2014 J.B. Cole, 1,* J.L. Hutchison, 1 D.J. Null, 1 P.M. VanRaden, 1 G.E. Liu, 1 S.G. Schroeder, 1 T.P. Smith, 2 T.S. Sonstegard, 1 C.P. Van Tassell, 1 and D.M. Bickhart 1 1 Animal Genomics & Improvement Laboratory and 2 US Meat Animal Research Center Agricultural Research Service, USDA 1 Beltsville, MD and 2 Clay Center, NE The hunt for a functional mutation affecting conformation and calving traits on chromosome 18 in Holstein cattle

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (2) Cole et al. Overview l What do we know about chromosome 18? l How can sequencing help us learn more? l What did we learn when we looked at the data? l How did we approach these new challenges? l Where are we now? Source: Ianuzzi (Chromosome Res., 4:448–456)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (3) Cole et al. Introduction l Several studies (Kuhn et al., 2003; Cole et al., 2009; Seidenspinner et al., 2009) have reported QTL on BTA 18 associated with dystocia l Bioinformatic analysis using SNP data has not identified the causal variant l Next generation sequencing (NGS) has recently been used to find causal variants for novel recessive disorders

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (4) Cole et al. Chromosome 18 is different l Markers on chromosome 18 have large effects on several traits: w Dystocia and stillbirth: sire and daughter calving ease and sire stillbirth w Conformation: rump width, stature, strength, and body depth w Efficiency: longevity and net merit l Large calves contribute to reduced cow lifetimes and decreased profitability

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (5) Cole et al. Marker effects for dystocia complex AR-BFG-`GS Cole et al., 2009 (J. Dairy Sci. 92:2931–2946) ARS-BFGL-NGS Source:

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (6) Cole et al. Correlations in dystocia complex

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (7) Cole et al. Maltecca et al., 2011 (Animal Genet. 42: ) The QTL also affects gestation length

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (8) Cole et al. The dystocia complex l The key marker is ARS-BFGL-NGS at (rs ) 57,589,121 Mb on BTA18 l Intronic to Siglec-12 (sialic acid binding Ig-like lectin 12) l Recent results indicate effects on gestation length (Maltecca et al., 2011) and calf birth weight (Cole et al., 2014), as well as calving traits (Purfield et al., 2014)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (9) Cole et al. Where did it come from? Source: Source:

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (10) Cole et al. Who popularized it? Source: 57,861 daughters >2 million granddaus Source: Maternal haplotype from Ivanhoe

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (11) Cole et al. This is a gene-rich region raphics&list_uids= Discussed on Tuesday (Abstract 288, Mao).

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (12) Cole et al. Copy number variants are present l ARS-BFGL-NGS is flanked by CNV w There’s a loss and a gain to the left (8 SNP region) w There’s a gain to the right (10 SNP region) l This can result in assembly problems Hou et al (BMC Genomics,12:127)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (13) Cole et al. What if we look at a different trait? l Cole et al. (2009) proposed the following mechanism: w Siglec-12 may sequester circulating leptin w This increases gestation length w Calf birth weight (BW) is higher because of increased gestation length w Higher BW is associated with dystocia

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (14) Cole et al. We don’t have birth weight data l Birth weights are not routinely recorded in the US l Collaborated with Hermann Swalve’s group to develop a selection index prediction of BW PTA l Performed GWAS and gene set enrichment analysis to search for interesting associations (Cole et al., 2014, JDS 97: )

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (15) Cole et al. GWAS for birth weight PTA h Cole et al., 2014 (J. Dairy Sci., 97:3156–3172)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (16) Cole et al. Are we measuring anything new? l Identified a SNP on BTA16 intronic to LHX4, which is associated with cow body weight and length (Ren et al., 2010, Mol. Bio. Reprod., 37: ). l 4 SNP in the QTL region on BTA 18 had large effects l Several other SNP with large effects intronic or adjacent to genes with unknown functions

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (17) Cole et al. KEGG pathways for birth weight What does regulation of the actin cytoskeleton have to do with birth weight in cattle? That is, do these results make sense? Maybe…these pathways may be involved in establishment & maintenance of pregnancy, as well as coordination of growth and development. Cole et al. (2014)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (18) Cole et al. Pedigree & haplotype design Arlinda Chief AA, SCE: 8 Chief AA, SCE: 7 MGS CMV Mica Aa, SCE: 14 Leduc Aa, SCE: 18 Melwood Aa, SCE: 8 Jed Aa, SCE: 15 Arlinda Rotate AA, SCE: 8 δ = 10 Tradition Aa, SCE: 10 MGS Rockman Ivanhoe Aa, SCE: 6 Delegate Aa, SCE: 15 Laramie aa, SCE: 15 These bulls carry the haplotype with the largest, negative effect on SCE: Combination ??, SCE: 7 Couldn’t obtain DNA:

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (19) Cole et al. How many scientists does it take… You went to her poster on Tuesday (Abstract 799, Cooper et al.), right? You just missed his talk (Abstract 164, Bickhart et al.)! He’s back in Maryland, working.

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (20) Cole et al. Sequencing coverage Bull nameSCE 1 Genotype 2 Total readsCoverage Pawnee Farm Arlinda Chief7AA333,628, Glendell Arlinda Chief8AA981,726, Sweet Haven Tradition10Aa390,387, Arlinda Rotate8AA~476,000, Arlinda Melwood8Aa~448,000, Juniper Rotate Jed15Aa656,190, CMV Mica14Aa433,353, Lystel Leduc18Aa767,440, Willow-Farm Rockman Ivanhoe6Aa195,769, Cass-River Select Delegate15Aa377,380, Wedgwood Laramie15aa371,477, Predicted transmitting ability (PTA) for sire calving ease, the percentage of offspring born with difficulty. Small values are desirable and large values are undesirable. 2 The genotype of the tag SNP for the QTL, where “A” and “a” are the major and minor alleles, respectively.

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (21) Cole et al. Results from Illumina sequencing l Data analyzed using paired-end read alignments and split-read mapping l Portions of two exons and a connecting intron within the Ig-like protein domains may have been duplicated l Some heterozygotes with desirable SCE also have deletions near the N-terminal end of the protein

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (22) Cole et al. Possible assembly problem on BTA18 This could be a GC-rich region (bias in Illumina chemistry). More reads than expected may align here because repetitive elements were combined during assembly.

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (23) Cole et al. Genome assembly (simplified) Reads must be assembled into chromosomes Assembly is a computational process (Liu et al., 2009; Zimin et al., 2009) This process is imperfect – repetitive regions are hard to assemble correctly! Sometimes, this… should be this.

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (24) Cole et al. Can it be corrected using long reads? l BTA18 genomic DNA extracted from CHORI-240 BAC library (L1 Domino 99375) at AGIL l Sequencing libraries constructed at USDA MARC, pooled, and run on PacBio RS II BAC IDInsert size (bp)StartEnd CH P14174,68256,954,65457,129,335 CH E12178,61857,058,24857,236,865 CH L6175,83157,092,23757,268,067 CH240-34N7158,84157,129,38357,288,223 Source: Pacific Biosystems

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (25) Cole et al. Processing of PacBio reads l BAC DNA was pooled at MARC to have enough material to construct a sequencing library l Reads were assembled into contigs using HGAP in SMRTanalysis v2.2.0 l 44 contigs with an N 50 of 31 kb were constructed

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (26) Cole et al. Analysis of alignments l PacBio contigs aligned against UMD3.1 contigs using MUMmer 3.0 l Short (Illumina) reads aligned against PacBio contigs using BWA 0.7.5a-r405 l Paired-end discordancy interrogated using custom scripts (Bickhart, unpublished data)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (27) Cole et al. Alignment of BAC contigs with UMD3.1 A line with a slope of 1 indicates that a segment is conserved between the two sequences – this contig is almost identical between our PacBio assembly and the UMD3.1 reference assembly.

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (28) Cole et al. Discordancy analysis l Illumina reads aligned w/PacBio contigs l Reads with lengths ±4σ were counted l Discordancies may indicate w Problems in the PacBio assembly w The presence of repetitive elements w Structural differences between the Holstein and Hereford (unlikely)

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (29) Cole et al. DNA in PacBio and not in UMD3.1 ~10 kbp of DNA in PacBio contig that doesn’t map to UMD3.1! Reads map to PacBio and UMD3.1— ARS-BFGL-NGS is placed here. Vector DNA – nothing to see here! Reads map to PacBio and UMD3.1 contigs.

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (30) Cole et al. There are clearly assembly problems PacBio sequence duplicated on UMD3.1 contig

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (31) Cole et al. What have we learned? l This is more complex than SNP genotyping, and unsuccessful experiments are expected l You needs lots of high-quality DNA for constructing PacBio libraries l Overlapping BACs should not be pooled (some people already know this) l Data editing and error-correction are critical

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (32) Cole et al. Next steps l Re-assemble raw reads following more stringent edits and data cleaning l Re-sequence single BACs or pooled, non- overlapping BACs l Sequence the RPCI-42 Holstein BACs (Monsanto calf) w Are structural differences between Holstein and Angus in this region

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (33) Cole et al. Conclusions l Structural variants in and around the Siglec-12 gene are associated with differences in SCE l SNP are misplaced on the UMD3.1 assembly l A region ~8 kb downstream of ARS-BFGL- NGS appears to be misassembled l The causal variant on BTA18 has not yet been conclusively identified

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (34) Cole et al. Acknowledgments l Reuben Anderson and Alexandre Dimitchev, AGIL, ARS, USDA l Renee Godtel, US Meat Animal Research Center, ARS, USDA l USDA-ARS appropriated projects (DMB, JBC, JLH, DJN, PMV), (GEL, SGS, TSS, CPV), and (TPS) l Cooperative Dairy DNA Repository and Council on Dairy Cattle Breeding

10 th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (35) Cole et al. Questions?