Applica'ons  of  Personal  Genome  Machine  (PGM™)  in  SNP-­‐based  Human  Iden'fica'onSharon  Chao  Woo*on,  PhDSenior  Bioinforma3cs  Scien3stHuman  Iden3fica3onLife  Technologies

07/02/12

Agenda

•Overview  of  PGM™  applica2ons

•SNPs  in  human  iden2fica2on

•PGM™  Technology

•Development  of  a  SNP  panel

•Future  plans

07/03/12

Applica'ons  on  the  PGM  

STRGenotype

SNPGenotype

mtDNAHaplotype

Y-­‐STRGenotype

MicrobialForensics

07/03/12

Applica'ons  on  the  PGM  

STRGenotype

SNPGenotype

mtDNAHaplotype

Iden%ty  SNPs

Lineage-­‐informa%ve

SNPs

Ancestry-­‐informa%ve  

SNPs

•  High  heterozygosity•  Low  popula'on  heterogeneity

PhenotypicSNPs

•  Haplotype  markers  for  kinship  analysis•  Mitochondrial  genome  or  control  region•  Y-­‐chr  SNPs•  Mini  haplogroups

•  High  popula'on  heterogeneity

•  Hair,  eye,  skin  color

Y-­‐STRGenotype

MicrobialForensics

07/02/12

PGM™  Applica'ons  -­‐  SNP  Genotyping

•  Abundant  in  the  human  genome  (~9  million)• 90%    of  human  gene2c  varia2on  comes  from  SNPs• SNPs  occur  about  every  300  bp;  coding  and  non  coding  regions•Most  SNPs  are  bi-­‐allelic• Low  muta2on  rate  (10  -­‐8  -­‐  10-­‐9  per  locus  per  genera2on)• Small  amplicon  size

STRGenotype

SNPGenotype

mtDNAHaplotype

Y-­‐STRGenotype

MicrobialForensics

07/02/12

•  Missing  person  iden2fica2on•  Paternity•  DVI•  Ancestral  haplotyping•  Molecular  “phenotype”

PGM™  Applica'ons  -­‐  SNP  Genotyping

07/03/12

Current  SNP  Technologies

•  Allele  discrimina2on  methods• Sequencing• Primer  extension• Liga2on•Hybridiza2on• Enzyma2c  cleavage

Homozygote 2

Homozygote 1Heterozygote

SNaP

shot

®O

ligo

ligat

ion

assa

y (O

LA)

TaqM

an®

Ass

ay

07/03/12

Current  SNP  Technologies

•  Allele  discrimina2on  methods• Sequencing• Primer  extension• Liga2on•Hybridiza2on• Enzyma2c  cleavage

Homozygote 2

Homozygote 1Heterozygote

SNaP

shot

®O

ligo

ligat

ion

assa

y (O

LA)

TaqM

an®

Ass

ay

Limitation on number of SNPs and samples run simultaneously

07/02/12

PGM™  for  SNP  Genotyping

•  Allows  combina2on  of  large  number  of  SNPs  in  one  mul2plex

• Simultaneous  sequencing  of  autosomal,  Y-­‐chr,  X-­‐chr,  mitochondrial  SNPs  possible

•  Barcode  up  to  96  individuals  and  sequence  on  one  chip•  Output  is  sequence,  not  an  indicator

07/02/12

PGM™  Instrument

07/02/12

 Sequencing  Instrument

 One  Touch™  Instruments  Emulsion  PCR  and  Enrichment

 Semiconductor  Chip

 Sequencing  Chemistry•  Natural  nucleo3des•  Natural  enzymes

 Sample  Prep•  Libraries•  Clonal  beads

 Torrent  Server

The  Ion  Torrent  PGM™  Instrument  System

INSTRUMENTS REAGENTS DATA  ANALYSIS

07/02/12

▲ Ion 314 >150 Mb

▲ Ion 316 >850 Mb

▲ Ion 318 >1400 Mb

Ion  chip  scalability

07/03/12

Leveraging  Semiconductor  Technology  

WAFER  SEMICONDUCTOR  MANUFACTURING

CHIP  SEMICONDUCTOR  PACKAGING

CHIP  CROSS  SECTION  SEMICONDUCTOR  DESIGN

07/03/12

Sequence  detec'on  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+

07/02/12

•  Must  be  read  “up-­‐and-­‐down”  along  with  “le`-­‐to-­‐right”

•  Height  of  bar  indicates  how  many  nucleo2des  incorporated  during  flow

• “Negative” or “zero” flows indicate no nucleotide incorporation

•“Nega2ve”  or  “zero”  flows  indicate  no  nucleo2de  incorpora2on• These  observa2ons  are  omibed  when  conver2ng  to  nucleo2de  space

Data  Output  is  an  Ionogram

Key Sequence

Sequence: …AATCTTCTGAATTTCTGCAA…. (TTT)

(AA) (AA)

07/02/12

PGM™  Applica'ons

STRGenotype

SNPGenotype

mtDNAHaplotype

Y-­‐STRGenotype

MicrobialForensics

07/02/12

PGM™  Applica'ons

Iden%ty  SNPs

Lineage-­‐informa%ve

SNPs

Ancestry-­‐informa%ve  

SNPs

PhenotypicSNPs

•  High  heterozygosity•  Low  popula'on  heterogeneity

•  Haplotype  markers  for  kinship  analysis•  Mitochondrial  genome  or  control  region•  Y-­‐chr  SNPs•  Mini  haplogroups

•  High  popula%on  heterogeneity

•  Hair,  eye,  skin  color

STRGenotype

SNPGenotype

mtDNAHaplotype

Y-­‐STRGenotype

MicrobialForensics

07/02/12

HID  SNP  Panel  v0.1

•Based  on  published  SNPs  with  high  heterozygosity  and  low  Fst

•Genotype  match  probabili2es  of  10-­‐31  -­‐  10-­‐35  136 SNPs

33 Y - SNP

70 - Ken Kidd SNPs

36 - SNPforID SNPlex

07/02/12

Popula'on  data

Kidd  Lab’s  ALFRED  (the  ALlele  FREquency  Database)  

ALFRED  allele  frequencies  by  popula7on  for  rs7704770

07/02/12

SNP  Panel  Development  -­‐  Ampliseq™

Construct Library Prepare Template Run Sequence Analyze DataCustomize Panel

AMPLISEQ™ CUSTOM PANEL

• Up to 6,144 primer pairs

• 10 ng DNA input

• Up to 200 bp targets

MULTIPLEX -CLONAL BEADAMPLIFICATION

Generic

MULTIPLEX -SEQUENCING ON SINGLE CHIP

Generic

SNP GENOTYPE PLUGIN

Analysis custom to SNP panel

07/03/12

Library  Prepara'on

Short Amplicon Method Long Amplicon Method

Target amplicons 75 - 200 bp Target amplicons > 200 bp

Genomic DNA

FWD

REV

FWD

REV

Short amplicon pool 75 - 200 bp

PCR, pool amplicons, end-repair

PCR, pool amplicons, fragment with Ion Shear™ Kit

Fragmented long amplicon pool 50 - 500 bp

Genomic DNA

Adaptor ligation

OR

Barcode adaptors ligation

P1IA

P1IA-BCx

Nick-translation and PCR

Final barcoded libraryIA BCx Target amplicon P1

Construct Library Prepare Template Run Sequence Analyze DataCustomize Panel

AMPLISEQ™ CUSTOM PANEL

• Up to 6,144 primer pairs

• 10 ng DNA input

• Up to 200 bp targets

MULTIPLEX -CLONAL BEADAMPLIFICATION

Generic

MULTIPLEX -SEQUENCING ON SINGLE CHIP

Generic

SNP GENOTYPE PLUGIN

Analysis custom to SNP panel

07/03/12

Library  Prepara'on

Short Amplicon Method Long Amplicon Method

Target amplicons 75 - 200 bp Target amplicons > 200 bp

Genomic DNA

FWD

REV

FWD

REV

Short amplicon pool 75 - 200 bp

PCR, pool amplicons, end-repair

PCR, pool amplicons, fragment with Ion Shear™ Kit

Fragmented long amplicon pool 50 - 500 bp

Genomic DNA

Adaptor ligation

OR

Barcode adaptors ligation

P1IA

P1IA-BCx

Nick-translation and PCR

Final barcoded libraryIA BCx Target amplicon P1

Construct Library Prepare Template Run Sequence Analyze DataCustomize Panel

AMPLISEQ™ CUSTOM PANEL

• Up to 6,144 primer pairs

• 10 ng DNA input

• Up to 200 bp targets

MULTIPLEX -CLONAL BEADAMPLIFICATION

Generic

MULTIPLEX -SEQUENCING ON SINGLE CHIP

Generic

SNP GENOTYPE PLUGIN

Analysis custom to SNP panel

07/02/12

Data  analysisConstruct Library Prepare Template Run Sequence Analyze DataCustomize Panel

AMPLISEQ™ CUSTOM PANEL

• Up to 6,144 primer pairs

• 10 ng DNA input

• Up to 200 bp targets

MULTIPLEX -CLONAL BEADAMPLIFICATION

Generic

MULTIPLEX -SEQUENCING ON SINGLE CHIP

Generic

SNP GENOTYPE PLUGIN

Analysis custom to SNP panel

07/02/12

Construct Library Prepare Template Run Sequence Analyze DataCustomize Panel

AMPLISEQ™ CUSTOM PANEL

• Up to 6,144 primer pairs

• 10 ng DNA input

• Up to 200 bp targets

MULTIPLEX -CLONAL BEADAMPLIFICATION

Generic

MULTIPLEX -SEQUENCING ON SINGLE CHIP

Generic

SNP GENOTYPE PLUGIN

Analysis custom to SNP panelSNP  genotype  calling

rs891700

coverage

alignments

ATCG

Het  G/A

reference  

0"

100"

200"

300"

400"

500"

600"

700"

800"

900"

T"G"C"A"

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0"

100"

200"

300"

400"

500"

600"

T"G"C"A"

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0"

200"

400"

600"

800"

1000"

1200"

T"G"C"A"

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0"

50"

100"

150"

200"

250"

300"

350"

400"

T"G"C"A"

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

BC6

BC7

BC8

BC9

BC10

BC6

BC7

BC8

BC9

BC10

0"

100"

200"

300"

400"

500"

600"

700"

rs1490413"

rs10495407"

rs891700"

rs1413212"

rs876724"

rs907100"

rs1357617"

rs1355366"

rs2046361"

rs1979255"

rs717302"

rs251934"

rs1029047"

rs727811"

rs917118"

rs737681"

rs763869"

rs2056277"

rs1015250"

rs1463729"

rs1360288"

rs735155"

rs964681"

rs901398"

rs2076848"

rs2107612"

rs2111980"

rs1335873"

rs1886510"

rs354439"

rs1454361"

rs873196"

rs8037429"

rs1528460"

rs729172"

rs1382387"

rs740910"

rs938283"

rs1493232"

rs1024116"

rs719366"

rs1031825"

rs1005533"

rs722098"

rs2831700"

rs914165"

rs733164"

rs2040411"

rs1028528"

T"G"C"A"

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

rs1490413#

rs10495407#

rs891700#

rs1413212#

rs876724#

rs907100#

rs1357617#

rs1355366#

rs2046361#

rs1979255#

rs717302#

rs251934#

rs1029047#

rs727811#

rs917118#

rs737681#

rs763869#

rs2056277#

rs1015250#

rs1463729#

rs1360288#

rs735155#

rs964681#

rs901398#

rs2076848#

rs2107612#

rs2111980#

rs1335873#

rs1886510#

rs354439#

rs1454361#

rs873196#

rs8037429#

rs1528460#

rs729172#

rs1382387#

rs740910#

rs938283#

rs1493232#

rs1024116#

rs719366#

rs1031825#

rs1005533#

rs722098#

rs2831700#

rs914165#

rs733164#

rs2040411#

rs1028528#

T#

G#

C#

A#

Reads per allele Reads per allele normalized

Coverage  needed  to  overcome  undercalling

100x

100x

100x

100x

100x

SNPs  by  rs  ID SNPs  by  rs  ID

Depth  of  coverag

e  for  e

ach  allele

Allele  alloca7on

0"

100"

200"

300"

400"

500"

600"

700"

800"

900"

rs1490413(

rs7520386(

rs560681(

rs10495407(

rs891700(

rs1413212(

rs876724(

rs12997453(

rs1357617(

rs9866013(

rs1872575(

rs1355366(

rs6444724(

rs13134862(

rs1554472(

rs6811238(

rs1979255(

rs717302(

rs159606(

rs13182883(

rs7704770(

rs251934(

rs338882(

rs1029047(

rs13218440(

rs2811231(

rs1478829(

rs1358856(

rs2503107(

rs2272998(

rs214955(

rs727811(

rs6955448(

rs917118(

rs1019029(

rs321198(

rs737681(

rs10092491(

rs4288409(

rs2056277(

rs4606077(

rs2270529(

rs7041158(

rs1463729(

rs10776839(

rs735155(

rs3780962(

rs1410059(

rs740598(

rs964681(

rs10768550(

rs10500617(

rs1498553(

rs901398(

rs6591147(

rs590162(

rs2107612(

rs2255301(

rs2269355(

rs2111980(

rs10773760(

rs1886510(

rs9546538(

rs1058083(

rs354439(

rs1454361(

rs873196(

rs4530059(

rs1821380(

rs729172(

rs2342747(

rs430046(

rs1382387(

rs2175957(

rs8070085(

rs1004357(

rs1027895(

rs8078417(

rs2291395(

rs4789798(

rs689512(

rs3744163(

rs2292972(

rs1493232(

rs9951171(

rs7229946(

rs985492(

rs521861(

rs1736442(

rs1024116(

rs719366(

rs576261(

rs12480506(

rs2567608(

rs1005533(

rs1523537(

rs722098(

rs464663(

rs2833736(

rs914165(

rs9606186(

rs5746846(

rs2073383(

rs733164(

rs987640(

rs2040411(

rs1028528(

rs1800865(

rs2075640(

rs2299942(

rs2267801(

rs2267802(

rs2071394(

rs1865680(

rs2075182.3(

rs2075181(

rs1515817(

rs2032595(

rs2032598(

rs2032599(

rs2032601(

rs2032600(

rs2032607(

rs2032604(

rs2032624(

rs2020857(

rs2032668(

rs2032666(

rs2032658(

rs2072422(

rs2032653(

rs1864258(

rs3897(

rs3900(

rs891407(

rs2032611(

rs2032631(

rs2032673(

rs2032626(

rs1558843(

rs1276035(

rs1276034(

T"G"C"A"

0"

100"

200"

300"

400"

500"

600"

700"

800"

T"G"C"A"

0"

100"

200"

300"

400"

500"

600"

700"

800"

900"

T"G"C"A"

0"

200"

400"

600"

800"

1000"

1200"

1400"

1600"

T"G"C"A"

0"

50"

100"

150"

200"

250"

300"

350"

T"G"C"A"

LT01

LT03

LT04

LT05

LT06

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

T#

G#

C#

A#

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

rs1490413(

rs7520386(

rs560681(

rs10495407(

rs891700(

rs1413212(

rs876724(

rs12997453(

rs1357617(

rs9866013(

rs1872575(

rs1355366(

rs6444724(

rs13134862(

rs1554472(

rs6811238(

rs1979255(

rs717302(

rs159606(

rs13182883(

rs7704770(

rs251934(

rs338882(

rs1029047(

rs13218440(

rs2811231(

rs1478829(

rs1358856(

rs2503107(

rs2272998(

rs214955(

rs727811(

rs6955448(

rs917118(

rs1019029(

rs321198(

rs737681(

rs10092491(

rs4288409(

rs2056277(

rs4606077(

rs2270529(

rs7041158(

rs1463729(

rs10776839(

rs735155(

rs3780962(

rs1410059(

rs740598(

rs964681(

rs10768550(

rs10500617(

rs1498553(

rs901398(

rs6591147(

rs590162(

rs2107612(

rs2255301(

rs2269355(

rs2111980(

rs10773760(

rs1886510(

rs9546538(

rs1058083(

rs354439(

rs1454361(

rs873196(

rs4530059(

rs1821380(

rs729172(

rs2342747(

rs430046(

rs1382387(

rs2175957(

rs8070085(

rs1004357(

rs1027895(

rs8078417(

rs2291395(

rs4789798(

rs689512(

rs3744163(

rs2292972(

rs1493232(

rs9951171(

rs7229946(

rs985492(

rs521861(

rs1736442(

rs1024116(

rs719366(

rs576261(

rs12480506(

rs2567608(

rs1005533(

rs1523537(

rs722098(

rs464663(

rs2833736(

rs914165(

rs9606186(

rs5746846(

rs2073383(

rs733164(

rs987640(

rs2040411(

rs1028528(

rs1800865(

rs2075640(

rs2299942(

rs2267801(

rs2267802(

rs2071394(

rs1865680(

rs2075182.3(

rs2075181(

rs1515817(

rs2032595(

rs2032598(

rs2032599(

rs2032601(

rs2032600(

rs2032607(

rs2032604(

rs2032624(

rs2020857(

rs2032668(

rs2032666(

rs2032658(

rs2072422(

rs2032653(

rs1864258(

rs3897(

rs3900(

rs891407(

rs2032611(

rs2032631(

rs2032673(

rs2032626(

rs1558843(

rs1276035(

rs1276034(

T#

G#

C#

A#

Higher  depth  of  coverage  precludes  undercalling

100x

100x

100x

100x

100x

SNPs  by  rs  ID SNPs  by  rs  ID

Depth  of  coverag

e  for  e

ach  allele

Allele  allo

ca7o

n

07/03/12

Amplicon  coverageConstruct Library Prepare Template Run Sequence Analyze DataCustomize Panel

AMPLISEQ™ CUSTOM PANEL

• Up to 6,144 primer pairs

• 10 ng DNA input

• Up to 200 bp targets

MULTIPLEX -CLONAL BEADAMPLIFICATION

Generic

MULTIPLEX -SEQUENCING ON SINGLE CHIP

Generic

SNP GENOTYPE PLUGIN

Analysis custom to SNP panel

Y  -­‐  SNPS

Female  individual Male  individual

SNPs  sorted  by  depth  of  coverage SNPs  sorted  by  depth  of  coverage

depth  of  coverag

e

depth  of  coverag

e

07/02/12

07/02/12

allele  coverage

alleles  represented

HID-­‐SNP  Genotyper  plugin

SNPs  by  rs  ID

07/02/12

314  chip:  ~32  individuals316  chip:  >96  individuals

12,762 124x

07/02/12

HID  Ion  Community  Homepage

07/03/12

Summary

•SNPs  are  valuable  iden2fiers  when  small-­‐amplicon  PCR  based  detec2on  is  necessary

•Not  intended  to  supplant  STRs  for  forensic  typing.

•Next-­‐genera2on  sequencing  technologies  allow  for  high  mul2plexed  capabili2es  -­‐  of  SNPs  and  individuals  

•Iden2ty  SNP  panel  on  PGM™  using  well-­‐characterized  polymorphisms

07/03/12

Future  Plans

•Poten2al  external  collabora2ons  for  research  applica2ons  on  the  PGM

•Ancestral  and  phenotypic  SNP  panel

•Mini  haplogroups,  Y  and  Mito  haplotyping  panel  

•Microbial  forensics

07/03/12

Acknowledgments  

•Robert  Lagacé

•Lori  Hennessy  

•Reina  Langit  

•  Joe  Chang

•  Chien-­‐wei  Chang

•Narasimhan  Rajagopalan  Sharon.Wootton@lifetech.com

07/03/12

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