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.