WES analysis.
Each exome was purified, hybridized and captured using DNA Prep Tagmentation PCR Reagents(Illumina, Santiago, USA),Nextera DNA Flex Pre-Enrichment Library Prep-Buffers(Illumina, Santiago, USA),DNA Fast Hyb Enrichment Beads and Exome Panel(45Mb) and IDT for Illumina Nextera DNA UD Indexes SetA(Illumina, Santiago, USA)kit according to the manufacturer’s laboratory procedures, the approximate DNA fragment size was determined by glycolipid gel electrophoresis. The enriched exomes were sequenced using Illumina Nova-6000 platform(Illumina, Santiago, USA). The paired end sequences were converted to FASTQ format. Reads were mapped against the human reference genome Grch37/hg19 using Burrows-Wheeler Aligner. The single nucleotide variants were called by Genome Analysis Toolkit (GATK https://software.broadinstitute.org/gatk/), Berry Genomics authorized ANNOVAR on SNV filtering. Using the Genome Aggregation Database (gnomAD),Exome Aggregation Consortium (ExAC),1000 Genomes on variation annotation and explanation. Variant classification followed current standards and guidelines developed by the American College of Medical Genetics and Genomics (ACMG) .the variants were categorized as pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign variants. The mutation sequences of the positive cases reported at trio WES were validated by Sanger sequencing.