Development of a Global STR Multiplex for Human Identification Analysis. Julio Mulero, Ph. D. R&D Group Human Identification, Life Technologies

Transcription

1 Development of a Global STR Multiplex for Human Identification Analysis Julio Mulero, Ph. D. R&D Group Human Identification, Life Technologies

2 Agenda Why a global multiplex? Multiplex Configuration Strategy Application-Specific Development and Testing Results Direct amplification for single source samples Casework samples Summary 10/12/2012 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 10/12/2012 3

4 MINIMUM REQUIRED LOCI D13S317 Identifiler Kit D7S820 Identifiler Kit D5S818 Identifiler Kit CSF1PO Identifiler Kit D1S1656 NGM SElect Kit D12S391 NGM SElect Kit D2S441 NGM SElect Kit D10S1248 NGM SElect Kit D18S51 Identifiler & NGM SElect Kits FGA Identifiler & NGM SElect Kits D21S11 Identifiler & NGM SElect Kits D8S1179 Identifiler & NGM SElect Kits VWA Identifiler & NGM SElect Kits D16S539 Identifiler & NGM SElect Kits TH01 Identifiler & NGM SElect Kits D3S1358 Identifiler & NGM SElect Kits AMEL Identifiler & NGM SElect Kits D2S1338 Identifiler & NGM SElect Kits D19S433 Identifiler & NGM SElect Kits DYS391 Yfiler Kit PENTA E 10/12/ Recommended CODIS Core Loci (April 2011) New recommendation drops TPOX from the list of required loci The new list of required loci includes all the markers from the NGM SElect & Identifiler Series Kits HIGHLY RECOMMENDED LOCI (1) TPOX Identifiler Kit (2) D22S1045 NGM SElect Kit (3) SE33 NGM SElect Kit (4) PENTA D

5 What is GlobalFiler? The GlobalFiler Kits are our response to the CODIS Core Loci Working Group s recommendation to expand the CODIS Core Loci from 13 to 20 required loci and 3 optional loci Originally communicated as a letter to the editor (FSI Genetics) and accepted for publication on April 2011 (revised on Jan 2012) Authored by Doug Hares from the FBI Expectation of a single amplification kit 10/12/2012 5

6 The GlobalFiler Kits Configuration D3S1358 VWA D16S539 CSF TPOX Y indel Amel D8S1179 D21S11 D18S51 DYS391 *D2S441 D19S433 TH01 FGA *D22S1045 D5S818 D13S317 D7S820 SE33 *D10S1248 *D1S1656 *D12S391 D2S Mini-STR Combination PI*= 5.73 x Mini-STRs PI* = 9.2 x MiniFiler Kit 8 mini-strs < 220 bp PI* = 1.05 x /12/2012 6

7 The GlobalFiler Kits Configuration D3S1358 VWA D16S539 CSF TPOX Y indel Amel D8S1179 D21S11 D18S51 DYS391 *D2S441 D19S433 TH01 FGA *D22S1045 D5S818 D13S317 D7S820 SE33 *D10S1248 *D1S1656 *D12S391 D2S1338 Y indel DYS p Amelogenin q Y-indel/DYS391 10/12/2012 7

8 The GlobalFiler Kits Allelic Ladder Improvements D2S1338 D19S433 GlobalFiler Kit range: GlobalFiler Kit range: Identifiler Kit range: NGM SElect Kit range: /12/2012 8

9 The GlobalFiler Kits Allelic Ladder 343 alleles and 245 virtual bins 10/12/2012 9

10 Minimizing False Homozygosity Literature reports indicate instances of false homozyosity in the Identifiler /NGM SElect Kits as a result of primer site SNP mutations Eight new SNP-specific primers have been included in the GlobalFiler Kits to minimize the occurrence of null alleles in future data sets A series of SNP-specific primers already exist in the Identifiler /NGM SElect Kits to account for other primer site mutations Locus References D3S1358 Kline et al. FSI Genetics 5 (2011) vwa Delamoye et al. FSI 143 (2004) D18S51 Delamoye et al. FSI 143 (2004) D19S433 Kline et al. FSI Genetics 5 (2011) TH01 Vanderheyden et al. Int J Legal Med 121 (2007) FGA Ricci et al. JFS 52 (2007) D5S818 Kline et al. FSI Genetics 5 (2011) SE33 Heinrich et al. Int J Legal Med 118 (2004) /12/

11 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 2.6 x NGM SElect Kit X X X X X X X X X X X X X X X X 1.12 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 * 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 10/12/

12 GlobalFiler Kit Instrument Compatibility 3500xL 3130xl Variable binning settings for 3130 series instruments adjusted to gain optimal color balance 10/12/

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 Optimized for use with the 3500/3500xL 10/12/

14 GlobalFiler Kit Comparison Master Mix GlobalFiler Express Kit Optimized to overcome inhibitors found in unpurified single source blood and buccal samples GlobalFiler Kit Optimized to provide optimal sensitivity and intracolor balance for casework samples Primer Mix* 2.5X formulation 10X formulation PCR Protocol PCR protocol optimized for speed of processing for single source samples PCR protocol optimized for high sensitivity and high performance on casework samples Control DNA ng/µl 0.1 ng/µl Allelic Ladder Identical Allelic Ladder * Both kits use identical primer sequences 10/12/

15 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 x 94 C/ 3 sec 60 C/ 30 sec 60 C/ 8 min Identifiler Direct Kit NGM SElect Express Kit GlobalFiler Express Kit Total Time 2 hr 35 min 45 min 40 min 10/12/

16 Optimizing Primer Mix for the GlobalFiler Express Kit N=6 Blood on FTA Blood on Untreated Bloodstain Card Buccal on Bode Buccal Collector Buccal on FTA Buccal on Copan 4N6FLOQSwab Buccal on Omniswab 10/12/

17 GlobalFiler Express Kit Population Study Sample Type N PCR Cycle Number Success Rate (175 RFU) Number of Off- Scale Profiles Blood on FTA % 4 Blood on Bloodstain Card (903 Paper) % 0 Buccal on FTA (EasiCollect Device) % 3 Buccal on Bode Buccal Colector % 0 Buccal on Copan 4N6FLOQSwab % 3 Intralocus Balance Intracolor Balance N=3552 N=1000 N=761 N=1545 N=774 Blood on FTA Buccal on FTA Blood on Untreated Bloodstain Card Buccal on Bode Buccal Collector Buccal on Copan FLOQSwab Blood on FTA Buccal on FTA Blood on Untreated Bloodstain Card Buccal on Bode Buccal Collector Buccal on Copan FLOQSwab 10/12/

18 GlobalFiler Express Kit Data Examples Buccal on Copan FLOQSwab Blood on FTA Card Buccal on Bode Buccal Collector The GlobalFiler Express Kit is optimized to deliver high quality results on a wide range of substrates Prep-n-Go Buffer facilitates cell lysis in non-treated paper and swabs. 10/12/

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 Efficient thermal cycling conditions 10/12/

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 Greater flexibility of sample input volume to facilitate analysis of low level samples PCR Conditions 95 C/ 1 min 94 C/ 20 sec 29 x 59 C/ 90 sec 60 C/ 10 min 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 10/12/

21 Mean % Alleles Recovered GlobalFiler Kit Performance Example: Sensitivity The GlobalFiler Kit delivers comparable sensitivity to the NGM SElect Kit (pg) TM NGM Select TM Kit GlobalFiler TM Kit N=4 10/12/

22 Alleles Recovered GlobalFiler Kit Performance Example: Sensitivity TM NGM Select TM Kit GlobalFiler TM Kit The GlobalFiler Kit amplifies more alleles than the NGM SElect Kit (pg) N=4 GlobalFiler Kit amplifies 43 alleles in a full profile of the control DNA 007 NGM Select Kit amplifies 34 alleles in a full profile of the control DNA /12/

23 GlobalFiler Kit Performance Example: Inhibitor Tolerance Humic Acid Inhibition Hematin Inhibition (ng/ul) (um) TM NGM Select TM Kit GlobalFiler TM Kit The GlobalFiler Kit buffer has been optimized to deliver enhanced inhibition tolerance compared to the NGM Select Kit 10/12/

24 Stutter Percentage (%) Intracolor Balance (%) Intralocus Balance GlobalFiler Kit Performance Examples 500 pg 1 ng GlobalFiler Kit Identifiler Plus Kit GlobalFiler Kit Identifiler Plus Kit Blue Identifiler Plus Kit Red GlobalFiler Kit The GlobalFiler Kit maintains the high levels of intracolor balance, intralocus balance and stutter percentage established with previous next generation kits such as the Identifiler Plus Kit 10/12/

25 Prototype GlobalFiler Kit vs. Identifiler Plus Kit Test Site data: Bone Sample; Organic Extraction Identifiler Plus Kit: Partial Profile (21 alleles) GlobalFiler Kit: Partial Profile (28 alleles) Identifiler Plus Kit: 50 RFU PAT (3130xl) GlobalFiler Kit: 175 RFU PAT (3500xL) 10/12/

26 Prototype GlobalFiler Kit vs. NGM SElect Kit Test Site data: Cigarette Butt, Organic Extraction NGM SElect Kit: Partial Profile (18 alleles) GlobalFiler Kit: Full Profile (43 alleles) NGM SElect Kit: 50 RFU PAT (3130xl) GlobalFiler Kit: 175 RFU PAT (3500xL) 10/12/

27 Prototype GlobalFiler Kit Baseline Quality Read Region 300 Test Site #1 300 Test Site #2 300 Test Site #3 Non-template Controls 10/12/

28 GlobalFiler Kit Feature Summary GlobalFiler Express Kit GlobalFiler Kit Amplifies the 20 required and 3 recommended loci in the new CODIS Core Loci recommendations Amplifies 3 Y chromosomal markers to ensure accurate gender determination Improves genotyping accuracy and automation through expanded allelic ladders and virtual bin sets Compatible with the Veriti & 9700 thermal cyclers and the 3500 & 3130 series genetic analyzers Developed specifically for the direct amplification of single-source samples Delivers rapid throughput on blood and buccal samples on treated paper, untreated paper and swabs Amplification Time ~ 40 min Developed specifically for the amplification of casework samples Delivers increased sensitivity, improved intralocus and intracolor balance, enhanced performance on inhibited and degraded samples Amplification Time ~ 80 min 10/12/

29 Acknowledgements R&D Dennis Wang, Lori Hennessy, Siddhita Gopinath, Wilma Norona, Matthew Ludeman, Robert Lagace, Chien-Wei Chang, Chang Zhong Tech Transfer Larry Wang, Anthony Matthews, Vanessa Chan, Linda Liu, Yenyoung Lei Software Allan Minn, Gloria Lam, Sharada Viyachander, Tony Hatch, Makesh Kumar, Ravi Gupta, Doug Greig, Babu Yama Product Management Karen Cormier, Bob Kays, Larry Joe, Jacki Benfield, Guido Sandulli 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 10/12/

30 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. 10/12/