Clearseq inherited disease panel kit

A protocol24 (link) using the Agilent (Santa Clara, California, USA) ClearSeq Inherited Disease panel kit for enrichment followed by NGS targeted on 2742 genes was adapted for the clinical testing of every enrolled proband. For variant calling, GATK best practice was employed for SNV/small indels. CANOES25 (link) and HMZDelFinder12 (link) were separately applied for CNV detection, and the results were merged. The annotation and filtrations of both SNVs and CNVs followed those reported in published works.26 27 Detailed descriptions of the sequencing, variant calling, annotation and filtering processes can be found in the online supplementary notes and online supplementary figure S1.

Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. DNA fragments were enriched for CES using the Agilent ClearSeq Inherited Disease panel kit (covering 2,742 genes, test in #2, #3, #5, #6, #7, #8, #9, #11, #12) or proband ES using the Agilent SureSelect XT Human All Exon 50 Mb kit (tested in #1, #4, #10). For details on the sequencing and analysis, please see the published paper by Wang et al. and Yang et al. (21 (link),22 (link)). The test method was decided by the physicians’ and parents’ choices based on the test price and year.
Raw data were mapped to the human reference genome (GRCh37/hg19). Variants were annotated by ANNOVAR and VEP software, with a minor allele frequency of less than 3% according to either the 1,000 Genomes Project or the Exome Aggregation Consortium (ExAC) and in-house database (23 (link)). The pathogenicity of the candidate variant was analyzed according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics (ACMG) (24 (link)) and also described in our previous paper (25 (link)).