Agilent sureselect human exon v5 kit

Whole‐exome sequencing (WES) was performed by parent's request. DNA library was constructed by Agilent SureSelect Human Exon V5 kit (Agilent Technologies) according to the manufacturer's protocols. Sequencing was processed on Illumina HiSeq X Ten System (Illumina, Inc) based on the manufacturer's protocols. The sequencing reads were mapped to the Genome Reference Consortium Human genome build 37 (GRCh37). The Genome Analysis Toolkit (GATK) was used for variant calling. Candidate single nucleotide variants (SNVs) and insertion‐deletions (indels) were saved in VCF files and uploaded to the online variation annotation tool TGex (https://tgex.genecards.cn/#/) for further filtering and prioritizing. Common variants were filtered based on the frequencies in the Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org), the Exome Sequencing Project (https://esp.gs.washington.edu), the 1000G (http://www.1000genomes.org), genomAD (http://gnomad.broadinstitute.org/) and our local database. The variant pathogenicity was assessed according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015).

Genomic DNA was extracted from dried blood spot and captured to create the library for whole‐exome sequencing by Agilent SureSelect Human Exon V5 kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. The libraries were sequenced by HiSeq X Ten with PE150 strategy (Illumina, San Diego, CA) with a read depth over 120X and more than 95% of the targeted regions covered over 20X. The sequencing reads were mapped to the Genome Reference Consortium Human genome build 37 (GRCh37). The Genome Analysis Toolkit (GATK) was used for variant calling. TGex software (LifeMap Sciences, Alameda, CA) was used to annotate the variants. Transcript NM_018006 was used as the reference sequence. The TRMU variants and their origins were verified by Sanger sequencing. All the variants were classified according to ACMG/AMP guidelines.