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.