Torrent server

Torrent Server (Thermo Fisher Scientific) was used for signal processing, base calling, read alignment, and generation of results files. Specifically, following sequencing, reads were mapped against the human reference genome hg19 using the Torrent Mapping Alignment Program. Variants were identified by using Torrent Variant Caller plugin with the optimized parameters for AmpliSeq exome-sequencing recommended by Thermo Fisher. The variant call format (VCF) files from all samples were combined using GATK (3.2-2) [27 (link)], and all variants were annotated using ANNOVAR [28 (link)]. Only non-silent exonic variants including non-synonymous single nucleotide variations (SNVs), stop-codon gain SNVs, stop-codon loss SNVs, splice site SNVs, and In-Dels in coding regions were kept if they were supported by more than ten reads and had allele frequency higher than 10%. To obtain somatic variants, we filtered against dbSNP build 138 (non-flagged only) and the matched adjacent benign or blood samples sequenced in this study. Putative variants were manually scrutinized on the Binary Alignment Map (BAM) files through Integrative Genomics Viewer version 2.3.25 [29 (link)].

For the NextSeq 550 and HiSeq 1500 platforms, the first step of sequencing analysis generated fastq files. The GRCh37 reference genome was chosen for the alignment of paired-end reads. We used the custom-designed pipeline based on GATK 3.8 (22 ) for data processing and quality control evaluation. ENSEMBL Variant Effect Predictor (23 (link)), ClinVar (2021/01/10) (16 (link)), gnomAD (v2.1.1) (24 (link)), and dbSNP (25 (link)) databases were applied for the annotation of single-nucleotide variants and short indels. PLINK v1.90 (26 (link)) was used to get identity by state (IBS) values and identity by descent (IBD) proportion to estimate relatedness for all pairs of individuals.
For the Ion S5 platform, sequencing and bioinformatic analysis resulted in bam files. We used the Torrent Server (Thermo Fisher Scientific, Waltham, MA, USA) with default parameters to obtain the vcf files. For the annotation of the vcf files Ion Reporter (Thermo Fisher Scientific, Waltham, MA, USA) with the Annotate Variants analysis tool was applied.

Base calling, de-multiplexing, quality control (QC) and alignment pre-processing [19 (link)] was completed using Torrent Suite 4.6 on a Torrent Server (ThermoFisher Scientific). Briefly, polyclonal and uniformly low-quality reads were removed, and the remaining reads were trimmed from the 3′ end only. Mapping was also performed within Torrent Suite 4.6, using the Torrent mapping alignment program (TMAP). Individual libraries were mapped to the sheep reference genome Oar v3.1 (University of California, Santa Cruz (UCSC)). For each of the sheep breeds, all individual BAM files were merged and sorted using SAMtools v0.1.19-44,428 cd [20 (link)], and coverage analysis was performed for both the individual and combined datasets through automated plugins in TorrentSuite 4.6. Duplicate reads were removed using Picard Tools v1.122.

Genomic DNA was extracted from whole blood or saliva using the QIAGEN QIAamp DNA blood kit or tissue kit (QIAGEN, Hilden, Germany). Custom amplification primers were designed, using Ion AmpliSeq Designer (Thermo Fisher Scientific, Waltham, MA, USA), to cover coding exons and flanking intron regions of the selected genes. Sample amplification and equalization were achieved using Ion AmpliSeq Library Kits 2.0 and the Ion Library Equalizer Kit, respectively (Thermo Fisher Scientific). Amplified sequences were ligated with Ion Xpress Barcode Adapters (Thermo Fisher Scientific). Emulsion PCR and subsequent enrichment were performed using the Ion OneTouch Template Kit v2.0 on Ion OneTouch 2 and Ion OneTouch ES, respectively (Thermo Fisher Scientific). The final product then was sequenced on the Ion PGM sequencing platform (Thermo Fisher Scientific). Raw data output from the sequencer was deposited in the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) under Accession Number DRA004490, and uploaded to the Torrent Server (Thermo Fisher Scientific) for variant calling, with NCBI GRCh37 as a reference. The resulting VCF (variant call format) files were analyzed by Ingenuity Variant Analysis (QIAGEN) for annotation and visualization. Combined Annotation Dependent Depletion (http://cadd.gs.washington.edu/) was applied for annotation of genetic variants.