CREATION OF A SNP CHIP IN SHEEP Rudiger Brauning and John McEwan on behalf of the International Sheep Genomics Consortium MapNet Workshop 27 August 2009.

CREATION OF A SNP CHIP IN SHEEP

Rudiger Brauning and John McEwanon behalf of the International Sheep Genomics Consortium

MapNet Workshop 27 August 2009

Skim sequencing

ScottishBlackface

Romney

Texel

Otago University Baylor College HGSC

Merino

Poll Dorset

Awassi

~3x coverage(9.74 Gbp),Roche 454 FLX

0.5x

0.5x0.5x

0.5x

0.5x0.5x

Sequence assembly pipeline

lower case masked 454 reads

454 sequencesrepeat mask

Sheep 454 contigs

Bovine genomemap uniquely

(normal / sensitivesensitive megablast)

Uniquely mapped 454 sequences

assemble with Newbler / CAP3CAP3

Sheep genome assembly v1.0 / v.2.0v.2.0

position sheep contigs on vsg2, replace cow sequence with sheep sequence, remove remaining cow sequence

vsheep

order, orientate and space

Ordered sheep contigs

Genome coverage - total

Genome coverage – non-repetitive fraction

Sheep assembly - coverage

91.06%

73.80% 66.29%

Turning a cow into a sheep - low resolutionOAR1

BTA3

BTA1

OAR2

BTA8

BTA2

OAR3

BTA11

BTA5

OAR4

BTA4

OAR5

BTA7

OAR6

BTA6

OAR7

BTA10

OAR8

BTA9

OAR9BTA9

BTA14

OAR10

BTA12

OAR11

BTA19

OAR12

BTA16

OAR13

BTA13

OAR14

BTA18

OAR15

BTA15

OAR16

BTA20

OAR17

BTA17

OAR18

BTA21

OAR19BTA22

OAR20BTA23

OAR21BTA29

OAR22BTA26

OAR23BTA24

OAR24BTA25

OAR25BTA22

OAR26BTA27

OARX

BTAX 5 chromosome inversions4 chromosome fusions1 split chromosome

The sheep genome assembly

Is comprised of 454 sequence contigs organised into sheep chromosomes

• 2.5 million contigs

– average length ~480 bases

• ordered and oriented using vsheep framework

– cover ~52% of the sheep genome

– cover ~100% of the unique fraction of the genome

15 sheep contigs – 1 bovine contig

How close is the assembly to the real sheep?The local order of sheep contigs is heavily dependant on the bovine genome assembly

• Used a light pass of 454 sequencing paired end reads as a quality control check of the assembly

– short paired ends ~30 bp

» ~89% tail-to-tail

– long paired ends ~70-100 bp

» ~98% tail-to-tail

• BAC-end sequences

– ~93% tail-to-tail

CNVs

assembly

SNP chip - numbers

600,000 putative SNPs454

59,454 SNPs

SNP distribution

1 SNP per 50 kbp

Illumina Infinium OvineSNP50 BeadChip - performance

average MAF = 0.30

> 20,000 animals genotyped

Quality control

59,454 SNPs

Future work – simulation results

simulation approach read length (bp) insert sizes (kb) coverage depth*A single BAC 100 1 20X B pooled BACs^ 100 1+5 23X+5XC pooled BACs^ 75 0.175 30X D seeded assembly` 100 1+5 23X+5X

A B C D

Acknowledgments• AgResearch NZ

– John McEwan– Gemma Payne– Nessa O’Sullivan– Tracey Van Stijn– Theresa Wilson– Rudiger Brauning– Alan McCulloch– Russell Smithies– Benoit Auvray

• University of Otago– Jo Stanton– Chrissie– Mark

• Baylor College of Medicine– Richard Gibbs– Donna Muzny– Michael E. Holder– Lynne Nazareth– Rebecca L. Thornton– Christie Kovar

• CSIRO Livestock Industries– Brian Dalrymple – James Kijas – David Townley– Abhirami Ratnakumar – Wes Barris– Sean McWilliam

• Genesis Faraday– Chris Warkup

• sheepGENOMICS– Rob Forage– Terry Longhurst

• TIGR– Ewen Kirkness

• Uni Melbourne– Jill Maddox

• USDA– Tim Smith– Curt van Tassell

• UNE– Hutton Oddy

• Uni Sydney– Frank Nicholas– Herman Raadsma

• Utah State University– Noelle Cockettwww.sheephapmap.org

www.livestockgenomics.csiro.auwww.sheepgenomics.com

isgcdata.agresearch.co.nz

THANKS