Next-Generation Sequencing Applications for STRs, SNPs and Mitochondrial DNA in Human Identification
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Transcript of Next-Generation Sequencing Applications for STRs, SNPs and Mitochondrial DNA in Human Identification
1 Life Technologies™ |
Next-Generation Sequencing Applications for STRs, SNPs and Mitochondrial DNA in Human
Identification
HID PGM Research Team Applied Markets Life Technologies
Special Acknowledgments
Dr. Walther Parson, Institute for Legal Medicine Innsbruck, Innsbruck, Austria
Dr. Kenneth Kidd, Department of Genetics, Yale University School of Medicine
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Ion Torrent PGM for Forensics § Life Technologies is very interested in developing applications that demonstrate the unique
value of Next-Generation Sequencing in Forensics.
§ We believe the technology would complement existing STR-based kits: − (1) as an investigative tool when there is no match in existing databases − (2) as an identification tool when working with challenging or degraded materials (as is regularly seen in
casework and disaster victim identification) − (3) as a high-throughput and less laborious alternative in Mitochondrial sequencing.
§ HID PGM Solutions: panels, protocols and software plugins for analysis of SNPs, Mitochondria and STRs.
§ We are currently validating these prototypes at select early access sites and anticipate availability to the greater community soon.
§ Benefits: Greater confidence when identifying challenging or degraded samples, a simple modular sequencing workflow, and informative annotation tools that can be easily utilized by a forensic analyst.
§ If interested in collaborating contact your local account management representative or Nnamdi Ihuegbu at [email protected] (add “PGM” to subject line)
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Agenda
• Next-Generation Sequencing Technology on PGM™ • HID Applications using PGM™
§ Identity SNPs
§ Ancestral informative and phenotypic SNPs
§ Mitochondrial Sequencing
§ STRs • Future plans
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NGS Technology on PGM™
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Sequencing Instrument
Ion OneTouch™ Instruments Emulsion PCR and Enrichment
Semiconductor Chip
Sequencing Chemistry • Natural nucleo?des • Natural enzymes
Sample Prep • Libraries • Clonal beads
Torrent Server
The Ion Torrent PGM™ Instrument System
INSTRUMENTS CONSUMABLES DATA ANALYSIS
http://ioncommunity.lifetechnologies.com/community/intro
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Library and Template Preparation
Clonal amplifica,on using emulsion PCR
ISP
Primer dNTPs
Polymerase MgCl2
Isolate templated ISPs
Final Templated ISPs ready for sequencing
Emulsion droplet
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Sequence Detection by pH
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Data Output is an Ionogram
• Must be read “up-and-down” along with “left-to-right” • Height of bar indicates how many nucleotides incorporated
during flow
Key Sequence
Sequence: …AATCTTCTGAATTTCTGCAA…. (TTT)
(AA) (AA)
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Why PGM™ Sequencing
• Amenable to degraded DNA (down to 75 bp)
• Multiplex individuals – Barcodes (up to 96)
• Faster library construction
• Automated enrichment system
• Very fast sequencing time
• Less total hands-on time
• Higher throughput (runs per week)
• Natural chemistry (less bias, less maintenance)
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HID Applications for PGM™
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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HID Applications on PGM™
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HID Applications for PGM™ SNP Applications
Iden?ty SNPs Lineage-‐
informa?ve SNPs
Ancestry-‐informa?ve
SNPs
• High heterozygosity • Low popula?on heterogeneity
Phenotypic SNPs
• Haplotype markers for kinship analysis
• High popula?on heterogeneity
• Hair, eye, skin color
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
Butler et al. Report on ISFG SNP panel discussion. Forensic Science International (2008) Kidd et al. Developing a SNP panel for forensic identification of individuals, Forensic Science International 164 (1) (2006)
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HID Applications for PGM™ SNP Applications
Iden?ty SNPs Lineage-‐
informa?ve SNPs
Ancestry-‐informa?ve
SNPs
• High heterozygosity • Low popula?on heterogeneity
Phenotypic SNPs
• Haplotype markers for kinship analysis
• High popula?on heterogeneity
• Hair, eye, skin color
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
Butler et al. Report on ISFG SNP panel discussion. Forensic Science International (2008) Kidd et al. Developing a SNP panel for forensic identification of individuals, Forensic Science International 164 (1) (2006)
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HID Applications for PGM™ SNP Panel v2.2 (Identity Panel)
§ Based on published SNPs with high heterozygosity and low Fst
§ Genotype match probabili?es of 10-‐31 -‐ 10-‐35
175 SNPs
35 Y - SNPs
92 - Ken Kidd SNPs
52 - SNPforID SNPlex
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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Iden?ty SNPs Lineage-‐
informa?ve SNPs
Ancestry-‐informa?ve
SNPs
• High heterozygosity • Low popula?on heterogeneity
Phenotypic SNPs
• Haplotype markers for kinship analysis
• High popula?on heterogeneity
• Hair, eye, skin color
HID Applica?ons for PGM™ Ancestral and Phenotypic SNP Panel
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
Butler et al. Report on ISFG SNP panel discussion. Forensic Science International (2008) Kidd et al. Developing a SNP panel for forensic identification of individuals, Forensic Science International 164 (1) (2006)
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Applica?on: Subject exclusion and inves?ga?ve leads • high discrimina?on between ancestral groups • hair and eye color determina?on
Based on panels validated on a wide range of popula?on datasets
hair and eye color
SNPs
245 SNPs
Ancestral SNPs
202
45
HID Applica?ons for PGM™ Ancestral and Phenotypic SNP Panel
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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HID Applications for PGM™ Data Analysis
Torrent Server Torrent Browser Run Analysis Application specific plugins
Third-party desktop tools
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Y-SNPs
Panel Development Tools: Amplicon Coverage
Female Individual Male Individual
SNP Panel v2.2
Mean 1 StdDev from Mean
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HID-‐SNP Genotyper Plugin
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allele coverage
alleles represented
SNPs by rs ID
HID-‐SNP Genotyper Plugin
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Concordance for 74 Ken Kidd SNPs, 30 Individuals
0%#
10%#
20%#
30%#
40%#
50%#
60%#
70%#
80%#
90%#
100%#
KK1# KK2# KK3# KK4# KK5# KK6# KK7# KK8# KK9# KK10# KK11# KK12# KK13# KK14# KK15# KK16# KK17# KK18# KK19# KK20# KK21# KK22# KK23# KK24# KK25# KK26# KK27# KK28# KK29# KK30#
Genotype#disparity#
Haplotype#disparity#
Concordant#
% Con
cordance
Individuals
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SNP Application Summary
HID-SNP Genotyper Plugin utility built to help display and evaluate data Next-generation sequencing technologies allow for high multiplexed capabilities - of SNPs and individuals Identity SNP panel on PGM™ sequencer using well-characterized polymorphisms HID SNP Panel v0.1 (Identity Panel) 99% concordant with Ken Kidd controls
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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Whole Genome Protocol Data and Results Courtesy of Walther Parson’s Laboratory
PGM™ Mitochondrial Sequencing
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Whole Genome Mitochondrial DNA Library Preparation
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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Summary Update Walther Parson 40 mitochondrial genomes bioinforma?c improvements: • Substantially higher through-put of mtGenome sequencing compared to Sanger • High concordance between Sanger and PGM
• 176 differences in 1,060,437 bps – 0.017%; 20% variance call threshold
• Roughly two thirds of differences observed in homopolymeric C tracts (> 7Cs); physical PGM sequence data seems to be there, as modified rCRS or other alignment algorithms reveal concordant calls to Sanger (up to 11 consecutive Cs) • Some deletions outside of C tracts observed together with (concordant) substitutions (alignment algorithm) • Software constantly improving; TMAP shows generally good performance, other de novo aligners perform better when indels are present in homopolymeric regions with respect to the rCRS. Possible hybrid reference & de novo alignment fix.
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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HID Applications for PGM™ STRs
Ability to determine genotypes by sequence length and composi?on Addi?onal resolu?on between individuals with the same allele repeat structure using sequence dissimilarity in the repeat and flanking sequence
Individual A 15 repeats: TCTA [TCTG]4 [TCTA]10 Individual B 15 repeats: TCTA [TCTG]3 [TCTA]11
10 11 10 15 8 9.3
STRs II-SNPs AI-SNPs
LI-SNPs MITO D-LOOP
mtGENOME
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Future Plans
• Continue external collaborations for research applications on the PGM™ sequencer • Small amplicon mitochondrial control region
assay • Small amplicon mitochondrial assay, forensic
SNPs • Lineage SNP panel, haplogroups • Microbial forensics
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http://ioncommunity.lifetechnologies.com/community/applications/hid/
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Acknowledgments
Robert Lagacé Reina Langit Sharon Woodon Chien-‐Wei Chang Joseph Chang Narasimhan Rajagopalan
Dr. Walther Parson Dr. Kenneth K. Kidd Dr. Bruce Budowle
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09/23/12
Thank You
© 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. For Forensic or Paternity Use Only The content provided herein may relate to products that have not been officially released and is subject to change without notice.