutoff before and after upgrade occurred mainly with lower DNA input amounts (Figures 20 and 21).
Low heterozygote peak height ratios can be caused by:
• Low DNA input, which results in low overall relative fluorescence
• Stochastic PCR effects, which result in:– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3100-to-3130xl Instrument Upgrade
30 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 18 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
Figure 19 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 31
Figure 20 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by input amount for all samples before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
Figure 21 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by input amount for all samples before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
Results of a 3100-to-3130xl Instrument Upgrade
32 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.2.2 Mixture Study
Mixed DNA profiles were evaluated and the number of alleles falling below 50 RFU was recorded before and after instrument upgrade.
Number of AllelesBelow Detection
Threshold
For a description of how the number of expected alleles was determined, see page 8.
Table 6 shows the number of alleles that fell below the detection threshold before and after upgrading the 3130 to a 3130xl:
• Before upgrade – Twenty one (21) alleles (out of a total of 4536) fell below the detection threshold (Figure 22).
• After upgrade – Forty two (42) alleles (out of a total of 5832) fell below the detection threshold (Figure 23).
The limit of detection of the minor contributor was approximately 9:1 before and after upgrading the 3100 instrument to a 3130xl instrument.
Table 6 Mixture study – Number of alleles below detection threshold – 3100-to-3130xl upgrade (data for 1:1 mixture not included)
Test 4 (before upgrade) Test 5 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3100-006 8 1620 3130xl-006 14 1944
3100-015 0 972 3130xl-015 14 1944
3100-028 13 1944 3130xl-028 14 1944
Total 21 4536 Total 42 5832
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 33
Figure 22 Mixture study – Identifiler® Kit electropherograms for a 3100 instrument before upgrade (Y-scale is constant in each of the upper panels)
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9 FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
Results of a 3100-to-3130xl Instrument Upgrade
34 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 23 Mixture study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 22) after 3100-to-3130xl instrument upgrade
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9 FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 35
7.2.3 Precision Study
The standard deviation in the size value for each allele of the allelic ladder was calculated within a run (sixteen samples per run for the 3100 instrument, 16 samples per run for the 3130xl instrument) before and after upgrading each of the three instruments:
• All Identifiler® Kit and Profiler Plus® Kit runs before and after instrument upgrade yielded standard deviation values <0.15 bp (Figure 24).
• A small overall decrease in the standard deviation was observed after upgrade in the Identifiler® Kit data (0.003 bp) and Profiler Plus® Kit data (0.011 bp) (data not shown).
Figure 24 Precision study – Sizing data for Identifiler® Kit and Profiler Plus® Kit before (Test 4) and after (Test 5) 3100-to-3130xl instrument upgrade
7.2.4 Resolution Study
The TH01 locus of the Identifiler® Kit allelic ladder was examined to evaluate the resolution and detection of alleles differing in size by 1 base pair. The 9.3 and 10 alleles of TH01 were resolved and individually detected for all injections before and after upgrading all instruments.
7.2.5 Concordance StudyAmplification of the 9947A, 9948, and 007 DNA samples produced correct genotypes for all loci for both Identifiler® Kit and Profiler Plus® Kit before and after upgrading.
Results of a 3100-Avant™-to-3130xl Instrument Upgrade
36 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.3 Results of a 3100-Avant™-to-3130xl Instrument Upgrade
For this upgrade path:• Data was collected on an 3100-Avant instrument (Test 1).• The 3100-Avant instrument was upgraded to a 3130 instrument.• The 3130 instrument was upgraded to a 3130xl instrument.• Data was collected on the 3130xl instrument (Test 3).
7.3.1 Sensitivity Study
The sensitivity study evaluated the following before and after upgrade of each instrument:
• The ability to detect a full genotype profile in a range of DNA input amounts, determined by evaluating the number of alleles that fell below the detection threshold
• Average peak heights• Average heterozygote peak height ratios
Number of AllelesBelow Detection
Threshold
Table 7 shows the number of alleles that fell below the detection threshold before and after upgrading the 3100-Avant instrument to a 3130xl instrument:
• Before upgrade – Sixty four (64) alleles (out of a total of 8725) fell below the detection threshold (Figure 25).
• After upgrade – Thirty four (34) alleles (out of a total of 8795) fell below the detection threshold (Figure 26).
As expected, instrument-to-instrument variation was observed between instruments of the same type before and after upgrade.
Table 7 Sensitivity study – Number of alleles below detection threshold – 3100-Avant™-to-3130xl instrument upgrade
Test 1 (before upgrade) Test 3 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50
RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50
RFU)
Total Number of
Alleles‡
3100-Avant-003 22 3678 3130xl-003 34 34133100-Avant-040 5 3082 3130xl-040 0 35903100-Avant-019 37 1965 3130xl-019 0 1792Total 64 8725 Total 34 8795
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 37
Full genotypes were consistently obtained with 250 pg DNA input amounts for all samples amplified with the Identifiler® and Profiler Plus® kits before and after upgrading the 3100-Avant instrument to a 3130xl instrument. All alleles that fell below the detection limit were obtained from the 125 pg amplification.
Alleles that fall below the threshold can be caused by:• Low DNA input, which results in low overall relative
fluorescence • Stochastic PCR effects, which result in:
– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3100-Avant™-to-3130xl Instrument Upgrade
38 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 25 Sensitivity study – Identifiler® Kit electropherograms for a 3100-Avant™ instrument before upgrade (Y-scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
Serial dilutions of 9947A DNA
0.125 ng
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 39
Figure 26 Sensitivity study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 25) after 3100-Avant™-to-3130xl instrument upgrade (Y-scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
Serial dilutions of 9947A DNA
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
0.125 ng
Results of a 3100-Avant™-to-3130xl Instrument Upgrade
40 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Peak Height A comparison of peak heights for each DNA input amount before and after instrument upgrade showed some variation in sensitivity (for a description of how peak heights were evaluated, see page 7):
• Overall, a 28.7% increase in peak heights was observed after upgrading the 3100-Avant instruments to 3130xl instruments.
• Identifiler® Kit data showed a 29.8% increase in overall peak height after upgrade (Figure 27).
• Profiler Plus® Kit data showed a 27.6% increase in overall peak height after upgrade (Figure 28).
Figure 27 Sensitivity study – Comparison of Identifiler® Kit peak heights by input amount for all samples before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 41
Figure 28 Sensitivity study – Comparison of Profiler Plus® Kit peak heights input amount for all samples before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
HeterozygotePeak Height
Ratio
Heterozygote peak height ratios were calculated and plotted for each DNA sample and concentration in the sensitivity study (for a description of how peak heights were evaluated, see page 7).
Heterozygote peak height ratios for all loci in Identifiler® Kit (Figure 29) and Profiler Plus® Kit (Figure 30) kits averaged 80% or greater before and after upgrading the 3100-Avant instruments to 3130xl instruments.
Heterozygote peak height ratios below the 70% cutoff before and after upgrade occurred mainly with lower DNA input amounts (Figures 31 and 32).
Low heterozygote peak height ratios can be caused by:
• Low DNA input, which results in low overall relative fluorescence
• Stochastic PCR effects, which result in:– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3100-Avant™-to-3130xl Instrument Upgrade
42 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 29 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
Figure 30 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 43
Figure 31 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by input amount for all samples before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
Figure 32 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by input amount for all samples before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
Results of a 3100-Avant™-to-3130xl Instrument Upgrade
44 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.3.2 Mixture Study
Mixed DNA profiles were evaluated and the number of alleles falling below 50 RFU was recorded before and after instrument upgrade.
Number of AllelesBelow Detection
Threshold
For a description of how the number of expected alleles was determined, see page 8.
Table 8 shows the number of alleles that fell below the detection threshold before and after upgrading the 3100-Avant instrument to a 3130xl instrument:
• Before upgrade – Thirty five (35) alleles (out of a total of 5832) fell below the detection threshold (Figure 33).
• After upgrade – Thirty seven (37) alleles (out of a total of 5562) fell below the detection threshold (Figure 34).
The limit of detection of the minor contributor was approximately 9:1 before and after upgrading the 3100-Avant instrument to a 3130xl instrument.
Table 8 Mixture study – Number of alleles below detection threshold – 3100-Avant™-to-3130xl instrument upgrade (data for 1:1 mixture not included)
Test 1 (before upgrade) Test 3 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3100-Avant-003 12 1944 3130xl-003 0 1782
3100-Avant-040 33 1944 3130xl-040 13 1863
3100-Avant-019 12 1944 3130xl-019 24 1917
Total 35 5832 Total 37 5562
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 45
Figure 33 Mixture study – Identifiler® Kit electropherograms for a 3100-Avant™ instrument before upgrade (Y-scale is constant in each of the upper panels)
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9 FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
Results of a 3100-Avant™-to-3130xl Instrument Upgrade
46 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 34 Mixture study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 33) after 3100-Avant™-to-3130xl instrument upgrade
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 47
7.3.3 Precision StudyThe standard deviation in the size value for each allele of the allelic ladder was calculated within a run (four samples per run for the 3100-Avant instrument, 16 samples per run for the 3130xl instrument) before and after upgrading each of the three instruments
• All Identifiler® Kit and Profiler Plus® Kit runs before and after instrument upgrade gave standard deviation values <0.15 bp (Figure 35).
• A small overall increase in the standard deviation was observed after upgrade in the Identifiler® Kit data (0.045 bp) and Profiler Plus® Kit data (0.030 bp) (data not shown).
Figure 35 Precision study – Sizing data for Identifiler® Kit and Profiler Plus® Kit before (Test 1) and after (Test 3) 3100-Avant™-to-3130xl instrument upgrade
7.3.4 Resolution Study
The TH01 locus of the Identifiler® Kit allelic ladder was examined to evaluate the resolution and detection of alleles differing in size by 1 base pair. The 9.3 and 10 alleles of TH01 were resolved and individually detected for all injections before and after upgrading all instruments.
7.3.5 Concordance Study
Amplification of the 9947A, 9948, and 007 DNA samples produced correct genotypes for all loci for both the Identifiler® Kit and the Profiler Plus® Kit before and after upgrading all instruments.
Results of a 3130-to-3130xl Instrument Upgrade
48 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.4 Results of a 3130-to-3130xl Instrument Upgrade
7.4.1 Sensitivity Study
The sensitivity study evaluated the following before and after upgrade of each instrument:
• The ability to detect a full genotype profile in a range of DNA input amounts, determined by evaluating the number of alleles that fell below the detection threshold
• Average peak heights• Average heterozygote peak height ratios
Number of AllelesBelow Detection
Threshold
Table 9 shows the number of alleles that fell below the detection threshold before and after upgrading the 3130 instrument to a 3130xl instrument:
• Before upgrade – Forty one (41) alleles (out of a total of 9851) fell below the detection threshold (Figure 36).
• After upgrade – Thirty four (34) alleles (out of a total of 8795) fell below the detection threshold (Figure 37).
As expected, instrument-to-instrument variation was observed between instruments of the same type before and after upgrade.
Table 9 Sensitivity study – Number of alleles below detection threshold – 3130-to-3130xl instrument upgrade
Test 2 (before upgrade) Test 3 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3130-003 2 3257 3130xl-003 34 3413
3130-040 9 3264 3130xl-040 0 3590
3130-019 30 3330 3130xl-019 0 1792
Total 41 9851 Total 34 8795
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 49
Full genotypes were consistently obtained with 250 pg DNA input for all samples amplified with the Identifiler® Kit and Profiler Plus® Kit before and after upgrading the 3130 instrument to a 3130xl instrument. All alleles that fell below the detection limit were obtained from the 125 pg amplification.
Alleles that fall below the threshold can be caused by:• Low DNA input, which results in low overall relative
fluorescence • Stochastic PCR effects, which result in:
– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3130-to-3130xl Instrument Upgrade
50 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 36 Sensitivity study – Identifiler® Kit electropherograms for a 3130 instrument before upgrade (Y-scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
Serial dilutions of 9947A DNA
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
0.125 ng
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 51
Figure 37 Sensitivity study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 36) after 3130-to-3130xl instrument upgrade (Y scale is constant in each of the upper panels)
1.5 ng
1.0 ng
0.5 ng
0.25 ng
0.125 ng
Serial dilutions of 9947A DNA
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
0.125 ng
Results of a 3130-to-3130xl Instrument Upgrade
52 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Peak Height A comparison of peak heights for each DNA input amount before and after instrument upgrade showed some variation in sensitivity (for a description of how peak heights were evaluated, see page 7):
• Overall, a 27.4% increase in peak heights was observed after upgrading the 3130 to 3130xl instruments.
• Identifiler® kit data showed a 32.7% increase in overall peak height after upgrade (Figure 38).
• Profiler Plus® Kit data showed a 19.5% increase in overall peak height after upgrade (Figure 39).
Figure 38 Sensitivity study – Comparison of Identifiler® Kit peak heights by DNA input amount for all samples before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 53
Figure 39 Sensitivity study – Comparison of Profiler Plus® Kit peak heights by DNA input amount for all samples before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
HeterozygotePeak Height
Ratio
Heterozygote peak height ratios were calculated and plotted for each DNA sample and concentration in the sensitivity study (for a description of how peak heights were evaluated, see page 7). Heterozygote peak height ratios for all loci in Identifiler® Kit (Figure 40) and Profiler Plus® Kit (Figure 41) kits averaged 80% or greater before and after upgrading the 3130 instruments to 3130xl instruments.
Heterozygote peak height ratios below the 70% cutoff before and after upgrade occurred mainly with lower DNA input amounts (Figures 42 and 43).
Low heterozygote peak height ratios can be caused by:
• Low DNA input, which results in low overall relative fluorescence
• Stochastic PCR effects, which result in:– Peak imbalance between the heterozygote alleles – Allele dropout
Results of a 3130-to-3130xl Instrument Upgrade
54 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 40 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
Figure 41 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by locus for all samples and DNA input amounts before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 55
Figure 42 Sensitivity study – Comparison of Identifiler® Kit heterozygote peak height ratio values by DNA input amount for all samples before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
Figure 43 Sensitivity study – Comparison of Profiler Plus® Kit heterozygote peak height ratio values by DNA input amount for all samples before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
Results of a 3130-to-3130xl Instrument Upgrade
56 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.4.2 Mixture Study
Mixed DNA profiles were evaluated and the number of alleles falling below 50 RFU was recorded before and after instrument upgrade.
Number of AllelesBelow Detection
Threshold
For a description of how the number of expected alleles was determined, see page 8.
Table 10 shows the number of alleles that fell below the detection threshold before and after upgrading the 3130 to a 3130xl:
• Before upgrade – Forty one (41) alleles (out of a total of 9851) fell below the detection threshold (Figure 44).
• After upgrade – Thirty four (34) alleles (out of a total of 8795) fell below the detection threshold (Figure 45).
The limit of detection of the minor contributor was approximately 9:1 before and after upgrading the 3130 to a 3130xl.
Table 10 Mixture study – Number of alleles below detection threshold – 3130-to-3130xl upgrade (data for 1:1 mixture not included)
Test 2 (before upgrade) Test 3 (after upgrade)
Instruments and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
Upgraded Instruments
and ID Numbers
Number of Alleles Below
Detection Threshold (50 RFU)
Total Number of
Alleles‡
3130-003 2 3257 3130xl-003 34 3413
3130-040 9 3264 3130xl-040 0 3590
3130-019 30 3330 3130xl-019 0 1792
Total 41 9851 Total 34 8795
‡. Data from failed injections not included
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 57
Figure 44 Mixture study – Identifiler® Kit electropherograms for a 3130 instrument before upgrade (Y-scale is constant in each of the upper panels)
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9FGA 26
Results of a 3130-to-3130xl Instrument Upgrade
58 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
Figure 45 Mixture study – Identifiler® Kit electropherograms generated on the same instrument (see Figure 44) after 3130-to-3130xl instrument upgrade
1:0
1:1
4:1
9:1
0:1
Mixtures9947A:9948(female:male)
9:1 mixture – Alleles from the minor contributor highlighted
TPOX 9FGA 26
GeneMapper® ID Software v3.2 Detection Threshold 50 RFU
7 Results and Discussion
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 59
7.4.3 Precision Study
The standard deviation in the size value for each allele of the allelic ladder was calculated within a run (four samples per run for the 3130 instrument, 16 samples per run for the 3130xl instrument) before and after upgrading each of the three instruments:
• All Identifiler® Kit and Profiler Plus® Kit runs before and after instrument upgrade yielded standard deviation values <0.15 bp (Figure 46).
• A small overall increase in the standard deviation was observed after upgrade in the Identifiler® Kit data (0.040 bp) and Profiler Plus® Kit data (0.023 bp) (data not shown).
Figure 46 Precision study – Sizing data for Identifiler® Kit and Profiler Plus® Kit allelic ladders before (Test 2) and after (Test 3) 3130-to-3130xl instrument upgrade
7.4.4 Resolution Study
The TH01 locus of the Identifiler® Kit allelic ladder was examined to evaluate the resolution and detection of alleles differing in size by 1 base pair. The 9.3 and 10 alleles of TH01 were resolved and individually detected for all injections before and after upgrading all instruments.
Results of a 3130-to-3130xl Instrument Upgrade
60 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
7.4.5 Concordance Study
Amplification of the 9947A, 9948, and 007 DNA samples produced correct genotypes for all loci for both the Identifiler® Kit and the Profiler Plus® Kit before and after upgrading all instruments.
8 ConclusionThe performance of the upgraded 3100-Avant™ and 3100 instruments to 3130 and 3130xl instruments respectively is consistent with the expected performance of the previously validated 3100-Avant™ and 3100 platforms.
9 ReferencesScientific Working Group on DNA Analysis Methods. Revised validation guidelines (approved July 2003). Available at: http//www.fbi.gov/hq/lab/fsc/backissu/july2004/standards/2004_03_standards02.htm.
DNA Advisory Board. Quality assurance standards for forensic DNA testing laboratories (approved October 1998). Forensic Science Communications 2000;2(3). Available at: http//www.fbi.gov/programs/lab/fsc/backissu/july2000/codispre.htm.
Applied Biosystems Quantifiler® Kit User’s Manual, PN 4344790, Rev. B, Applied Biosystems, 11/2003.
User Bulletin ABI PRISM® 3100/3100-Avant™ Genetic Analyzers Using Data Collection v2.0: Protocols for Processing AmpFlSTR® PCR Amplification Kit PCR Products, PN 4350218, Rev. A., 12/2003.
User Bulletin Applied Biosystems 3130/3130xl Genetic Analyzers Using Data Collection Software v3.0, PN 4363787, Rev. A, 02/2005.
ABI PRISM® 3100 Genetic Analyzer User's Manual, PN 4315834, Rev. B., 01/2001.
9 References
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 61
Results of a 3130-to-3130xl Instrument Upgrade
62 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin
9 References
Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin 63
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07/2006
Part Number 4376468 Rev. AStock Number 106UB49-01
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