Groenen Madison Porcine 60K [Read-Only]€¦ · Greger Larson et al. (2005) Science 307:1618 ... zMax Rothschild zZhiliang Hu zJames Reecy Sanger Institute, UK zCarol Churcher zRichard

Animal Breeding & Genomics Centre

Illumina Agriculture Seminar SeriesAugust 3, 2009, Madison WI

Martien Groenen

High throughput SNP discovery in the pig using the Illumina Genome Analyzer and characterization of the porcine HapMap

panel using the Illumina Porcine 60K iSelect TM Beadchip



Overview

SNP discovery and chip design

Statistics of the 60 K chip

Hapmap data: MDS and clustering

Hapmap data: Haploype diversity, haplotype

sharing and selective sweeps

Fom SNP genotyping towards resequencing



HistoryObjective

Design of a 60K Illumina BeadChip

ChallengeLimited number of SNPs availableOnly half of porcine genome sequence availableJob had to be done between April/August 2008

ApproachSNP identification by sequencing reduced representation libraries (RRL) on an Illumina Genome Analyzer



Overview sequencing approach

6x 6x 6x 6x

30x Highly reliable SNP identification

Rough estimate of MAF within breeds

5 Reduced representation libraries (RRL)Restriction digest of DNASeparate on acrylamide gelsIsolate 150-200 bp fragments

6x


RRL Genome%

HaeIII 160-200 1

AluI 100-150 2

AluI 150-200 3

MspI 100-200 1

DraI 180-260 2

TOTAL 9

Total No Reads(million)

67.1

88.6

145.9

69.1

100.3

481

No Reads after filtering (million)

57.8

83.4

95.8

52.3

67.7

357

RRLs results

=12.8 billion bp



Align to reference genome and identify SNPs using MAQ

C/T G/C

HaeIII HaeIIISNPs

SNP called if minor allele seen at least 3x


SNP distribution on Illumina GA reads

All

Transitions

Transversions


All porcine SNPs currently availableSNP source Number

HQ Solexa SNPs WU* 333,000

454 sequencing MARC 110,000

7K iSelect chip AU-RI 5,500

dbSNP 17,700

INRA (Sanger seq) 61,700

Cambridge University 14,900

Total No SNPs 543,000

Total Unique SNPs 510,000

TOTAL #SNPs submitted for the chip: 72,000

70 % mapped on build 77 % predicted23 % unmapped

*Plus additional 58,000 SNPs where minor allele seen twice


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SNP distributionBuild 7

Build 9(89 % of SNPs)


Porcine 60K BeadChip: Excellent performanceFinal total number of SNPs on the chip: 62,163Number of SNPs with MAF>0.05 in at least 1 breed: 59KConversion rate overall: 94.8% (Illumina GA SNPs: 96 %)Good correlation between allele count based on GA sequencing and Geotyping


The porcine HapMap project

576 individuals typed by Illumina991 individuals typed by WU


Phenotypic variation



Porcine Hapmap panelBreed Illumina WU TotalLandrace 79 33 112Large White 129 28 157Duroc 82 16 98Pietrain 95 17 1112Wild Boar 20 249 26912 Chinese breeds 31 233 2643 Synthetic 6 49 55Hampshire 69 8 77Berkshire 58 - 58Museum samples - 28 28Other suiformes 6 41 47Other european br. - 289 289Tobasco 1 - 1TOTAL 576 991 1567

Sus celebensisSus verrucosusSus barbatusPecari tajacuPotomochoerusBabyrousa Sus cebifrons

Including 165 animals of the discovery panel

Animal sequenced



Use of the 60K chip: European vs Chinese breeds

Biased towards common variantsBiased towards SNPs in European breedsSNP density of the 60K chip

Sufficient for GWA and GWS in european breedsToo low for Chinese breeds

Large White Ningxiang

Amaral et al. (2008) Genetics 179: 569



Domestication of the pig

Greger Larson et al. (2005) Science 307:1618

Gene flowSelective sweepsHaplotype diversityHaplotype sharingExtent of LD

mtDNA analysis


Multi dimensional scaling and clustering



MDS plot Axis1-2: Division Europe-Asia (PLINK)

Outgroup(other suidae)Asian breeds

European/US breeds

Asian WB

European WB

European WB



MDS plot Axis1-2: Division Europe-Asia (PLINK)

WB

Museum sampleCollected in turkey 1964

Museum sample from Indonesia (~ 100 y old)


OutgroupsOther suidae


Sus scrofa Sus barbatus (11)

Sus celebensis (2)

Sus verrucosus (10)

Other Suidae

Sus cebrifons (1)

~ 1 MY

~ 2 MY

~ 4 MYPotomochoerus p. (1)

Babyrousa (1)~ 10 MY

Potomochoerus l. (1)

Pecari tajacu (1) ~ 70 MY

Phacochoerus a. (2)



SNPs shared with other suidae

765438

578

--

5837

59167

1167

2306

Species Average heter.

Sus scrofa 23 %

Sus barbatus 3 %

Sus celebensis 2 %

Sus cebifrons 0.8 %

Sus verrucosus 0.6 %

Phacochoerus 0.8 %

Potomochoerus l. 0.7 %

Potomochoerus p. 0.9 %

Babyrousa b. 0.9 %

478

Ancestral allele identified for > 95% of the SNPs

496



Haplotype diversity, haplotype sharing and

selective sweeps


Sharing is high between populations of the same breed, but not uniform across chromosomes

Duroc/Meishan

Sharing is low between Chinese and European breeds, but local exceptions exist

3567 SNPs on SSC7, nearly complete, 133 Mb

Duroc/DurocPietrain/Pietrain

Haplotype sharing between pig populations

Large W/LandraceLarge W/Pietrain

Sharing is intermediate between populations of several 'white' breeds


High Low (1 haplotype)

Haplotype diversity and linkage disequilibrium10 MbSSC7

LWLRPI

MS

3.5 MbDu

SSC14

DULWLRPIMS



Identification of selective sweeps: Examples on SSC1-4LW27

LWxPI

HA01

DU24

DU22

DU20

SSC1 SSC4SSC3SSC2

Low heterozygosityHigh frequency of derived allele



From SNP typing to resequencing



(1) Site frequency spectrum analysis based on RRL sequence data

RRL cover 5-10 % of the genomeCoverage differs between breeds4 commercial white breeds + Wild BoarData from poolsWithin 500 Kb windows estimate:

– Watterson’s estimator: θ = f(S, n). – Tajima’s estimator: π = f(S, n, freqs.).– Tajima’s D: D = (π-θ) / sd.– Fst measures population differentiation

reference genome

breed 1

breed 2



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Reanalyse data per breed

SNP distributions

TransitionsTransversions

Minor allele count



Example: Site frequency spectrum of Ssc8 derived from deep sequencing of pools

Pietrain Wild Boar

Selective sweep around the KIT locus in white breeds

Variation of nucleotide diversity ( Wθ̂ )



Sequencing of Sus Verrucosus (Java warty pig)

6x on Illumina GA200, 500 and 3000 bp libraries

~2 My



FundingUSDA – CSREESEU (Sabre, PigSNP)Institute for Pig Genetics (IPG)

Acknowledgements (1)

Food Quality and Safety

DNA samplesChina Agricultural University

Ning LiUniversity of Sassari, Italy

Massimo ScanduraStaff Institute, Japan

Naohiko OkumuraINRA-Toulouse, France

Alain DuvroMARC, USA

Gerry RohrerRoslin Institute, UK

Alan ArchibaldEU PigBiodiv1

Wageningen University, The NetherlandsRichard Crooijmans

University of Illinois, USALarry Schook

Aristotle University of Thessaloniki, GreeceCostas Triantaphyllidis

Istituto Zootecnico per la sardegna, ItalySara Casu

Research Centre for Biology – LIPI, Indonesia

Gono SemiadiHendrix Genetics (HG)Institute for pg genetics (IPG)Pig Improvement Company (PIC)

PTP, ItalyElisabetta Guiffra

REPROGEN, AustraliaJaime Gongora

Universidade de Lisboa, PortugalDeodália Dias

Universitat Autonoma BarcelonaMiguel Perez-Enciso

University of Aarhus, DenmarkChristian Bendixen

Durham University, UKGreger Larsen

Iowa State University, USAMax Rothschild



Acknowledgements (2)Wageningen University, The Netherlands

Richard CrooijmansMarcos RamosAndreia FonsecaHinri KerstensHendrik-Jan Megens

University of Aarhus, DenmarkChristian BendixenJakob Hedegaard

USDA,ARS, MARC, USAGary RohrerTim SmithDan Nonneman

INRA , Toulouse, FranceDenis Milan

Roslin Institute, UKAlan ArchibaldAndy Law

Durham University, UKGreger Larsen

Purdue University, USABill Muir

University of Illinois, USALarry SchookJon Beever

USDA, ARS, Beltsville, USACurt Van Tassel

Iowa State University, USAMax RothschildZhiliang HuJames Reecy

Sanger Institute, UKCarol ChurcherRichard Clark

University of Missour-Columbia, USAJeremy TaylorBob Schnabel

Universitat Autonoma BarcelonaMiguel Perez-EncisoLucca Ferreti

Illumina Inc., San Diego, USAMark HansenMarylinn Munson

Documents

Groenen Madison Porcine 60K [Read-Only]€¦ · Greger Larson et al. (2005) Science 307:1618 ... zMax Rothschild zZhiliang Hu zJames Reecy Sanger Institute, UK zCarol Churcher zRichard