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1
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!
3
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
5
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!
6
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
176
* 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?
25
Data - Run Report
26
Torrent Server and Torrent Suite™ Software Web-based data delivery with integrated alignment, variant calling, and data analysis plugins
27
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)
34
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
38
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
45
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?