Whole-exome sequencing
Whole-exome sequencing and analysis protocols developed by Agilent Technologies (2017), Inc. (Santa Clara, USA) were adapted for the clinical test of whole-exome sequencing. Briefly, genomic DNA samples were fragmented with the use of M220 Focused-ultrasonicator (Covaris, Massachusett, USA). The fragment was ranging from 150 bp to 250 bp. The ends were repaired with Hieff NGS® UltimaTM Endprep Mix Kit (Yeasen, Shanghai, China), ligated to Illumina multiplexing paired end adapters with Hieff NGS® UltimaTM DNA Ligation Module (Yeasen, Shanghai, China), purified by AMPure XP Beads (Beckman Coulter, Illinois, USA), and amplified by means of a PCR assay with the use of primers with sequencing barcodes (indexes) by 2×Super Canace® II High-Fidelity Mix for Library Amplific ation Kit (Yeasen, Shanghai, China). Library enrichment for WES was conducted by Agilent SureSelect Human All Exon v6 (Santa Clara, CA, USA). Enriched samples were sequenced using an NovaSeq 6000 platform (San Diego, CA, USA). Mean coverage of the sequences was 95.6×, and on average 97.4% of base pairs with >10× coverage were successfully detected. Sequencing data were aligned to the hg38 reference genome.