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Development and Validation of the 3500 Series Genetic Analyzer for Human Identification
Jeff SailusForensic Science Applications Group
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Development of a Next Generation Genetic AnalyzerA Whole System Approach
New Laser and Polymer Pump
Block
Redesigned Consumables with Tracking
Real-time System and Consumable
Information
Predefined Protocols and HID
Templates
Improved Temperature
Control Shorter Run Times
Optional Signal Normalization
Data Analysis QC Tools and
Flags
Simplified Reinjection
Scheme
Instrument Hardware
Consumables
Data Collection
QC Analysis
GMID-X v1.2
ID-X 1.1.1 Upgrade Supports Windows XP
and Vista
Analyzes .fsa & New .hid File Format
Consumable Data Tracking
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Improved Data Quality and Sample Throughput
Smaller Oven
More Consistent Migration for Better
Sizing Precision
Faster Run Time Higher Throughput
Flat Oven Door Seal and New Locking
Mechanism
Detection cell heater
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Instrument Setup and Performance
Elimination of Lower Polymer Block and Polymer Delivery
Tubing
Reduced Polymer WasteDirect Channels
Promote Efficient Polymer Flow
Redesigned Array Port
Minimized Potential for Bubbles and Leaks
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Instrument Setup and Performance
• Pre-Filled, Quality-Controlled Reagents
• Information Recorded via RFID• Lot numbers• Part Numbers• Serial numbers• Dates (expiration and installation)• Capacity/Usage
• Per Sample Running Cost Comparable to 31xx
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Data Collection: QC Analysis Tools for Preliminary Data Analysis
Review data quality flags, electropherograms and sizing tables - Select samples for reinjection
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3500 Developmental Validation Studies
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Developmental 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
• 4 Dye Amplification Kits do not use the normalization functionality
• Polymer and Array• POP-4™ and 36 cm Capillary Array
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Developmental Validation ScopeSample Summary
Test Name Samples Input Replicates
Genotype Concordance and Reproducibility
• 40 male and 42 female gDNA samples• 4 racial groups• Controls
1ng 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
Kit dependent 0.125ng, 0.25ng, 0.5ng, 0.75, 1ng, 6ng
• 5 replicates for DNA samples• 3 replicates for NTC• 4 injections
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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Validation ScopeAmpFℓSTR® Kits
• Identifiler®
• Minifiler™
• SGM Plus®
• Yfiler®
• Sinofiler™
• SEfiler Plus™
• Profiler Plus®
• Cofiler®
• Identifiler® Direct• Identifiler® Plus• NGM™
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Precision and Accuracy Studies
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3500 Sizing Precision Study:Within an Injection of Identifiler Allelic Ladder
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3500 Sizing Precision Study:Across Multiple Injections of Identifiler Allelic Ladder Samples
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Resolution and Baseline Evaluation
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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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Baseline Noise - Developmental Evaluation G5 Average Noise (PAT=Avg 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 Studies were then used to confirm the level of pull up and an optimal peak amplitude threshold setting.
175 RFU
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Introduction to Normalization
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Normalization: What is it?
A method to attenuate signal variations associated with instrument, capillary array, sample salt load, and injection variability
• Factory Standardization: Hardware-based calibration to help enable more consistent instrument to instrument performance
• (Optional) Internal Standard Normalization:Chemistry and software based method to help enable more consistent performance across injections and instruments
• GeneScan™ 600 LIZ® Size Standard v2.0• 3500 Data Collection and GMID-X v1.2
Before normalization
After normalization
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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
Factory + Internal Standard
Normalization greatest reduction
in variation
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Sensitivity and Internal Standard Normalization Studies
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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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Size Standard Peak Height Consistency Study: Five AmpFℓSTR® PCR Amplification Kits GeneScan™ 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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Size Standard Peak Height Consistency Study:Injection to Injection Peak Height Consistency
% 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
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Size Standard Peak Height Consistency Study:%CV for 5 Different AmpFℓSTR® KitsGeneScan™ 600 LIZ® Size Standard v2.0
% CV
Factory + Internal Std Normalization · 6 instruments · 3 lots of size standard
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Additional Developmental Validation Studies
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Additional Developmental Studies
• Genotype Concordance• 100% genotype concordance with samples run on the 3130xL
• GeneMapper® ID-X v1.2 Verification• 100% Concordant with historical data set collected from legacy CE
instruments using a variety of AmpFℓSTR® kits analyzed and GeneMapper® ID-X v1.1 software versions (including mixtures)
• Normalization and RFID documentation functionality performed as expected
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In Summary• 100% genotype concordance between the 3500(xl) and 3130xl instrument and
GMID-X software
• Average Sizing precision data with GeneScan™ 600 LIZ® Size Standard v2.0 was 0.05 or less within an injections and across injections
• Linear relationship between input amounts and peak heights, with average peak heights ~6,000 rfu for 1ng input reactions Identifiler® Plus and NGMTM
• Internal Standard Normalization significantly reduced injection-to-injection and instrument-to-instrument variation, but not the absolute values of the allele peak heights
• No reproducible PCR artifacts were observed within the read regions - spectral pull up of less than 5% was observed
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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 your Applied Biosystems representative.
Legal
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Thank you!
www.appliedbiosystems.com/3500HID