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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
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
09/23/12
An Overview of PGM™ Technology
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
2/14/2013 | © Life Technologies 5
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
2/14/2013 | © Life Technologies 6
Sequence Detection by pH
2/14/2013 | © Life Technologies 7
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
2/14/2013 | © Life Technologies 8
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
2/14/2013 | © Life Technologies 9
Personal Genome Machine® (PGM™) Workflow
2/14/2013 | © Life Technologies 10
Workflow and Technology Summary
10
2/14/2013 | © Life Technologies 11
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™
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™
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™
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™
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
09/23/12
Development of the PGM™ HID SNP Panel
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
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
2/14/2013 | © Life Technologies 19
Library Preparation
2/14/2013 | © Life Technologies 20
Data Analysis
Torrent Server Torrent Browser Run Analysis Application-Specific Plugins
Third-Party Desktop Tools
2/14/2013 | © Life Technologies 21
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
2/14/2013 | © Life Technologies 23
HID-SNP Genotyper Plugin
2/14/2013 | © Life Technologies 24
Allele Coverage
Alleles Represented
SNPs by rs ID
HID-SNP Genotyper Plugin
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
2/14/2013 | © Life Technologies 26
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
2/14/2013 | © Life Technologies 27
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:
2/14/2013 | © Life Technologies 28
Sunshin
e
Ion Community Web Page
ioncommunity.lifetechnologies.com\
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
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.