Variant Identification and Annotation Pipeline

After sequencing, raw data were saved in FASTQ format. Illumina sequencing adapters and low quality reads (< 80 bp) were filtered by Cutadapt [14 ]. Clean reads were aligned to UCSC hg19 human reference genome using the Burrows-Wheeler Alignment [15 (link)] tool. Duplicated reads were removed using Picard (http://broadinstitute.github.io/picard). Insertions, deletions and SNP variants were detected and filtered using the Genome Analysis Toolkit [16 (link)]. Then the identified variants were annotated using ANNOVAR [17 (link)] and associated with the following databases: 1000 genomes, Exome Aggregation Consortium, The Human Gene Mutation Database, and predicted by Mutation Taster (MT) [18 (link)], Sorting Intolerant From Tolerant (SIFT) [19 (link)], PolyPhen-2 (PP2) [20 (link)] and Genomic Evolutionary Rate Profiling (GERP++) [21 (link), 22 (link)]. Splice-site were predicted by Human Splicing Finder [23 (link)]. All variants identified by the Illumina HiSeq X Ten sequencer were confirmed by Sanger sequencing. The pathogenicity of mutations was assessed in accordance with American College of Medical Genetics and Genomics guideline (ACMG) [9 (link)–11 (link)].