Exome Sequencing Library Preparation

Extracted from 1.0 mL of each blood sample, each genomic DNA sample was quantified by using a spectrofluorometer (Gemini™ XPS Microplate Reader, Molecular Devices, USA), and about 200 ng of each DNA sample were randomly fragmented by Covaris sonication (LE220-plus Focused-ultrasonicator, Covaris, USA). The DNA fragments with the size mainly distributed between 150 and 250 bp were repaired with an "A" base added at the 3′-end of each strand, thereafter adapters were ligated to both ends of the end repaired/dA tailed DNA fragments, which were selected by size, amplified by ligation-mediated PCR (S1000 Thermal Cycler, Bio-Rad, USA), purified [QIAquick PCR Purification Kit, QIAGEN China (Shanghai)], and hybridized to the exome array for enrichment. After washing out non-hybridized fragments, captured products were circularized and the rolling circle amplification was performed to produce DNA nanoballs. Each resulting qualified captured library was then loaded on BGISEQ-500 sequencing platforms (MGI Tech, China) to perform high-throughput sequencing [15 (link), 16 (link)]. Sequencing-derived raw image files were processed by BGISEQ-500 base calling software with default parameters to generate the sequence data of each case as paired-end reads.