20B8173733
The genomic DNA from the peripheral blood of the patient and his parents was extracted using the QIAamp Blood DNA Mini kit (Qiagen, Germany). The concentration and purity of the sample DNA were detected using a UV-240 UV spectrophotometer. The fragmented DNA was then screened by magnetic beads, and the screened fragments measured 280 bp320 bp, with the ends filled and the base "A" added at the 3 end to make the fragments compatible with the 3 end of the fragment. After amplification and purification by ligation-mediated PCR, the library of a single subject sample was constructed. A customized gene capture probe (MGIV4 59M, MGI) was used to hybridize 1020 labeled patient DNA libraries at 47C for 1624 h. Probe washing and elution reactions were performed after hybridization, followed by post-PCR reactions of the captured samples. The libraries were tested for fragment size and concentration using Agilent 2100 Bioanalyzer and a BMG microplate reader; after passing the test, libraries requiring different data amounts were used for pooling and quantification. Then, the pooling libraries were subjected to single-stranded cyclization. After making a DNA nanoball (DNB), the libraries were sequenced using a high-throughput sequencer, MGISEQ-2000 with PE100+10 type, and the raw sequencing data were obtained after sequencing.