hid validation: the 3500 series genetic...

HID Validation: The 3500 Series Genetic Analyzers

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HID Validation: The 3500 Series Genetic Analyzers

Outline

• Scope• Materials and Reagents

• Validation Elements• Selection of Validation Data

• Additional Sources of Validation Information• User Bulletin

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HID Validation: The 3500 Series Genetic Analyzers

Validation ScopeHardware and Reagents

• Instruments• (3) 8 Capillary Instruments (3500)• (3) 24 Capillary Instruments (3500xL)

• Size Standard• GeneScan™ 600 LIZ® Size Standard v2.0• GeneScan™ 500 ROX™ Size Standard

• Profiler Plus® / Cofiler® do not use normalization

• Polymer and Array• POP-4™ and 36 cm Capillary Array

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HID Validation: The 3500 Series Genetic Analyzers

Validation ScopeSample Summary

Test Name Samples Input Replicates

Genotype Concordance and Reproducibility

•40 male and 42 female gDNA samples•4 racial groups•Controls

1-2 ng total DNA •1 replicate per sample•2 injections

Sizing Precision and Accuracy

Allelic ladder 1 µL per well •See below

Sensitivity and Normalization

•007 control DNA•3 gDNA samples

•STR: 0.125ng, 0.5ng, 1ng, 6ng

•5 replicates for DNA samples•3 replicates for NTC•4 injections•Minifiler: 0.125ng,

0.25ng, 0.5ng, .75ng

Mixture Analysis •4 pairs of male and female gDNA•Mixture ratios•1:0, 1:1, 1:3, 1:5, 1:9, and 0:1

•Recommended concentrations per kit

1 replicate per sample

Resolution •Allelic Ladder•Research material

•1 µL per well 24 replicates

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HID Validation: The 3500 Series Genetic Analyzers

Validation ScopeAmpFℓSTR® Kits

• Identifiler®

• Minifiler™

• SGM Plus®

• Yfiler®

• Sinofiler™

• SEfiler Plus™

• Profiler Plus®

• Cofiler®

• Identifiler® Direct• Identifiler® Plus• NGM™

Precision and Accuracy

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HID Validation: The 3500 Series Genetic Analyzers

Precision and AccuracyExample Configurations for Standard Deviation

Within Injection - 3500

• 8 data points per mean• 12 injections per plate• 12 columns

• 3 Instruments

• 12 data points per mean• 4 injections per plate• 8 rows

• 3 Instruments

Across Injections – 3500xL

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HID Validation: The 3500 Series Genetic Analyzers

3500 Sizing Precision Study:Within an Injection of Identifiler® Allelic Ladder

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HID Validation: The 3500 Series Genetic Analyzers

3500 Sizing Precision Study:Across Multiple Injections of Identifiler® Allelic Ladder Samples

96 Replicates of Identifiler® Allelic Ladder

3500 -12 Injections3500xL - 4 Injections

Introduction to Normalization

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HID Validation: The 3500 Series Genetic Analyzers

Sources of CE Variation (3100)

• Numerous optical components, capillary array

• Electrophoresis, injection

• Sample and sample preparation

Raman Intensity, 3100file: INSTRUMENT DATA Mod2B.xls

y = -2.7202x + 6042.8R2 = 0.0059

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 20 40 60 80 100 120 140

Instrument

A/D

Units

Series1Linear (Series1)

median= 6026.50Stdev = 808.54Average = 6054.11Stdev/ average = 0.13

Almost 2-fold difference in signal from over 100 instruments from the factory floor

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HID Validation: The 3500 Series Genetic Analyzers

Normalization: What is it?

• This feature normailzes signal variations associated with instrument, capillary array, sample salt load, and injection variability among capillaries and instruments

• Normalization can be applied during primary analysis of the data

Before normalization After normalization

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HID Validation: The 3500 Series Genetic Analyzers

• Normalization Target (NT)• The expected average RFU for eleven peaks in the

GeneScan 600 LIZ® v2 size standard used for normalization

• Normalization Factor• Normalization Target divided by the Average peak height of

the subset of peaks in the GeneScan 600 LIZ® v2 size standard used for normalization

• Normalization Factor Threshold• The passing range for Normalization Factor (default range is

0.3 to 3.0)

Normalization: Definitions

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HID Validation: The 3500 Series Genetic Analyzers

Sources of Variation by Normalization Method

No NormFactory only

Int Std onlyFactor+IntStd

Injection to Injection

Cap to Cap

Within

Instrument to Instrument

Total

39%

33%

25%

14%

31%

24% 24%

13%

20%19%

5%5%

12%11%

4%4%3%

3%

0%0%

0%

5%

10%

15%

20%

25%

30%

35%

40%

%CV

Levels of Normalization

Sources of Variation

• Instrument to Instrument largest source of variation

• Internal standard Normalization greatest reduction in variation

Sensitivity and GeneScan™ 600 LIZ® Size Standard v2.0

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HID Validation: The 3500 Series Genetic Analyzers

Sensitivity Study:Average Peak Height across a Range of DNA Input Amounts AmpFℓSTR®

MiniFilerTM PCR Amplification Kit

Normalization MethodFactory OnlyFactory + Internal Std

0 750 50 250 125

H1126

H1152

H1161 OO7

10000

5000

15000

00.125ng 0.25ng 0.50ng 0.75ng

10000

5000

15000

0

0.125ng 0.25ng 0.50ng 0.75ngGenomic DNA #2

Control DNA Standard 007

Genomic DNA #1

Genomic DNA #3

Peak

Hei

ght

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HID Validation: The 3500 Series Genetic Analyzers

Sensitivity Study:Average Peak Height by Dye for 1ng DNA Input AmpF ℓSTR® Identifiler®

PCR Amplification Kit

2000

4000

8000

10000

6000

12000

14000

Internal Standard Normalization

20002000

Disabled Enabled

Peak

Hei

ght

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HID Validation: The 3500 Series Genetic Analyzers

Size Standard Peak Height Consistency Study:Five AmpFℓSTR® PCR Amplification KitsGeneScan™ 600 LIZ® Size Standard v2.0

6000

5000

4000

3000

2000

1000

0

Lot 1 Lot 2 Lot 3

GS600 Peak Height

Identifiler®Identifiler® DirectIdentifiler® PlusMinifilerTM

NGMTM

7000

GS600 Lot Number

Peak

Hei

ght

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HID Validation: The 3500 Series Genetic Analyzers

Size Standard Peak Height Consistency Study:%CV for 5 Different AmpFℓSTR® KitsGeneScan™ 600 LIZ® Size Standard v2.0

% CV

Size Standard Peak Height Consistency · 6 instruments · 3 lots of size standard

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HID Validation: The 3500 Series Genetic Analyzers

Internal Standard Normalization: Injection to Injection Peak Height ConsistencyGeneScan™ 600 LIZ® Size Standard v2.0

% CV

Normalization MethodFactory OnlyFactory + Internal Std

40

30

20

10

0Lot 1 Lot 2 Lot 3

Size Standard Peak Height Consistency · 6 instruments · 3 lots of size standard

Resolution and Baseline Evaluation

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HID Validation: The 3500 Series Genetic Analyzers

Resolution

• SEfiler Plus™ (SE33 triplet) and Identifiler® (TH01 9.3/10) Allelic Ladders

• 160 injections per instrument on 6 instruments

• Research STR markers greater than 300 base pairs were also developed with single base pair differences (data not shown)

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HID Validation: The 3500 Series Genetic Analyzers

Baseline Noise - Developmental Evaluation G5 Average Noise (10x SD)

0

20

40

60

80

100

120

140

160

180

200

FAM VIC NED PET LIZ TOTAL

10x

Ave

rage

Std

. Dev

.

Data generated during the Sensitivity experiment was then used to confirm the level of pull up and the peak amplitude threshold setting.

175 RFU

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HID Validation: The 3500 Series Genetic Analyzers

Pull-up360 Samples at 6 ng template

5.0%

20000

4000

8000

12000

16000

24000

Additional Studies and Validation Documentation

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HID Validation: The 3500 Series Genetic Analyzers

Identifiler® Direct

• 82 gDNA, 25 cycles• Three 3500xl and three 3500 instruments.• Mother plate split into 6 daughter plates.

8000

8000

8 Capillary 3500

24 Capillary 3500xL

14000

14000

0

0

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HID Validation: The 3500 Series Genetic Analyzers

Additional Studies

• Genotype Concordance• 100% Concordant with 3130xL

• GeneMapper® ID-X v1.2 Evaluation• Normalization and RFID documentation functionality performed as

expected• Average Mx values obtained in the Mixture Study were similar to

the 3130xl

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HID Validation: The 3500 Series Genetic Analyzers

Validation Documentation

• AAFS and ISHI Posters

• Bode Conference Presentations

• User Bulletin containing developmental validation in progress

• www.appliedbiosystems.com/3500hid

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HID Validation: The 3500 Series Genetic Analyzers

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

©2009 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

For those who require IVD-marked devices, the 3500 Dx and 3500xL Dx Genetic Analyzers and system accessories meet the requirements of the In Vitro Diagnostic Medical Devices Directive (98/79/EC). The 3500 Dx and 3500xL Dx systems are for distribution and use in specific European countries only. For more information about the 3500 Dx Series Systems, contact yourApplied Biosystems representative.

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