Human Identification Applications on Ion Torrent PGM™

1/10/2013 | © Life Technologies 1

Human Identification Applications on Ion Torrent PGM™

Lisa Calandro Director, Product Management Human Identification, Life Technologies Special Acknowledgments Dr. Walther Parson, Institute of Legal Medicine, University of Innsbruck, Austria Dr. Kenneth Kidd, Department of Genetics, Yale University School of Medicine, USA


An Overview of PGM™ Technology

Development of the PGM™ HID SNP Panel

Mitochondrial Sequencing on the PGM™

Future Development Opportunities

Agenda

09/23/12

An Overview of PGM™ Technology


Sequencing Instrument

Ion OneTouch™ Instruments Emulsion PCR and Enrichment

Semiconductor Chip

Sequencing Chemistry Natural nucleotides Natural enzymes

Sample Prep Libraries Clonal beads

Torrent Server

The Ion Torrent PGM™ Instrument System

INSTRUMENTS REAGENTS DATA ANALYSIS


Sequence Detection by pH


Workflow and Technology Summary

Confidential and Proprietary—DO NOT DUPLICATE 6


Ion AmpliSeq™ Technology: As Simple As PCR Ultra-high multiplex PCR for targeted resequencing

Up to 3, 072 primer pairs per tube

Starting from just 10ng DNA

From DNA to annotated variants in as little as 10 hours

7


8

Ion AmpliSeq™ Technology How does it work?

Up to 3000 primers

10ng/rxn or X ng for x pools Ultra-Multiplex

PCR

Amplification

Phosphorylation

Adaptaters ligation Template Sequence Analysis Library


Demonstration of the Chemistry


Sequencing: Flows and Cycles

Confidential and Proprietary -- Do Not Duplicate

• A “flow” is the event of exposing the chip to one particular dNTP (T, A, C, or G), followed by a washing step • A “cycle” is four consecutive dNTP flows: T-A-C-G = 1 cycle

Ion Sphere -----Primer------ A G T C A A G C G T C C C A T G

Sequence of Interest Key Sequence

T A C G T A C G T A C G T A C G T A C G

Cycle 1 Cycle 3 Cycle 2 … etc

T

A

C

G

Cycle 1

...---Ion Sphere™


T






C



T

A

C

G

Cycle 2

...---Ion Sphere™


T






C



T

A

C

G

T

Cycle 3+

A G ...---Ion Sphere™


T






A C G



T

A

C

G

T

T T C G C A G G G T A C

And so on…

...---Ion Sphere™


Must be read “up-and-down” along with “left-to-right”

Height of bar indicates how many nucleotides incorporated during flow

Data Output: Ionogram


Amplicon-based sequencing more sensitive than other assay systems

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) – no laser or camera

Why PGM™ Sequencing?

09/23/12

Development of the PGM™ HID SNP Panel


SNP Classes

Identity SNPs Lineage-

informative SNPs

Ancestry-informative

SNPs

High Heterozygosity Low Population Heterogeneity

Phenotypic SNPs

Haplotype Markers for Kinship Analysis

High Population Heterogeneity

Hair, Eye, Skin Colour

Budowle BioT 2008

RUO v0.1 released 7/2012


Based on published SNPs with high heterozygosity and low Fst

Genotype match probabilities of 10-31 - 10-35

HID SNP Panel v0.1 (Identity Panel)

136 SNP panel

33 Y - SNP

70 - Ken Kidd

36 - SNPforID


Library Preparation


Data Analysis

Torrent Server Torrent Browser Run Analysis Application-Specific Plugins

Third-Party Desktop Tools


Panel Development Tools: Amplicon Coverage

Total Number of Amplicon Reads (Sorted) [Grey bar denoted 1 SD from the mean (red line)]

Sorted Amplicon

Num

ber o

f Rea

ds

Total Number of Amplicon Reads (Sorted) [Grey bar denoted 1 SD from the mean (red line)]

Sorted Amplicon

Num

ber o

f Rea

ds

Female Individual Male Individual

Y SNPs Y SNPs


SNP Genotype Call Viewer: Integrative Genomics Viewer (IGV)

rs891700

Coverage

Alignments

A

Het G/A

Reference

T C G


HID-SNP Genotyper Plugin


Allele Coverage

Alleles Represented

SNPs by rs ID

HID-SNP Genotyper Plugin


Next-generation sequencing technologies provide allow high levels of multiplexing for both SNPs and individuals

Development of the HID SNP Panel v0.1, an Identity SNP panel on the PGM™ sequencer using well-characterized polymorphisms

HID SNP Panel v0.1 demonstrates 99% concordance with Ken Kidd controls

HID-SNP Genotyper Plugin utility built to assist display and data evaluation

Summary


Sunshine

Ion Community Web Page

ioncommunity.lifetechnologies.com\


Continue external collaborations to research potential applications on the PGM™ sequencer

Develop ancestral/phenotypic and lineage SNP panels

Develop small amplicon mitochondrial control region assay

Investigate applications within Microbial Forensics

Future Plans


Acknowledgments

Robert Lagacé Sharon Wootton Reina Langit Joe Chang Chien-Wei Chang Narasimhan Rajagopalan

1/10/2013 | © Life Technologies 36 09/23/12

Thank You

© 2012 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.