Le220 plus focused ultrasonicator

After PicoGreen quantification and quality control by Agilent BioAnalyzer, 500 ng aliquots of genomic DNA were sheared using a LE220-plus Focused-ultrasonicator (Covaris catalog #500569). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) with modifications. DNA libraries were subjected to size selection by mixture with 0.5 vol of aMPure XP beads (Beckman Coulter catalog # A63882) after post-ligation cleanup. Libraries were not amplified by PCR and were pooled equivolume for sequencing. Samples were run on a NovaSeq 6000 in a 150-bp/150-bp paired-end run using the NovaSeq 6000 SBS v1 Kit and an S1 flow cell (Illumina). The average number of read pairs per sample was 10 million.

After PicoGreen quantification and quality control by Agilent Bio-Analyzer, 500 ng of gDNA were sheared using a LE220-plus Focused-ultrasonicator (Covaris catalog # 500569) and sequencing libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) with modifications. Briefly, libraries were subjected to a 0.5× size select using aMPure XP beads (Beckman Coulter catalog # A63882) after post-ligation cleanup. Libraries not amplified by PCR (07652_C) were pooled equivolume and were quantitated based on their initial sequencing performance. Libraries amplified with 5 cycles of PCR (07652_D, 07652_F, 07652_G) were pooled equimolar. Samples were run on a NovaSeq 6000 in a 150 bp/150 bp paired-end run, using the NovaSeq 6000 SBS v1 Kit and an S4 flow cell (Illumina).

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.