Thermo Fisher Scientific Performance Verification After Owner's manual

Brand: Thermo Fisher Scientific

Model: Performance Verification After

Type: Owner's manual

User Bulletin

Applied Biosystems 3130 Series Genetic Analyzers

July 2006

SUBJECT: Performance Verification After Upgrade from

ABI PRISM® 3100 Series Genetic Analyzers

In This User

Bulletin

This user bulletin includes the following topics:

1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Before Upgrading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

3.2 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

4 Experimental Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5 Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

6 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

7.1 Results of a 3100-Avant-to-3130 Instrument Upgrade . . . . . . . . .12

7.2 Results of a 3100-to-3130xl Instrument Upgrade. . . . . . . . . . . . .24

7.3 Results of a 3100-Avant-to-3130xl Instrument Upgrade . . . . . . .36

7.4 Results of a 3130-to-3130xl Instrument Upgrade. . . . . . . . . . . . .48

8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

9 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

2 Applied Biosystems 3130 Series Genetic Analyzers Performance Verification User Bulletin

1 Introduction

Instrument

Upgrade 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:

Instrument

Components

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 AmpF

STR PCR

Amplification Kit PCR Products User Bulletin (PN 4363787).

Performance

Verification

Studies

Performed

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 Upgrading

IMPORTANT! 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 Reagents

To 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 Instruments

Three 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

Tes t 1

Upgrade to

3100-Avant-040

3100-Avant-019

3130-003

Tes t 2

3130-040

3130-019

3130xl-003

Tes t 3

3130xl-040

3130xl-019

3100-006

Tes t 4

3100-015

3100-028

3130xl-006

Tes t 5

3130xl-015

3130xl-028

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)

Tes t In s tr u m en t Instrument ID

Numbers Purpose

13100-Avant™ before

upgrade

003, 040, and 019 To generate baseline data on the

3100-Avant Genetic Analyzer before

upgrade.

23130:

• After upgrade from

3100-Avant

and

• 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.

33130xl 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.

43100 before upgrade 006, 015, and 028 To generate baseline data on the

3100 Genetic Analyzers before upgrade.

53130xl 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 Kit

50 4303326

4Experimental 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 0.00

1:1 0.75 0.75

4:1 1.20 0.30

9:1 1.35 0.15

0:1 0.00 1.50

AmpFlSTR

Profiler Plus

PCR

Amplification

Kit

1:0 3.00 0.00

1:1 1.50 1.50

4:1 2.40 0.60

9:1 2.70 0.30

0:1 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

1base pair.

Concordance

Study

The genotype results from the sensitivity study were evaluated for

concordance.

5 Data Collection

Data 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 2.0

040 1.0

019 1.0

3100 006 1.1

015 1.1

028 2.0

3130 003, 040, and 019 3.0

3130xl 003, 040, 019,

006, 015, and 028

3.0

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 Data

Compiling 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 Data

Analysis

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 Discussion

Electropherograms 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 Alleles

Below 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)

Tot al

Number of

Alleles‡

Upgraded

Instruments

and ID

Numbers

Number of

Alleles

Below

Detection

Threshold

(50 RFU)

Tot al

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

Heterozygote

Peak 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 Alleles

Below 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)

Tot al

Number of

Alleles‡

Upgraded

Instruments

and ID

Numbers

Number of

Alleles

Below

Detection

Threshold

(50 RFU)

Tot al

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

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Thermo Fisher Scientific Performance Verification After Owner's manual

Brand: Thermo Fisher Scientific

Model: Performance Verification After

Type: Owner's manual

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