Development of a Global STR Multiplex for Human Identification Analysis
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1 Development of a Global STR Multiplex for Human Identification Analysis ISHI 2012 Dennis Wang Sr. Staff Scientist Human Identification Group 1
2 Agenda Why a Global Multiplex? Multiplex Configuration Strategy Application-Specific Development and Testing Results Direct amplification for single source samples Casework samples Summary 2
3 Why a Global Multiplex? New CODIS Recommendations Expanded ESSL Missing Persons Investigations Why? Increase international data compatibility Increase discrimination power Reduce the likelihood of adventitious matches How? Increase the number of loci Incorporate loci contained in global databases 3
4 What is GlobalFiler? Locus CODIS (Current) Europe CODIS (Proposed) GlobalFiler Kit D13S317 X X X D7S820 X X X D5S818 X X X CSF1PO X X X D1S1656 X X X D12S391 X X X D2S441 X X X D10S1248 X X X D18S51 X X X X FGA X X X X D21S11 X X X X D8S1179 X X X X The GlobalFiler Kits are our response to the worldwide expansion of forensic locus sets, recently captured in the CODIS Core Loci Working Group s recommendation to expand the CODIS Core Loci from 13 to 20 required loci and 3 highly recommended loci VWA X X X X D16S539 X X X X TH01 X X X X D3S1358 X X X X AMEL X X X X D2S1338 X X X X D19S433 X X X X DYS391 X X TPOX X X X D22S1045 X X X 1. D.R. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012), e52-e D.R. Hares, Addendum to expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012), e135. Life Technologies SE33 4 X X X
5 5-Dye Configuration Containing All Required and Highly Recommended Loci Primer Redesigns less concordant Fewer Mini-STRs poor performance for degraded DNA Multiple loci > 400 bp poor performance for degraded DNA poor sizing and resolution 5
6 The GlobalFiler Kits: 6-Dye Configuration Only 2 loci require primer redesign Mini-STRs Combination PI*= 5.73 x GlobalFiler Kit 10 Mini-STRs PI* = 9.2 x MiniFiler Kit 8 Mini-STRs PI* = 1.05 x % of alleles in allelic ladder <400bp 6 * Preliminary calculations based on a US Caucasian Database
7 The GlobalFiler Kits: Configuration Y indel Amel DYS391 All gender markers located in the same dye channel for ease of interpretation p Amelogenin q DYS391 / Y indel Deletion Insertion 7
8 The GlobalFiler Kits Allelic Ladder Improvements D2S1338 GlobalFiler Kit range: D19S433 GlobalFiler Kit range: Identifiler Kit range: NGM SElect Kit range:
9 The GlobalFiler Kits: Allelic Ladder 31 alleles added to 9 GlobalFiler Kit marker ladders New virtual bins added to improve genotyping efficiency 343 alleles 246 virtual bins 589 total alleles 9
10 D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vwa TPOX D18S51 D5S818 FGA D10S1248 D22S1045 D2S441 D1S1656 D12S391 SE33 Maximizing Discrimination Kit PI Identifiler Kit X X X X X X X X X X X X X X X 5.01 x NGM SElect Kit X X X X X X X X X X X X X X X X 2.35 x GlobalFiler Kit X X X X X X X X X X X X X X X X X X X X X *7.12 x GlobalFiler Mini s X X X X X X X X X X *9.20 x * Preliminary calculations based on a US Caucasian Database The GlobalFiler Kits will: Deliver a new level of discrimination Reduce the risk of adventitious matches Facilitate international DNA profile comparisons The GlobalFiler Kits offer a power of discrimination times greater than the total population that has ever lived on Earth! 10
11 GlobalFiler Kits: Instrument Compatibility GeneAmp 9700 Thermal Cycler with Silver or Gold-Plated Silver 96-Well Block Veriti Thermal Cycler The GlobalFiler Kits utilize the DS36 (J6) Matrix Standard Set 3130 Series Genetic Analyzers 3500 Series Genetic Analyzers 3730 Genetic Analyzer (GlobalFiler Express Kit only) GeneMapper ID-X Software v1.4 11
12 GlobalFiler Kit Instrument Compatibility 3500xL 3130xl 12
13 Kit Development Strategy The GlobalFiler Kits GlobalFiler PCR Amplification Kit GlobalFiler Express PCR Amplification Kit A high performance, 6-dye chemistry for casework samples An ultra-high throughput, 6- dye chemistry for single source samples 13
14 The Diverse Demands of Forensic Testing Databasing The Samples Casework Predictable Unpredictable Standardized collection Highly variable substrates High yield, high quality DNA Low yield, low quality DNA Single source Mixed source Fresh Aged Key Workflow Requirements Medium - high throughput Low - medium throughput High, 1st pass success rate Minimal sample transfer and processing steps Maximum results from compromised samples High sensitivity Eliminate rate limiting steps Maximum specificity Automated processing Simplified interpretation 14
15 GlobalFiler Express Kit: Workflow Concepts 15
16 Optimizing Reaction Time for the GlobalFiler Express Kit Protocols developed on Veriti thermal cyclers using 100% ramp speed Performance verified on the 9700 thermal cyclers using MAX mode PCR Conditions 95 C/ 1 min 94 C/ 3 sec 60 C/ 30 sec x 60 C/ 8 min Identifiler Direct Kit NGM SElect Express Kit GlobalFiler Express Kit Total Time 2 hr 35 min 45 min min 16
17 Optimizing Reaction Volume for the GlobalFiler Express Kit Success Rate 25 µl volume 15 µl volume Bode Buccal DNA Collector Blood on FTA 10/10 10/10 Bode DNA Collector 19/19 19/19 Copan 4N6FLOQSwab 11/11 11/11 15 μl 25 μl Buccal on Copan FLOQSwab Blood on FTA Performance and profile quality were maintained at 15 μl with a shorter and faster amplification time 15 μl 25 μl μl 25 μl
18 GlobalFiler Express Kit Population Study Sample Type N PCR Cycle Number 1 st Pass Success Rate (175 RFU) Blood on FTA % Blood on Bloodstain Card (903 Paper) % Buccal on FTA (EasiCollect Device) % Buccal on Bode Buccal Collector % Buccal on Copan 4N6FLOQSwab % GlobalFiler Express Kit External Testing Test Site Sample Type N PCR Cycle Number 1 st Pass Success Rate (175 RFU) A Blood & Buccal on FTA % B Buccal on FTA % C Buccal on FTA % D Buccal on FTA % E Buccal on FTA and Swabs 40 26/ % Total % 18
19 GlobalFiler Kit: Casework Design Considerations Casework Kit Performance Requirements: High sensitivity for trace/low level DNA samples Good intracolor balance for mixture samples Sufficient tolerance to PCR inhibitors Maintain high species specificity Efficient thermal cycling conditions 20
20 Optimization of GlobalFiler Kit Reaction Conditions Reagent Volumes 3.3X Master Mix 7.5 µl 10X Primer Set 2.5 µl Sample Input 15.0 µl 25.0 µl PCR Conditions 95 C/ 1 min 94 C/ 20 sec 59 C/ 90 sec 60 C/ 10 min 29 x Greater flexibility of sample input volume to facilitate analysis of low level samples Optimized thermal cycling conditions shorten amplification conditions by > 1 hr Identifiler Plus & NGM SElect Kits GlobalFiler Kit Total Time 2 hr 25 min 1 hr 20 min 21
21 GlobalFiler Kit Performance Example: Sensitivity [DNA] 1 ng 500 pg 250 pg 125 pg 62.5 pg pg pg GlobalFiler Kit Yind SE33 DY3 D22 D2(S4) D12 D10 D1 FGA D5 Amel D18 TPOX vwa D19 D2(S1) D16 D13 TH01 D3 CSF D7 D21 D8 Identifiler Plus FGA D5 Amel D18 TPOX vwa D19 D2 D16 D13 TH01 D3 CSF D7 D21 D8 Greater sensitivity was observed with the GlobalFiler Kit compared to the Identifiler Plus Kit 22 Greater sensitivity observed with the GlobalFiler Kit compared to the Identifiler Plus Kit
22 GlobalFiler Kit Performance Example: Inhibitor Tolerance Humic Acid Inhibition Hematin Inhibition Identifiler Plus Kit GlobalFiler Kit The GlobalFiler Kit buffer has been optimized to deliver enhanced inhibition tolerance compared to the Identifiler Plus Kit 23
23 GlobalFiler Kit Performance on Casework Samples Intracolor Balance (%) Intralocus Balance 500 pg 1 ng GlobalFiler Kit Identifiler Plus Kit GlobalFiler Kit Identifiler Plus Kit The GlobalFiler Kit maintains the high levels of intracolour and intralocus balance established by other Applied Biosystems next generation STR kits 24
24 GlobalFiler Kit Performance on Casework Samples Stutter Percentage (007 DNA) 70bp Species Specificity 470bp 400 RFU 10 ng Cat DNA 10 ng Dog DNA 10 ng Horse DNA 10 ng Bacteria DNA Identifiler Plus Kit GlobalFiler Kit Reduced mean stutter percentage for the majority of loci and high specificity delivered by optimised buffer chemistry and established primer design methods simplify interpretation of GlobalFiler Kit data 25
25 GlobalFiler Kit Test Site Results Identifiler Plus Kit: Partial Profile (21 alleles) GlobalFiler Kit: Partial Profile (28 alleles) Test Site #2: Bone Sample; Organic Extraction Identifiler Plus Kit: 50 RFU PAT (3130xl) GlobalFiler Kit: 175 RFU PAT (3500xL) 26
26 GlobalFiler Kit Test Site Results NGM SElect Kit: Partial Profile (18 alleles) GlobalFiler Kit: Full Profile (43 alleles) Test Site #2: Cigarette Butt, Organic Extraction NGM SElect Kit: 50 RFU PAT (3130xl) GlobalFiler Kit: 175 RFU PAT (3500xL) 27
27 Test Site Results: 900 Year Old Bone Sample Medieval Austrian, Agnes of Waiblingen was the daughter of Salian King Henry IV and was married to Leopold III, one of the most important Babenberg Margraves She died in 1143 and is reported to have had 17 children 28
28 GlobalFiler Kit Baseline Quality Read Region 300 Test Site #1 300 Test Site #2 300 Test Site #3 Non-template Controls 29
29 Development of a Next Generation Global Multiplex Go Faster! Up to 5x faster amplification time than previous generation kits Go Further! > GlobalFiler Express Kit: ~40 minutes; GlobalFiler Kit: ~80 minutes > Enables sample to answer in <2 hours 24 loci, including 10 mini-strs, increasing discrimination power by up to 9 orders of magnitude Enhanced buffer system and sensitivity for even better performance on inhibited and low-level samples Go Global! The only kit with all markers recommended for inclusion by the CODIS Core Loci Working Group, including all markers commonly used in Europe Maximizes global database compatibility 30
30 Acknowledgements R&D Julio Mulero, Lori Hennessy, Siddhita Gopinath, Wilma Norona, Matthew Ludeman, Robert Lagace, Chien-Wei Chang, Chang Zhong, Nigel Beard Tech Transfer Larry Wang, Anthony Matthews, Vanessa Chan, Yenyoung Lei Software Allan Minn, Gloria Lam, Sharada Viyachander, Tony Hatch, Makesh Kumar, Ravi Gupta, Doug Greig, Babu Yama Product Management Karen Cormier, Larry Joe, Jacki Benfield Forensic Science Applications Jennifer Schroeder, Lisa Calandro, Jeff Sailus, Nicola Oldroyd Quality Assurance Liwei Qi, Yurah Yen Marketing Ariana Wheaton, Jonathan Tabak, Shereen Darwish, Lisa Schade Oligo Development Sarah Ruhe, Kathy Wang, Hong Ji UK Manufacturing Catherine Duke Project Management Wendy Coffey, Linda Kauffman 31
31 Thank You 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Refer to product page on the Life Technologies website for Limited Use License. The content provided herein may relate to products that have not been officially released and is subject to change without notice. FOR FORENSIC AND PATERNITY USE ONLY
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