Dna shearing system

Genomic DNA was sheared using a DNA shearing system (Covaris, Cat. #S220) prior to library preparation. Genomic DNA (200 ng) from six Japanese Black cattle (one wild/wild homozygote and five CNVR_221/CNVR_221 homozygotes) was used to generate sequence libraries, which were prepared using TruSeq Nano DNA library prep kit (Illumina, Cat. #20015964) and IDT for Illumina TruSeq DNA UD Indexes (Illumina, Cat. # 20020590) according to the manufacturer’s instructions. The sequence data were generated as 2 × 101 bp reads using the NovaSeq 6000 SP Reagent Kit (Illumina, Cat. # 20027465) and Novaseq6000, and processing and base calling were performed using Illumina Real-Time Analysis 3.
The sequence reads (fastq file) for each animal were aligned to ARS-UCD1.2 genome assembly [42 ] using bwa-0.7.17 [45 (link)]. The bam files were created using Samtools [46 (link), 47 (link)], and duplicate reads were removed using Picard [48 ]. The realignment around indels and recalibration were performed using GATK-4.0.12.0 [49 ]. SNPs and indels were called using Haplotype caller by GATK. The data from the WGS were deposited in the Wagyu genome database (WGDB) of the Japan Livestock Technology Association (Yushima, Bunyouku, Tokyo 113–0034, Japan) and were managed by the WGDB consortium.

Following DNA extraction, 3 μg of each sample DNA was sheared to 150–200 bp using the Covaris DNA Shearing System. To capture the exonic DNA, we used the SureSelectXT Human All Exon V4 or V5 capture library (Agilent) for 50 Mb of exonic regions. The sequence library was constructed with the SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing Library kit (Agilent) according to the manufacturer's instructions. We performed DNA sequencing of 100 or 101-bp paired-end reads using the Illumina HiSeq 2000 sequencer.

The genomes of the ancestor R0 and the evolved cell population R7 were resequenced using a next-generation sequencer (Junior, Roche). Genomic DNA was purified with a Wizard Genomic DNA Purification kit (Promega) and fragmented using a DNA shearing system (Covaris), according to the manufacturer’s instructions. Whole-genome shotgun sequencing by the 454 GS Junior platform (Roche) was performed according to the manufacturer’s instructions. The sequence reads were assembled using Newbler 2.7 and aligned using the GS Reference Mapper software (ver. 2.6; Roche). Approximately 99% of the reads in each dataset (DRA003743, DDBJ) were uniquely mapped to the MDS42 genome (AP012306, DDBJ). The mutations were analyzed using the GS Reference Mapper software. The candidate mutations were further determined by Sanger methods of the genome samples subjected to a resequencing analysis using a genetic analyzer (ABI PRISM 3100, Applied Biosystems). Sequencing was performed to both the purified genomes and the cell pellets of these populations. In addition, the cell populations (R0–R7) acquired in the experiments of growth curves were repeatedly sequenced for further verification.