hid validation: the 3500 series genetic...
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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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