Development of a Global STR Multiplex for Human Identification Analysis Jonathan Tabak Life Technologies 1
Agenda Introduction Multiplex Configuration Strategy Application-Specific Development and Testing Results Direct amplification for single source samples Casework samples Summary 2
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-e54. 2. 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
CODIS Loci Recommendations Amelogenin D13S317 D7S820 D5S818 CSF1PO D1S1656 D12S391 MINIMUM REQUIRED LOCI Identifiler & NGM SElect Kits Identifiler Kit Identifiler Kit Identifiler Kit Identifiler Kit NGM SElect Kit NGM SElect Kit HIGHLY RECOMMENDED LOCI (1) TPOX Identifiler Kit (2) D22S1045 NGM SElect Kit (3) SE33 NGM SElect Kit Original recommendations (April 2011) included 21 loci required and 4 highly recommended for inclusion by manufacturers 1 Revised recommendations (January 2012) removed the Penta E and Penta D loci 2 D2S441 D10S1248 D18S51 FGA D21S11 NGM SElect Kit NGM SElect Kit Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits The revised list includes all markers commonly used in U.S. and Europe, already found in the NGM SElect & Identifiler Series Kits D8S1179 VWA D16S539 TH01 Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits GlobalFiler Kits include all required and recommended loci within a single multiplex D3S1358 AMEL D2S1338 D19S433 DYS391 Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits Identifiler & NGM SElect Kits Yfiler Kit 1. D.R. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. (2011), doi:10.1016/j.fsigen.2011.04.01 2. D.R. Hares, Addendum to expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. (2012), doi:10.1016/j.fsigen.2012.01.003 5
Customer Feedback With so many loci, isn t there a risk of losing sensitivity and having more drop out? Our lab is dealing with more low level trace and touch type samples, and analysis can be a nightmare, especially with mixtures. With even more loci, I think that difficulty could increase exponentially. We are already facing big backlogs and budget shortfalls. Having to purchase and implement new instrument platforms is an enormous additional hurdle. *Source: 2011 HID Future Trends in Forensic DNA Analysis Technology Seminar Series, Technical Lead Mtgs. 6
Multiplex Configuration Strategy 7
5-Dye Configuration Containing All Required and Highly Recommended Loci 1 2 3 1 2 4 5 6 4 3 7 8 Primer Redesigns less concordant Fewer Mini-STRs poor performance for degraded DNA Multiple loci > 400 bp poor performance for degraded DNA compromised sizing and resolution 8
The GlobalFiler Kits: 6-Dye Configuration 1 2 11 12 Only 2 loci require primer redesign 3 13 14 4 7 5 6 8 16 17 15 18 Mini-STRs Combination PI*= 5.73 x 10-21 9 10 18 GlobalFiler Kit 10 Mini-STRs PI* = 9.2 x 10-12 MiniFiler Kit 8 Mini-STRs PI* = 1.05 x 10-10 97% of alleles in allelic ladder <400bp 9 * Preliminary calculations based on a US Caucasian Database
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 10
What is an Indel? Indels are short length polymorphisms, consisting of the presence (INsertion) or absence (DELetion) of a short sequence (1-50 nucleotides) In the GlobalFiler Kits, an Indel marker on the Y chromosome was incorporated into the multiplex Deletion Insertion 12
Improved Accuracy of Gender Determination Gender identification redundancy helps remove the need for laboratories to maintain a second gender confirmation test protocol In addition, the Y-indel position (<90 bp) in the GlobalFiler Kit helps improve data recovery in degraded samples 13
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 14
The GlobalFiler Kits Allelic Ladder Improvements D2S1338 GlobalFiler Kit range: 11-28 D19S433 GlobalFiler Kit range: 6-19.2 Identifiler Kit range: 15-28 NGM SElect Kit range: 9-17.2 15
More Genotyping Accuracy: Minimizing False Homozygosity Literature reports indicate instances of false homozygosity 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 GlobalFiler Kit Loci with New SNP- Specific Primers D3S1358 vwa D18S51 D19S433 TH01 FGA D5S818 SE33 16
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 10-18 NGM SElect Kit X X X X X X X X X X X X X X X X 2.35 x 10-21 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 GlobalFiler Mini s X X X X X X X X X X *9.20 x 10-12 * 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 17
An Unprecedented Power of Discrimination GlobalFiler Kit PI = 7.12 x 10-26 = 1 in 1.4 x 10 25 Total Historical Human Population = 1.1 x 10 11 The GlobalFiler Kits offer a power of discrimination ~10 14 times greater than the total population that has ever lived on Earth! Source - www.prb.org/ 18
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 19
Instrument Compatibility 3500xL 3130xl 20
Instrument Compatibility 3500xL 3730 21
Improved Reagent Traceability Packaging Improvements for the GlobalFiler Kits New Peel and Stick kit labels can be removed from kit sleeves and placed on component boxes or recorded in laboratory logbooks Improves ability to link kit components with kit lot numbers 22
Application Specific Development & Testing 23
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 24
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 25
GlobalFiler Express Kit Workflows 26
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 25-27 x 60 C/ 8 min Identifiler Direct Kit NGM SElect Express Kit GlobalFiler Express Kit Total Time 2 hr 35 min 45 min 39-40 min 27
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 28
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 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 containing all markers recommended for inclusion by the CODIS Core Loci Working Group, including all markers commonly used in Europe Maximizes global database compatibility 30
Acknowledgements External Test Sites R&D 66 worldwide and growing! 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 Vijaychander, 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
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 http://www.slideshare.net/lifetech_hid http://www.lifetechnologies.com/globalfiler 32