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2/14/2013 | © Life Technologies 1 Human Identification Applications on Ion Torrent™ PGM System Lisa Calandro Director, Product Management Human Identification, Life Technologies Dr. Walther Parson Institute of Legal Medicine University of Innsbruck, Austria

Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Page 1: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 1

Human Identification Applicationson Ion Torrent™ PGM™ System

Lisa Calandro

Director, Product Management

Human Identification, Life Technologies

Dr. Walther Parson

Institute of Legal Medicine

University of Innsbruck, Austria

Page 2: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 2

An Overview of PGM™ Technology

Development of the PGM™ HID SNP Panel

Mitochondrial DNA Sequencing on the PGM™ System

Agenda

Page 3: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

09/23/12

An Overview of PGM™ Technology

Page 4: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 4

Next Generation Sequencing Increases the Amount of Information One Can Obtain from a Biological Sample

Human Forensics: – Determine multiple polymorphisms:

STRs, SNPs, Indels, etc.

– Develop investigative leads: phenotypic/ancestral SNPs

– Missing persons, DVI

Microbial Forensics:- Resequencing and de novo

sequencing with a high degree of accuracy

- Metagenomics for environmental monitoring

Page 5: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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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

Page 6: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Sequence Detection by pH

Page 7: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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

Custom design service− Bioinformatic pipeline− System-specific chemistry

Allows high levels of multiplexing− Several thousand targets

Starting from just 10ng DNA

From DNA to annotated variants in as little as 10 hours

7

Page 8: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Ion AmpliSeq™ Custom Solutions

8

Chip Type Wells Capacity Genome Coverage

500X(Somatic)

20X(Germline)

Ion 314™ 1M 50 Mb 1 kb 10 kb

Ion 316™ 6M 200-250 Mb 50 kb 100 kb

Ion 318™ 12M 1.2 Gb 500kb 1,000 kb

Size your panel to match the Ion Chip

Sample size = target length x depth of coverage

16 kb x 100 (1.6 Mb) 30 samples on 314 chip/96 on 316 chip

16 kb x 1000 (16 Mb) 3 samples on 314 chip / 15 on 316

Page 9: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Personal Genome Machine® (PGM™) Workflow

Page 10: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 10

Workflow and Technology Summary

10

Page 11: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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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 InterestKey Sequence

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

Cycle 1 Cycle 3Cycle 2… etc

T

A

C

GCycle 1

...---Ion

Sphere™

Page 12: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 12

T

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 InterestKey Sequence

C

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

Cycle 1 Cycle 3Cycle 2… etc

T

A

C

G

Cycle 2

...---Ion

Sphere™

Page 13: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 13

T

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 InterestKey Sequence

C

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

Cycle 1 Cycle 3Cycle 2… etc

T

A

C

G

T

Cycle 3+

A G

...---Ion

Sphere™

Page 14: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 14

T

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 InterestKey Sequence

AC G

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

Cycle 1 Cycle 3Cycle 2… etc

T

A

C

G

T

TT C G C A GGGT A C

And so on…

...---Ion

Sphere™

Page 15: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 15

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

Height of bar indicates how many nucleotides incorporated during flow

Data Output: Ionogram

Page 16: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

09/23/12

Development of the PGM™ HID SNP Panel

Page 17: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 17

SNP Classes

Identity SNPsLineage-

informativeSNPs

Ancestry-informative

SNPs

High HeterozygosityLow Population Heterogeneity

PhenotypicSNPs

Haplotype Markers for Kinship Analysis

High Population Heterogeneity

Hair, Eye, Skin Colour

Budowle BioT 2008

RUO v0.1 released 7/2012

Page 18: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 1809/23/12

HID SNP Panel v2.2 (Identity Panel)

Based on published SNPs with high heterozygosity and low Fst

Genotype match probabilities of 10-31 - 10-35

179 SNPs

35 Y -

SNPs

92 - Ken Kidd

SNPs

52 - SNPforID

SNPlex

Page 19: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Library Preparation

Page 20: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Data Analysis

Torrent Server Torrent Browser Run Analysis Application-Specific Plugins

Third-Party Desktop Tools

Page 21: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Panel Development Tools: Amplicon Coverage

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

Sorted Amplicon

Nu

mb

ero

f R

ead

s

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

Sorted Amplicon

Nu

mb

ero

f R

ead

s

Female Individual Male Individual

Y SNPs Y SNPs

Page 22: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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HID-SNP Genotyper Plugin

Page 23: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 24

Allele Coverage

Alleles Represented

SNPs by rs ID

HID-SNP Genotyper Plugin

Page 24: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 25

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

Development of the HID SNP Panel v2.2, an Identity SNP panel on the PGM™ sequencer using well-characterized polymorphisms

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

Summary

Page 25: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Future Plans: Ancestral informative and phenotypic SNP Panel

hair and eye colorSNPs

Application: Subject exclusion and investigative leads

1. high discrimination between ancestral groups

2. hair and eye color determination

Based on panels validated on a wide range of population datasets

245 SNPs

Ancestral SNPs

202

45

Page 26: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

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Future Plans – STR Analysis

Ability to determine genotypes by sequence length and composition

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

Additional resolution between individuals with the same allele repeat structure using sequence dissimilarity in the repeat and flanking sequence:

Page 27: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 28

Sunshin

e

Ion Community Web Page

ioncommunity.lifetechnologies.com\

Page 28: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 29

Acknowledgments

R&D Team: Collaborators & Advisors:Robert Lagacé Dr. Kenneth Kidd

Sharon Wootton Dr. Art Eisenberg

Reina Langit Dr. Bruce Budowle

Joe Chang Dr. Walther Parson

Chien-Wei Chang

Narasimhan Rajagopalan

Nigel Beard

Page 29: Human Identification Applications on Ion Torrent™ PGM™System – Lisa Calandro

2/14/2013 | © Life Technologies 3009/23/12

Thank You

© 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliates 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.