exploring human identification applications for the ion torrent personal genome machine sequencing...
TRANSCRIPT
Exploring Human Identification Applications for the Ion Torrent Personal Genome Machine
(PGM™) Sequencing Instrument Human Identification Group, Life Technologies
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Agenda • Overview of PGM™ instrument technology and chemistry
• Overview of PGM™ instrument workflow
• Potential applications of PGM™ instrument for human identification
• Early feasibility studies
• Future plans
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Genotyping by Fragment Analysis or Sequencing
3500 Genetic Analyzer Fragment or sequence analysis by fluorescent
detection
Y-STR Haplotype
SNP Genotype
mtDNA Haplotype
Microbial ID
STR Genotype
Ion Torrent PGM™ Sequence analysis by
proton detection
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Human Identification Applications on PGM™
STRs Mito SNPs
Short Tandem Repeats
Mitochondrial DNA
Single Nucleotide
Polymorphisms
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PGM™ Instrument Technology
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PGM™
One Touch Instruments Emulsion PCR and Enrichment
Instruments Semiconductor Chip
Sequencing Chemistry • Natural nucleotides • Natural enzymes
Sample Prep • Libraries • Clonal beads
Reagents Torrent Server
Informatics
The Ion Torrent PGM™ Instrument System
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Ion Technology Core Principles
• Scalability • Semiconductor technology • 40 years of Moore’s law
• Simplicity • Natural nucleotides • No lasers • No optics • No camera • No fluorescence • No enzyme cascade
• Speed • Rapid detection of sequence
extension The Chip is the Machine TM
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Scalable Semiconductor Technology
Wafer Semiconductor Manufacturing
Chip Semiconductor Packaging
Chip Cross Section Semiconductor Design
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Semiconductor Scalability
▲ Ion 314 >150 Mb
▲ Ion 316 >850 Mb
▲ Ion 318 >1400 Mb
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Sanger Sequencing Method for CE
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PGM™ Instrument uses Simple Natural Chemistry
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1 Nucleotide bases (dNTP’s) are sequentially flowed into well one at a time
2 Upon incorporation, the nucleotide releases a hydrogen ions which creates a pH change in the well
3 Sensing layer detects the change in pH
4 Sensing plate translates the chemical signal to a digital signal
5 Voltage detected is proportional to number of bases incorporated
Electric voltage measurement
Detection Chemistry on the PGM™ Instrument
Sensor Plate
Silicon Substrate
Drain Source Bulk
∆ pH
∆ V
Sensing Layer
Schematic cross-section of a single well on a Ion Torrent sequencing chip
Bead with clonally amplified template
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Demonstration of the Chemistry
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Data Output is an Ionogram • An “Ionogram” is the output of the signals in flow space
• Must be read “up-and-down” along with “left-to-right”
• Height of bar indicates how many nucleotides incorporated during flow
• “Negative” or “zero” flows indicate no nucleotide incorporation • These observations are omitted when converting to nucleotide space
Key Sequence
Sequence: …AATCTTCTGAATTTCTGCAA…. (TTT)
(AA) (AA)
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Summary of Technology
• Based on semiconductor technology • Simple, natural nucleotides • Nucleotides are flowed into chip one at a time • Nucleotide incorporation releases a H+ proton • Measure pH change by change in voltage • Voltage detected is proportional to number of bases
incorporated • Nucleotide sequence displayed as an Ionogram
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PGM™ Instrument Workflow
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PGM™ Workflow
OneTouch™ OneTouch™ES PGM™ AutoMate Express™ Torrent Server & Torrent Browser
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OneTouch™ and OneTouch ES™ Instruments
Biotinylated Template + ISP
MyOne Bead + Streptavidin
Non-Templated ISP
emPCR®
Enrichment
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Sequencing the Targeted Amplicons
Loading Port
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Ion Torrent™ Server for Data Analysis
Desktop Analysis
Plugin Store (Ion Community)
Plug-Ins Cloud Analysis
NextBioXfr (Plug-In)
Torrent Server Torrent Browser Run Reports Plug-Ins
HID_SNP_Genotyper variantCallerForMtDNAAlignment (TMAP) RNA-seq (IsoEM) U. Connecticut Variant Annotation (SNPeff) Edge Biosystems De novo assembly (MIRA)
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Ion Torrent™ Product Workflow
Individual Step Application Specific
Multiplex Step Generic
Multiplex Step Generic
Multiplex Step Application Specific
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Ion Community http://ioncommunity.iontorrent.com
PGM™ Users − Site prep − User guides − FAQs − Videos − Discussion Forum
Torrent Suite − Torrent Suite Guides − Release notes − File formats − Database schema
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Potential Human Identification Applications for PGM™ Instrument
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Human Identification Applications on PGM™
STRs Mito SNPs
Short Tandem Repeats
Mitochondrial DNA
Single Nucleotide
Polymorphisms
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Common STR Genotyping Technologies
• Capillary electrophoresis • Detection of STR loci by fragment length and fluorescence
• Mass spectroscopy • Sequence differences in STR alleles but not location
Identifiler® Kit
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PGM for STR Genotyping • Allows large number of STR loci to be sequenced simultaneously
• Combine autosomal, Y, X STR markers • Increased discrimination from SNPs in repeats or flanking regions
• Allows identification of up to 96 individuals simultaneously on one chip
• Requires 10 ng of DNA input- similar to most NGS platforms • Suitable for reference type samples • Optimization of lower input needed for forensic samples
• May require updated database information • Process/criteria for uploading sequence information and/or SNPs in the profiles • Process/criteria for query of profile
• Convert to length to query existing databases
• Use SNP information for further exclusion capability • Frequency information on SNPs detected
• For the purposes of statistical calculations for inclusion
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Human Identification Assays on PGM™ Mitochondrial DNA Sequencing
Missing person/verification of human remains
Genealogical research
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MOST COMMON APPROACH - Sanger sequence with BDT v1.1 chemistry
Mitochondrial DNA Sequencing Methods
SNaPShot for mito coding region SNPs
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PGM™ for Mitochondrial Sequencing • Able to sequence the whole 16 kb mito genome on one chip with high coverage
• CE requires 90 reactions (26 for PCR, 64 for sequencing) for whole genome sequencing using the MitoSeq protocol
• Able to sequence the control region plus targeted SNPs in the coding region on one chip
• Able to sequence up to 96 individuals on a single chip
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Mitochondrial Whole Genome Sequencing
Coverage across the mtGenome Number of reads mapped
Mitochondrial genome - 16,569 bases
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Mitochondrial Whole Genome Sequencing
Coverage
C T
Reference
Zoomed into Polymorphisms
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Mitochondrial Haplotypes H
AP
LOG
RO
UP
A
SS
EM
BLY
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Potential Human Identification Assays on PGM™
• SNP genotyping • Missing person identification • Paternity • DVI • Molecular “phenotype”
• Phenotypic SNPs for investigative leads
Eye color
Hair color
Facial reconstruction
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Features of SNPs • Abundant in the human genome (~9 million)
• 90% of human genetic variation comes from SNPs • SNPs occur about every 300 bp; coding and non-coding regions • Most SNPs are biallelic • Low mutation rate (1 X 10-9 per locus per generation) • High heterozygosity & low population heterogeneity • Small amplicon size
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Common SNP Technologies • Allele discrimination methods
• Sequencing • Primer extension • Ligation • Hybridization • Enzymatic cleavage
Limitation on number of SNPs detected simultaneously
SNaPshot® Assay
Oligo ligation assay (OLA)
Homozygote 2
Homozygote 1 Heterozygote
TaqMan® Assay
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PGM™ for SNP Genotyping
• Allows combination of large number of SNPs in one multiplex • Combine autosomal, Y-, X- chromosome and phenotypic SNPs simultaneously
• Allows identification of up to 96 individuals simultaneously on one chip
• Requires 10 ng of DNA input - similar to most NGS platforms • Suitable for reference type samples • Optimization of lower input needed for forensic samples
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PGM™ for SNP Genotyping
• 136 SNPs – 103 autosomal and 33 Y covered by amplicons < 150bp
• Based on published SNPs with high heterozygosity and low Fst
• Genotype match probabilities of 10-31 - 10-35
Human Identification SNP panel version 01
Each arrow represents one or more SNPs
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Library Preparation Protocol
Prepare library 10ng DNA input
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SNP Amplicon Coverage
.
Female - Individual
Y SNPs
Male - Individual
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HID on ION Community Website
Visit ION Community Website
What’s Available: Protocols, Plug ins, and other Tools Click HID icon Register/Login
ION COMMUNITY PAGE HID PAGE
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Genotype Calling on PGM™
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Genotype Calling on PGM™
SNPs
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Ion AmpliSeq™ Technology: Designer or Custom Panels
Single-tube ultra-high multiplex PCR, single day workflow More Info @ www.ampliseq.com
+ Up to 6,144 primer pairs per tube
10 ng gDNA
Construct Library Prepare Template Run Sequence Analyze Data Customize Panel
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Summary Demonstrated Applications for HID
• Whole mitochondrial genome of five individuals sequenced on 1 chip
• Autosomal and Y SNPs from multiple individuals sequenced on 1 chip
• 314 chip mean high quality coverage was 50x for mitochondrial genome and 1000x for SNPs
• Barcodes enable sequencing multiple samples on 1 chip
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Future Plans • Investigate potential external collaborators for research applications on
the PGM
• Explore potential research protocols • for whole mitochondrial genome sequencing for reference sample • for sequencing large SNP multiplexes for reference samples
• Investigate sequencing of other human or microbial identification markers
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Thank You
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