3130 Series Performance Verification User Bulletin (PN ...

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


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


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


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


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


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


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


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


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


7.1 Results of a 3100-Avant™-to-3130 Instrument Upgrade

7.1.1 Sensitivity Study




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



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



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



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



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



0.125 ng



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



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:





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



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



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.


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)





Total Number of

Alleles‡


and ID Numbers



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




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




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



TPOX 9FGA 26




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


7.2 Results of a 3100-to-3130xl Instrument Upgrade






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).


Table 5 Sensitivity study – Number of alleles below detection threshold – 3100-to-3130xl instrument upgrade





Total Number of

Alleles‡


and ID Numbers



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



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



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



0.125 ng



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



0.125 ng



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



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


Ratio


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.







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



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



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.


Threshold


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)





Total Number of

Alleles‡


and ID Numbers



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




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



TPOX 9 FGA 26




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



TPOX 9 FGA 26





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



7.2.5 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


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).






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).


Table 7 Sensitivity study – Number of alleles below detection threshold – 3100-Avant™-to-3130xl instrument upgrade




Detection Threshold (50

RFU)

Total Number of

Alleles‡


and ID Numbers


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




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.






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



0.125 ng



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



0.125 ng




• 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).


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



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


Ratio


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.







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



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



7.3.2 Mixture Study



Threshold


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)





Total Number of

Alleles‡


and ID Numbers



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




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



TPOX 9 FGA 26




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



TPOX 9FGA 26




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).


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







7.4 Results of a 3130-to-3130xl Instrument Upgrade






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).



Table 9 Sensitivity study – Number of alleles below detection threshold – 3130-to-3130xl instrument upgrade





Total Number of

Alleles‡


and ID Numbers



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




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.






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



0.125 ng



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



0.125 ng




• 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).


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



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


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.







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



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



7.4.2 Mixture Study



Threshold


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).


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)





Total Number of

Alleles‡


and ID Numbers



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




Figure 44 Mixture study – Identifiler® Kit electropherograms for a 3130 instrument before upgrade (Y-scale is constant in each of the upper panels)


1:0

1:1

4:1

9:1

0:1



TPOX 9FGA 26



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



TPOX 9FGA 26





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).


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







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


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Information in this document is subject to change without notice.

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AB (Design), Applied Biosystems, ABI PRISM, AmpFlSTR, GeneMapper, Identifiler, ProFiler Plus, and Quantifiler are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries.

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07/2006

Part Number 4376468 Rev. AStock Number 106UB49-01

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