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


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)


Agenda

• Next-Generation Sequencing Technology on PGM™ • HID Applications using PGM™

§  Identity SNPs

§  Ancestral informative and phenotypic SNPs

§  Mitochondrial Sequencing

§  STRs • Future plans


NGS Technology on PGM™


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


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


Sequence Detection by pH


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)


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)


HID Applications for PGM™

STRs II-SNPs AI-SNPs

LI-SNPs MITO D-LOOP

mtGENOME


HID Applications on PGM™


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


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)


HID Applications for PGM™ SNP Applications


informa?ve SNPs


SNPs


Phenotypic SNPs





LI-SNPs MITO D-LOOP

mtGENOME



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


LI-SNPs MITO D-LOOP

mtGENOME



informa?ve SNPs


SNPs


Phenotypic SNPs




HID Applica?ons for PGM™ Ancestral and Phenotypic SNP Panel


LI-SNPs MITO D-LOOP

mtGENOME



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


LI-SNPs MITO D-LOOP

mtGENOME


HID Applications for PGM™ Data Analysis

Torrent Server Torrent Browser Run Analysis Application specific plugins

Third-party desktop tools


Y-SNPs

Panel Development Tools: Amplicon Coverage

Female Individual Male Individual

SNP Panel v2.2

Mean 1 StdDev from Mean


HID-‐SNP Genotyper Plugin


allele coverage

alleles represented

SNPs by rs ID

HID-‐SNP Genotyper Plugin


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


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


LI-SNPs MITO D-LOOP

mtGENOME


Whole Genome Protocol Data and Results Courtesy of Walther Parson’s Laboratory

PGM™ Mitochondrial Sequencing


Whole Genome Mitochondrial DNA Library Preparation


LI-SNPs MITO D-LOOP

mtGENOME


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.


LI-SNPs MITO D-LOOP

mtGENOME


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


LI-SNPs MITO D-LOOP

mtGENOME


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


http://ioncommunity.lifetechnologies.com/community/applications/hid/



Acknowledgments

Robert Lagacé Reina Langit Sharon Woodon Chien-‐Wei Chang Joseph Chang Narasimhan Rajagopalan

Dr. Walther Parson Dr. Kenneth K. Kidd Dr. Bruce Budowle


09/23/12

Thank You

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