title: Whole-exome sequencing of high- and low-fertility Bos taurus bulls
design: Genomic DNA for 24 selected bulls was extracted using the Maxwell 16, per manufacturers instructions. Genomic DNA (gDNA) was purified using Zymo Researchs DNA Clean and Concentrator  kit and heated to 52 C for 2 min prior to concentration estimation. gDNA concentrations were estimated using the Qubit dsDNA BR Assay Kit for use with the Qubit 2.0 Fluorometer. Roche Nimblegen, SeqCap EZ Developer Library was purchased from Roche (Roche NimbleGen, WA, USA) to perform a custom-designed sequencing of the bovine exome. Liquid capture probes were designed to target all exons annotated in the Bos taurus UMD3.1 genome, plus 100 base pairs of 5UTR, per standard protocols. An in-house Perl script was used to identify target regions. Probes were manufactured by Roche (Roche NimbleGen). Magnetically labelled, oligonucleotide probes capture the target regions of DNA during library preparation. One hundred base pairs of 5 untranslated region (UTR) were also included for all genes. In total, there were 227,647 exons in the genome transfer file. Of these, 202,899 were targeted in this design, covering 56,671,697 bp. Less than five total matches and five mismatches were allowed. Of these probes, 92.5% were unique to a single genomic position, and 4.5% had only 2 possible matches within the genome. The length of probes was 200 bp with approximately 2.1 million probes made to cover the exome. Probes were also designed to target mitochondrial DNA and were 1/5th of the concentration of nuclear DNA. Using probes unique within the genome, that is probes that map exactly once in the genome, covers only 80% of the target regions; whereas using probes that map up to 5 times in the genome covers approximately 98.4% of the target regions. Library preparation for WES was commercially performed (Clinical Genomics, Canada) following the Roche Nimblegen SeqCap EZ Developer Library protocol, as per the manufacturers instructions. Briefly, 100 ng gDNA of each sample were prepared using the TruSeq Nano DNA Library Prep Kit. This step involved shearing of gDNA using Bioruptor DNA shearing and Agencourt AMPure XP beads, end-repair of fragments, adapter ligation, LM-PCR and quality control. The strategy to hybridise fragments of gDNA to the exome baits was to make one equimolar pool of all 24 gDNA libraries. This pool was then split into four separate pools and hybridisation captures were performed on each pool with oligonucleotide probe capture reagents, resulting in 1X capture for every 6 samples. All captures were then re-pooled and split into four groups for sequencing on four HiSeq. 2500 lanes. Pooling ensures that samples were mixed prior to capture, and to give an even distribution of sample to each sequencing lane.