Illumina NGS Workflow for Integrated HBV Sequence Capture

The Illumina NGS workflow we used to capture the integrated HBV sequences was described elsewhere [23 (link)]. Briefly, 1 ug of genomic DNA was fragmented by adaptive focused acoustic technology (AFA; Covaris) and then repaired; an ‘A’ was ligated to the 3’ end, Agilent adaptors were ligated to the fragments, and the adaptor-ligated product was PCR-amplified. We captured HBV using 250 ng of DNA library according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization of capture bait was performed at 65 °C using a heated cycler lid option at 105 °C for 24 h. The purified product was quantified according to the manufacturer’s instructions (qPCR quantification protocol guide) and qualified using the TapeStation DNA screentape D1000 (Agilent). Finally, we did paired-end 100-bp read-length sequencing of the purified captured DNAs by Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) following the manufacturer’s instructions.