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Development of a “Global” STR Multiplex for Human Identification Analysis

Development of a “Global” STR Multiplex for Human Identification Analysis






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    Development of a “Global” STR Multiplex for Human Identification Analysis Development of a “Global” STR Multiplex for Human Identification Analysis Presentation Transcript

    • Development of a “Global” STR Multiplex forHuman Identification AnalysisHuman Identification, Life Technologies
    • Agenda Why a global multiplex? Multiplex Configuration Strategy Application-Specific Development and Testing Results − Direct amplification for single source samples − Casework samples Summary11/13/2012 2
    • Why a Global Multiplex? New CODIS Expanded Missing Persons Recommendations ESSL Investigations Increase international data compatibility Why? Increase discrimination power Reduce the likelihood of adventitious matches Increase the number of loci How? Incorporate loci contained in global databases11/13/2012 3
    • D13S317 MINIMUM REQUIRED LOCI Identifiler® Kit Recommended CODIS CoreD7S820 Identifiler® Kit Loci (April 2011)D5S818 Identifiler® KitCSF1PO Identifiler® Kit  New recommendation drops TPOXD1S1656 NGM SElect™ Kit from the list of required lociD12S391 NGM SElect™ KitD2S441 NGM SElect™ Kit  The new list of required loci includesD10S1248 NGM SElect™ Kit all the markers from the NGM SElect™D18S51 Identifiler® & NGM SElect™ KitsFGA Identifiler® & NGM SElect™ Kits & Identifiler® Series KitsD21S11 Identifiler® & NGM SElect™ KitsD8S1179 Identifiler® & NGM SElect™ KitsVWA Identifiler® & NGM SElect™ KitsD16S539 Identifiler® & NGM SElect™ KitsTH01 Identifiler® & NGM SElect™ KitsD3S1358 Identifiler® & NGM SElect™ KitsAMEL Identifiler® & NGM SElect™ Kits HIGHLY RECOMMENDED LOCID2S1338 Identifiler® & NGM SElect™ Kits (1) TPOX Identifiler® KitD19S433 Identifiler® & NGM SElect™ Kits (2) D22S1045 NGM SElect™ KitDYS391 Yfiler® Kit (3) SE33 NGM SElect™ KitPENTA E 11/13/2012 4 (4) PENTA D
    • 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 kit11/13/2012 5
    • The GlobalFiler™ Kits Configuration 1 D3S1358 2 VWA 11 12 D16S539 CSF TPOX Y indel 3 Amel D8S1179 13 14 D21S11 D18S51 DYS391 4 *D2S441 5 Mini-STR Combination D19S433 6 15 TH01 PI*= 5.73 x 10-21 FGA 7 *D22S1045 8 16 D5S818 17 D13S317 D7S820 SE33 9 10 *D10S1248 *D1S1656 18 *D12S391 D2S1338 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 Mini-STRs MiniFiler ™ Kit PI* = 9.2 x 10-12 8 mini-STRs < 220 bp PI* = 1.05 x 10-1011/13/2012 6
    • The GlobalFiler™ Kits Configuration D3S1358 VWA D16S539 CSF TPOX Y indel Y indel Amel D8S1179 DYS391 D21S11 D18S51 DYS391 *D2S441 D19S433 TH01 FGA *D22S1045 D5S818 D13S317 D7S820 SE33 *D10S1248 *D1S1656 *D12S391 D2S1338 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 p q Amelogenin Y-indel/DYS39111/13/2012 7
    • The GlobalFiler™ Kits Allelic Ladder Improvements D2S1338 D19S433 GlobalFiler™ Kit range: 11-28 GlobalFiler™ Kit range: 6-19.2 NGM SElect™ Kit range: 9-17.2 Identifiler® Kit range: 15-2811/13/2012 8
    • The GlobalFiler™ Kits Allelic Ladder 343 alleles and 245 virtual bins11/13/2012 9
    • 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) 329-332. vWA Delamoye et al. FSI 143 (2004) 47-52. D18S51 Delamoye et al. FSI 143 (2004) 47-52. D19S433 Kline et al. FSI Genetics 5 (2011) 329-332. TH01 Vanderheyden et al. Int J Legal Med 121 (2007) 297-301. FGA Ricci et al. JFS 52 (2007) 393-396. D5S818 Kline et al. FSI Genetics 5 (2011) 329-332. SE33 Heinrich et al. Int J Legal Med 118 (2004) 361-363.11/13/2012 10
    • Maximizing Discrimination D10S1248 D22S1045 Kit PI* D8S1179 D3S1358 D13S317 D16S539 D2S1338 D19S433 D1S1656 D12S391 CSF1PO D21S11 D7S820 D18S51 D5S818 D2S441 TPOX TH01 vWA SE33 FGA Identifiler® Kit X X X X X X X X X X X X X X X 2.6 x 10-17 NGM SElect™ Kit X X X X X X X X X X X X X X X X 1.12 x 10-20 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 10-26 * 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 comparisons11/13/2012 11
    • GlobalFiler™ Kit Instrument Compatibility 3500xL 3130xl Variable binning settings for 3130 series instruments adjusted to gain optimal color balance11/13/2012 12
    • Kit Development Strategy The GlobalFiler™ Kits GlobalFiler™ GlobalFiler ™ Express PCR Amplification Kit PCR Amplification Kit A high performance, 6-dye An ultra-high throughput, chemistry for casework 6-dye chemistry for single samples source samples Optimized for use with the 3500/3500xL11/13/2012 13
    • GlobalFiler™ Kit Comparison GlobalFiler™ Express Kit GlobalFiler™ Kit Optimized to overcome inhibitors Optimized to provide optimal sensitivity Master Mix found in unpurified single source blood and intracolor balance for casework and buccal samples samples Primer Mix* 2.5X formulation 10X formulation PCR protocol optimized for high PCR protocol optimized for speed of PCR Protocol sensitivity and high performance on processing for single source samples casework samples Control DNA 007 2 ng/µL 0.1 ng/µL Allelic Ladder Identical Allelic Ladder* Both kits use identical primer sequences11/13/2012 14
    • 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 25-27 x 94°C/ 3 sec 60°C/ 30 sec 60°C/ 8 min Identifiler® Direct NGM SElect™ GlobalFiler™ Kit Express Kit Express Kit Total Time 2 hr 35 min 45 min 40 min11/13/2012 15
    • Optimizing Primer Mix for the GlobalFiler™ Express Kit Blood on Blood on Buccal on Buccal on Buccal on Buccal on FTA® Untreated Bode Buccal FTA® Copan Omniswab Bloodstain Collector ™ 4N6FLOQSwab ™ N=6 Card11/13/2012 17
    • GlobalFiler™ Express Kit Population Study Success Rate Number of Off- Sample Type N PCR Cycle Number (175 RFU) Scale Profiles Blood on FTA® 200 25 100% 4 Blood on Bloodstain Card (903 Paper) 45 25 100% 0 Buccal on FTA® (EasiCollect™ Device) 56 26 96.4% 3 Buccal on Bode Buccal Colector™ 90 26 96.6% 0 Buccal on Copan 4N6FLOQSwab™ 46 24 95.7% 3 Intralocus Balance Intracolor Balance N=3552 N=1000 N=761 N=1545 N=774 Blood on Buccal Blood on Buccal on Buccal on Blood on Buccal on Blood on Buccal on Buccal on FTA® on FTA® Untreated Bode Copan FTA® FTA® Untreated Bode Copan Bloodstain Buccal FLOQSwab™ Bloodstain Buccal FLOQSwab™ Card Collector™ Card Collector™11/13/2012 18
    • GlobalFiler™ Express Kit Data Examples Buccal on Bode Buccal Collector™Blood on FTA® CardBuccal on Copan FLOQSwab™  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. 11/13/2012 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 conditions11/13/2012 20
    • Optimization of GlobalFiler™ Kit Reaction Conditions Reagent Volumes 3.3X Master Mix 7.5 µL Greater flexibility of sample 10X Primer Set 2.5 µL input volume to facilitate Sample Input ≤15.0 µL analysis of low level samples 25.0 µL PCR Conditions 95°C/ 1 min Optimized thermal cycling 94°C/ 20 sec conditions shorten amplification 29 x conditions by > 1 hr 59°C/ 90 sec 60°C/ 10 min Identifiler® Plus & NGM GlobalFiler™ SElect™ Kits Kit Total 2 hr 25 min 1 hr 20 min Time11/13/2012 21
    • GlobalFiler™ Kit Performance Example: Sensitivity 100.0 Mean % Alleles Recovered 80.0 60.0 The GlobalFiler™ Kit delivers comparable 40.0 sensitivity to the NGM SElect™ Kit 20.0 0.0 250 125 62 31 15 (pg) N=4 TM NGM SelectTM Kit GlobalFilerTM Kit TM11/13/2012 22
    • GlobalFiler™ Kit Performance Example: Sensitivity 50.0 TM NGM SelectTM Kit 45.0 GlobalFilerTM Kit TM 40.0 35.0 Alleles Recovered 30.0 25.0 The GlobalFiler™ Kit amplifies more alleles 20.0 than the NGM SElect™ 15.0 Kit. 10.0 5.0 0.0 250 125 62 31 15 (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 00711/13/2012 23
    • GlobalFiler™ Kit Performance Example: InhibitorTolerance Humic Acid Inhibition Hematin Inhibition 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 0 120 150 180 0 300 340 380 (ng/uL) (uM) TM NGM SelectTM Kit GlobalFilerTM Kit TM The GlobalFiler™ Kit buffer has been optimized to deliver enhanced inhibition tolerance compared to the NGM Select™ Kit11/13/2012 24
    • GlobalFiler™ Kit Performance Examples 500 pg 1 ngIntracolor Balance (%) Intralocus Balance GlobalFiler™ Identifiler® GlobalFiler™ Identifiler® Kit Plus Kit Kit Plus Kit Blue – Identifiler® Plus Kit Red – GlobalFiler™ Kit  The GlobalFiler™ Kit maintains theStutter Percentage (%) high levels of intracolor balance, intralocus balance and stutter percentage established with previous next generation kits such as the Identifiler® Plus Kit11/13/2012 25
    • Prototype GlobalFiler™ Kit vs. Identifiler® Plus KitTest Site data: Bone Sample; Organic ExtractionIdentifiler® 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)11/13/2012 26
    • Prototype GlobalFiler™ Kit vs. NGM SElect™ KitTest 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)11/13/2012 27
    • Prototype GlobalFiler™ Kit Baseline Quality 74 444 Read Region 300 Test Site #1 300 Test Site #2 300 Test Site #3 Non-template Controls11/13/2012 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 Developed specifically for the amplification of of single-source samples casework samples Delivers rapid throughput on blood and buccal Delivers increased sensitivity, improved intralocus samples on treated paper, untreated paper and and intracolor balance, enhanced performance on swabs inhibited and degraded samples Amplification Time ~ 40 min Amplification Time ~ 80 min11/13/2012 29
    • Acknowledgements  R&D  Forensic Science Applications Dennis Wang, Lori Hennessy, Siddhita Jennifer Schroeder, Lisa Calandro, Jeff Gopinath, Wilma Norona, Matthew Sailus, Nicola Oldroyd Ludeman, Robert Lagace, Chien-Wei Chang, Chang Zhong  Quality Assurance Liwei Qi, Yurah Yen  Tech Transfer Larry Wang, Anthony Matthews, Vanessa  Marketing Chan, Linda Liu, Yenyoung Lei Ariana Wheaton, Jonathan Tabak, Shereen Darwish, Lisa Schade  Software Allan Minn, Gloria Lam, Sharada  Oligo Development Viyachander, Tony Hatch, Makesh Kumar, Sarah Ruhe, Kathy Wang, Hong Ji Ravi Gupta, Doug Greig, Babu Yama  UK Manufacturing  Product Management Catherine Duke Karen Cormier, Bob Kays, Larry Joe, Jacki Benfield, Guido Sandulli  Project Management Wendy Coffey, Linda Kauffman11/13/2012 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.11/13/2012 31