Developments in the detection of genetic variation (SNPs, CNVs and ...

Overview SNP typing: Current status• Examples in chicken SNP discovery using 2 nd generation sequencingtechnology (Solexa) Copy Number Variation (CNV) Re-sequencing of genomesAnimal Breeding &Genomics Centre

SNP genotyping assays10 6 SNPs100-500K SNPs60K SNPs1536 SNPs1-384 SNPscustom madehumanAnimal Breeding &Genomics Centre

The infinium assay12 samples / chipAnimal Breeding &Genomics Centre

High call rates Final chip contained 18,264 SNPs Genotytping calls for 17,790 SNPs (97.4 %)AAABBBAnimal Breeding &Genomics Centre

Many SNPs with multiple heterozygous clusters Example of a SNP with populationdependent clustering of allelesAnimal Breeding &Genomics Centre

Application: Population Genetics using pools Pools are a cost-effective way to identify whatSNPs are segregating in a specific breed Highly reliable estimates of allele frequencies Estimate Phylogeny Identify regions under selection (selectivesweeps)Animal Breeding &Genomics Centre

High correlation between individuals and poolsR 2 =0.97K-correction of MAF based on thesignals in the heterozygote clusterR 2 =0.99MAF calculated based on pools from30- 60 individualsFour populations typed both as poolsand individualsAnimal Breeding &Genomics Centre

High correlation between individuals and poolsGallus LafayettieIdentification of theancestral alleleData from individuallygenotyped animalscompared to a pooledDNA sample of thatbreedAnimal Breeding &Genomics Centre

Identification of selective sweepsChromosome 2Likelihood ratioLocation (bp)Animal Breeding &Genomics Centre

Overview SNP typing: Current status SNP discovery using 2 nd generation sequencingtechnology (Solexa) Copy Number Variation (CNV) Re-sequencing of genomesAnimal Breeding &Genomics Centre

Ultrahigh throughput sequencing (Solexa, Illumina GA)> 1 billion bp in a single run at ~€10,000Read length 36 bp3-8 million reads per lane8 lanes per flow cell1 sequence run = 3 daysTGCTACGAT …123456789TTTTTTTGT …Animal Breeding &Genomics Centre

SNP detection: two strategiesReference genome availablePigsChickenNo reference genome availableTurkeyDuckGreat titTilapiaSalmon (planned)ReferencegenomeSNPSNP assay primersAnimal Breeding &Genomics Centre

Strategy 1: With reference genomeReduced representationlibrary (RRL)Restriction digest of DNASeparate on acrylamidegelsIsolate 150-200 bpfragments6x 6x 6x 6x6xEstimate of MAFwithin breeds30xHighly reliable SNP identificationAnimal Breeding &Genomics Centre

Analysis overviewRaw GA sequences(Solexa)Pre-maq perl scriptFilteredGA sequences(Solexa)Alignment to referencegenome and SNPidentification using MAQPost-maq perl scriptHigh qualitySNPs with MAFAnimal Breeding &Genomics Centre

Alignment and SNP detection using MAQHaeIIISNPsHaeIII173 bpAnimal Breeding &Genomics Centre

Alignment and SNP detection using MAQHaeIIIHaeIIIC/TG/CAnimal Breeding &Genomics Centre

Strategy 1: With reference genome Pigs (finished) 300 million 36 bps reads (after QC, raw data=450 million) 5x5 different RRLs representing + 10% of the genome 350,000 high quality SNPs MAF 60K Ilumina iSelect BeadChip ordered Chicken 115 million reads (partly analyzed) 4x1 RRLs (2 Cobb, 2 HG) 6 % of genome detected 384,000 SNPs with MAF Also cover missing chromosomesAnimal Breeding &Genomics Centre

Porcine Infinium Beadchip SNPsA total of 60,212 SNPs were selected41,667 with coordinates on build 7 (average spacing of 44 Kbalong the genome)18,545 SNPs covering the remainder 30% of the genome4,497 of these 18,545 SNPs have location predicted by human-pigcomparative mapping57,996 SNPs had minor allele frequency calculatedAverage MAF=0.4Test chip analysed end of october3000 SNPs replacedFinal chip ready by beginning ofDecemberAnimal Breeding &Genomics Centre

The porcine HapMap projectAnimal Breeding &Genomics Centre

Porcine Hapmap panel typed by IlluminaBreed Discovery Panel Trio’s Total #Landrace 29 17 76Large White 36 25 135Duroc 34 15 79Pietrain 23 15 95Wild Boar 20 - 20Hampshire - 15 69Berkshire - 15 58Meishan - - 30Wild Boar 17 - 3Other Sus. sp. - - 6Sus celebensisSus verrucosusSus barbatusTobasco 1 Animal beingsequencedAnimal Breeding &Genomics Centre

Porcine Hapmap panel typed by WU Wild boar samples from all over the world Additional breeds from Europe Additional breeds from China Genotyping at Illumina: December Genotyping at WU: February Total number: ~ 1100Animal Breeding &Genomics Centre

Strategy 2: Without reference genomeReduced representation library (RRL)Separate on agarose gelIsolate 2000-4000 bp fractionRandom shearing of fragmentsIsolate 200-250 bp fragmentsAssembly of sequence contigs that can function as areference (using a number of assemblers e.g. SSAKE, SHARGS, VELVET)SNP assay primersAnimal Breeding &Genomics Centre

Analysis overview strategy 2Raw GA sequences(Solexa)Pre-maq perl scriptFilteredGA sequences(Solexa)De novo assemblyof contigse.g SSAKESHARGSVELVETAlignment to referencegenome and SNPidentification using MAQPost-maq perl scriptHigh qualitySNPs with MAFAnimal Breeding &Genomics Centre

Strategy 2: Without reference genome Turkey ~35 million 35 bp reads (after QC) Great Tit 2 RRLs done: 50 million 36 bp (10 % of the genome) 1 RRL not yet analyzed (paired end) Tilapia 30 milion reads Duck 30 milion reads paired end (not yet analyzed)Animal Breeding &Genomics Centre

Size distribution of contigsnr of contigs340000320000300000280000260000240000220000200000180000160000140000120000100000800006000040000200000SSAKE contig assembly Tilapia vs Turkey vs Gt300049_7576_100 151_200 301_400 501_600101_150 201_300 401_500 >601contig sizeTilapiaTurkeyGt3000Animal Breeding &Genomics Centre

SNPs identified Turkey 14,000 SNPs 50 % have sufficient flanking sequence for assaydesign 50 % have (unique) homology with chicken 384 SNP assay ordered Great tit (one RRL finished) 25,225 SNPs Will be compared to Zebra finch genomeAnimal Breeding &Genomics Centre

Overview SNP typing: Current status SNP discovery using 2 nd generation sequencingtechnology (Solexa) Copy Number Variation (CNV) Re-sequencing of genomesAnimal Breeding &Genomics Centre

Copy number variation (CNV) in chicken Agilent 244K chicken array Analysed 80 individuals from different breeds Included a few small pedigrees Used sequenced bird (UCD001) as the referenceAnimal Breeding &Genomics Centre

Example chicken chromosome 190 kb30 kbAnimal Breeding &Genomics Centre

Example 2: Late feathering locus 180 Kb duplication on Z-chromosome Partial duplication of PRLR and SPEF2 gene Affecting feather development Used for sexing 1 day old chicksElferink MG, Vallée AA, Jungerius AP, Crooijmans RP, Groenen MA. (2008) Partial duplicationof the PRLR and SPEF2 genes at the late feathering locus in chicken. BMC Genomics. 9:391Animal Breeding &Genomics Centre

Example 2: Late feathering locusZZfatherZWmotherk + / K3 copiesZWdaughterk + / -1 copyK / -2 copiesAnimal Breeding &Genomics Centre

Overview SNP typing: Current status SNP discovery using 2 nd generation sequencingtechnology (Solexa) Copy Number Variation (CNV) Re-sequencing of genomesAnimal Breeding &Genomics Centre

Future applications in functional genomics Increased ultra high throughput sequencing Shift from microarrays to direct sequencing of cDNA Re-sequencing the complete genome of individuals• Identify all variations in an individual’s genome Sequencing to identify regulatory elements• E.g. miRNAs, transcription factor binding sites etc.Animal Breeding &Genomics Centre

3 rd generation sequencer with capacity to produceup to 100 billion bp of sequenceSequencing singlemolecules28 Tb of data storage (sufficient for only 2 runs !)Animal Breeding &Genomics Centre

AcknowledgementsChicken genome sequencing consortiumChicken SNP consortiumSwine SNP consortiumWashington University, St. LouisUSDA, High-Density SNP Discovery, Validation andCharacterization in SwineUSDA, Genome-wide marker-assisted selection overmultiple generations in commercial poultryEU-SabreEU-EadgeneThank YouAnimal Breeding &Genomics Centre