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HID-Ion PGM™ System for Forensics

HID University Summer Seminar Series 2014

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Agenda

• Developments in Forensic Genetics • Current challenges • How can NGS help you get more information? • What is NGS? • What is the HID-Ion PGM™ System? • How does the HID-Ion PGM™ System work? • What are the HID-Ion PGM™ solutions? • How can HID-Ion PGM™ solutions help you crack tough cases? • How are others using HID-Ion PGM™ solutions? • We are here for you!

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Developments in Forensic Genetics

2014

Next Generation Sequencing

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Current challenges

• Partial or no profiles from highly degraded or trace samples • No suspect and no STR profile matches in DNA database • Laborious sequencing of mtDNA • Difficult mixture interpretations • Unknown tissue/body fluid origin • Complex kinship cases and/or familial searches

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More information can help resolve your tough cases

• Typical identification with autosomal STRs

• Small fraction of forensically-relevant markers

• CE limitations such as fragment size

There is much more forensically-relevant information available • SNP

• Identity SNPs • Ancestry SNPs • Phenotypic SNPs

• mtDNA • Lineage • DVI

• RNA • Body fluid ID

STR

NGS can provide more information, more efficiently than

conventional technologies!

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What is NGS?

Next Generation Sequencing: • Process of sequencing thousands to millions of DNA pieces

(reads) at the same time, typically followed by assembly and alignment.

• Sanger sequencing can be up to ~1000 bp • Ion Torrent™ next generation sequencing is ~200-400 bp; may

vary for others

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NGS Dictionary

• Library • A collection of amplicons with attached

sequencing adapters

• Barcode • A unique identifier attached to the ends

of a library of amplicons

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NGS Dictionary

• Read • A single stretch of continuous

nucleotides generated from DNA molecules

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Conventional technologies

Identifiler®

CE genotyping by fragment length and fluorescence

Mass spectroscopy sequence comparison in STR but not location

STR

Poly

mor

phis

ms

SNPs

Homozygote 2

Homozygote 1 Heterozygote

SNaP

shot

®

Olig

o lig

atio

n as

say

(OLA

)

TaqM

an®

Ass

ay

Limitation on

number of loci and samples run simultaneously

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NGS vs. Sanger Sequencing (CE)

• Sanger sequencing

Mixed bases: T & C

AC C170

T CCAANT C CC180

NC T AT CA T T T190

T T

Chrom Position* Genotype Coverage A Reads C Reads G Reads T Reads Deletions Freq

chr1 170 CC 3485 3 3480 0 2 0 99.85

chr1 176 CT 4776 2 2280 1 2484 9 52.01

• Next Generation Sequencing • Provide 1000X more data than Sanger sequencing*

Position Genotype

170 C

176 T/C

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* This statement is based on Ion PGM™ platform with Ion 314 chip.

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NGS has similar workflow as Sanger Sequencing

Sanger Sequencing workflow

NSG workflow

Isolate DNA and quantify

• Variant Reporter® Software

• 3500 Genetic Analyzer

• Cycle sequencing with BigDye® Terminator (fluorescent dye)

• Torrent Suite™ • Ion PGM™ system

• Ion OneTouch™ 2 • Ion Chef™ System

• Quantifiler® HP/Trio Kit

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Ion Torrent ™ Next Generation Sequencing technology

• Leveraging consumer technology for scientific breakthroughs

• Sequence detection by pH

Rothberg J.M. et al Nature doi:10.1038/nature10242

Sensor Plate

Silicon Substrate Drain Source Bulk

dNTP

To column receiver

∆ pH

∆ Q

∆ V

Sensing Layer

H+

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HID-Ion PGM™ Solutions enable more information, more efficiently

• More reads provides better sensitivity • Greater power to detect minor alleles

• Multiplexing is key • Multiplexing hundreds of SNPs or SNP+STR • Multiplexing samples (barcoding)

• Smaller SNP amplicon design for degraded DNA than CE STR • Duration from sample to result can be less than 24 hrs

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PrepFiler® Express

Quantifiler® Trio

Ion AmpliSeq™ Library Kit 2.0 Ion Library Quantitation Kit

Ion OneTouch™ 2

Ion OneTouch™ ES

Ion PGM™

Torrent Suite™ software

HID SNP Genotyper

Quantification

Library Preparation

Emulsion PCR

Data Analysis

Enrichment & Chip loading

Sequencing

Day 1 DNA Extraction

Day 2

Day 3

Plug-in analysis

1:30

7:00

2:00

3:00

2:00

1:00

6:00

Cycle Time

1:20 0:30

0:30

2:30

0:30

1:30

Hands-on

HID-Ion PGM™ Workflow (8 Samples Ion 314™ Chip)

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Ion PGM™ Sequencer

Ion OneTouch™ 2 Instruments Emulsion PCR and Enrichment

Semiconductor Chips

Sequencing Chemistry • Natural nucleotides • Natural enzymes

Sample Prep • Libraries • Clonal beads

Torrent Server

The Ion PGM™ System

INSTRUMENTS CONSUMABLES DATA ANALYSIS

Ion 314™ Chip

Ion 318™ Chip

Ion 316™ Chip

Automation available, minimizing hands-on time

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HID-Ion PGM™ System: How does it work?

It’s a Process… 1. Library Preparation 2. Quantify 3. Ion OneTouch™ 2 System* 4. Ion OneTouch™ ES System* 5. Sample on the Chip* 6. Chip on the Ion PGM™ System 7. Data! *The Ion Chef™ System can do Steps 3-5

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Library Preparation

1. Amplify the regions of interest

2. Carve back the primers

3. Add barcodes and adapters

All this in a single tube!

4. Clean up the reaction

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What happens to the Library next?

3. Ion OneTouch™ 2

4. Ion OneTouch ™ ES 5. Sample on Chip

Clonal Amplification

Load Chip Incorporate Nucleotide

Detect and Call

6. Chip on the PGM

Ion Chef™ system

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Clonal Amplification using Emulsion PCR

Isolate templated ISPs

ISP

Primer dNTPs

Polymerase MgCl2

Final Templated ISPs ready for sequencing

Emulsion droplet

P1 Barcode + A = X

ISP = DNA Taxi

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Ion OneTouch™ 2 Add Library and Ion Sphere™ Particles (ISP) PCR Part 2 – Emulsion PCR

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Post Amplification

Add Magnetic Streptavidin Bead

Immobilize to Magnet and Wash

Denature ISP with NaOH

*

*This species can be minimized through proper dilution. Proper DNA to Ion Sphere™ Particle ratio is critical!

Ion Sphere™ Particle (ISP) Enrichment

Biotin

ISP

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Ion OneTouch™ ES

• Liquid-handling robot • Enriches for ISPs with

template on them

8-well strip tube (flanked by magnets)

Pipet Tip

1) ISP sample 5) Wash Solution 2) MyOne™ Beads 6) Empty 3) Wash Solution 7) Melt-Off Solution 4) Wash Solution 8) Empty

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To the Chip!

Chips Chip Cross Section

One ISP per well…mostly

Pipet sample in through this port

Chip differences -look for the “football”

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The Ion PGM™ System • The Ion PGM™ System

• Flows a single type of nucleotide at a time over the chip

• Detects the voltage changes from the wells in the chip

• Washes the chip • Repeats a defined number of

times • Transfers data from each flow

to the Torrent Server

If the chip is the machine, then what is this thing for?

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Data - Run Report

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Torrent Server and Torrent Suite™ Software Web-based data delivery with integrated alignment, variant calling, and data analysis plugins

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Base Calling

unaligned

BAM

4.4 GB

Incorporation for 1 Flow (DAT)

Many flows (DAT)

242 GB

On Ion PGM™ System

Raw signals per flow (WELLS)

0.1 1.2 0.3 2.1 0.1 0.2 2.1 3.1 0.0 0.2 2.1 3.1 0.0 0.1 1.2 0.3 2.1 0.0 0.0 0.0 3.2 1.4 0.1 1.3 1.0 0.2 0.1

Signal Processing 12 GB On TS

~ KB

VCF Variant Caller

4.3 GB ~ 100 Mbp* (1000 X more than Sanger Sequencing)

BAM

Alignment

Torrent Suite™ Software Data Analysis Flow

HID SNP Genotyper report

~ KB

* 100Mbp is based on Ion 314 chip.

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How HID-Ion PGM™ Solutions fit into your workflow

Quantification Detection

Degradation Index • Not sufficient DNA • Highly degraded DNA

Search against CODIS database

• Partial STR profile • No matches

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HID-Ion PGM™ solutions are designed to resolve tough cases

Over Ion PGM™ Systems in use worldwide 1,000

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• About 15% of casework samples yield partial or no STR profiles due to degradation*

• Growing numbers of archived and touch samples

Why analyze highly degraded DNA?

* Based on survey results conducted by Life Technologies in Feb 2013.

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HID-Ion AmpliSeq™ Identity Panel (A25643)

Identify degraded casework samples in less than 24 hr • Ready-to-use panel consists of 124 SNPs • Small amplicon size (average132;141 nt) for degraded DNA • Only 1 ng DNA recommended • 99.99% of concordance for 43 Ken Kidd SNPs

Available now!

8 individuals per Ion 314™ chip 38 individuals per Ion 316™ chip 77 individuals per Ion 318™ chip

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HID-Ion AmpliSeq™ Identity Panel Markers

1 Pakstis, A. J., Speed, W. C., Fang, R., Hyland, F. C., Furtado, M. R., Kidd, J. R., & Kidd, K. K. (2010). SNPs for a universal individual identification panel. Human Genetics, 127(3), 315–324. 2 Phillips, C., Fang, R., Ballard, D., Fondevila, M., Harrison, C., Hyland, F., et al. (2007). Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel. Forensic Science International: Genetics, 1(2), 180–185. 3 Karafet, T. M., Mendez, F. L., Meilerman, M. B., Underhill, P. A., Zegura, S. L., & Hammer, M. F. (2008). New binary polymorphisms reshape and increase resolution of the human Y chromosomal haplogroup tree. Genome Research, 18(5), 830–838.

43 Ken Kidd (45 unlinked set 1)

48 SNPforID2

34 upper Y-clade3

Avg. marker size is141 nts

Markers

Size (nts)

90 autosomal markers Avg. size is 132 nts

33 85 unlinked autosomal SNPs from 1000 Genomes data, PI calculated from 1000 Genomes

Decrease in amplicon size (higher degradation)

The HID-Ion AmpliSeq™ Identity panel enables significant match probabilities at greater levels of degradation

Bet

ter R

MP

(rand

om m

atch

pro

babi

lity)

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Get the answer in HID SNP Genotyper

Automatic population statistic calculation and profile comparison against one another within run

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• No suspect and no STR profile matches in DNA database

• Ancestry or phenotype such as eye, hair, or skin color information can provide investigative leads to law enforcement officers

Why generate more investigative leads?

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Obtain biogeographic ancestry information for more investigative leads • Ready-to-use panel consists of 165 autosomal SNPs • Suitable for casework samples or degraded DNA • Small amplicon size (average122;130 nt) • Only 1 ng DNA recommended • 99.77% of concordance for 55 Ken Kidd SNPs

HID-Ion AmpliSeq™ Ancestry Panel (A25642)

*Autosomal SNPs ≥ 300x

Available now!

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Visualize the ancestry origins on a map

• Free HID SNP Genotyper plug in • Provides automatic calculation of likelihood ratio for population groups

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Analyze DNA mixtures more efficiently

Example: • A 15 is not always just a 15… • Individual A 15 repeats: TCTA [TCTG]4 [TCTA]10 • Individual B 15 repeats: TCTA [TCTG]3 [TCTA]11

• STR panel on HID-Ion PGM™ System can be a potential solution for difficult mixture analysis • Provides not only the allele number but also the

sequence for each repeat • Can be combined with other types of markers such as

SNPs

• Currently testing CODIS and non-CODIS STR panels

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HID-Ion PGM™ STR solution (in development)

Forensics or Paternity Use Only

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• Current Sanger sequencing is laborious • Sequencing the whole mitochondrial genome

offers more information • Ion AmpliSeq™ technology can streamline the

whole mt genome sequencing workflow

Analyze missing person or remains efficiently

File reproduced in accordance with creative commons public domain license: original work by jhc, who grants anyone the right to use this work for any purpose, without any conditions, unless such conditions are required by law, derivative work by Shanel is shown. http://commons.wikimedia.org/wiki/File:Mitochondrial_DNA_en.svg

• PGM™ Mitochondrial WGS protocol • Substantially higher throughput compared to Sanger • High concordance between Sanger and PGM

• Whole Mitochondrial Tiling path panel (In development)

• Small amplicon size design for degraded samples • Simple and fast workflow by Ion AmpliSeq technology

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First publication for mtDNA analysis on PGM in 2013

“These results are extremely promising, and are expected to significantly increase the random-match probability for mtDNA analysis when evaluating highly compromised samples.” Dr. Walther Parson, Institute of Legal Medicine, Innsbruck, Austria

How are others using HID-Ion PGM™ Solutions?

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How are others using HID-Ion PGM™ Solutions? cont'd

Professor Niels Morling MD DMSc Director and chairman of the Department of Forensic Medicine Faculty of Health and Medical Sciences University of Copenhagen

SNP analysis on PGM

STR analysis on Ion PGM

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The biggest impact is that we will be able to combine a large number of genetic markers of different types in a single run analysis, including markers such as STRs and SNPs (single base pair changes)—potentially even RNA that cannot be multiplexed with any other current technology. This will open up new possibilities, as various types of forensically relevant information can be obtained from a single sample analysis.” Dr. Manfred Kayser, Dept. of Forensic Molecular Biology, Erasmus University, Rotterdam, The Netherlands

How are others using HID-Ion PGM™ Solutions? cont'd

Bruce Budowle, Ph.D. Professor, Executive Director of Institute of Applied Genetics Dept: Molecular and Medical Genetics

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Are you facing any of these challenges?

No or Partial profile from degraded DNA

No matches in DNA database

Laborious mtDNA sequencing for missing person

Difficult mixture interpretations

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We are here for you!

Please contact your local Sales rep for any questions!

Learn More about NGS for Forensics at lifetechnologies.com/HID-NGS

Learn More or order our Ion AmpliSeq™ Ancestry or

Identity panel here lifetechnologies.com/ancestry lifetechnologies.com/identity Download HID SNP Genotyper Plugin at Ion Community

http://ioncommunity.lifetechnologies.com/docs/DOC-3138

Follow us at human_id

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• STR panels are for Forensics or Paternity Use Only • The rest of the panels are for Research, Forensic or Paternity

Use Only. When used for purposes other than Human Identification the instruments and modules of the cited software are for Research Use Only. Not for use in diagnostic procedures.

• © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

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Thank you & Questions?