HBV genome enriched sequencing
Full-length HBV genome of type B and type C were amplified by PCR using biotin-labeled dNTPs. PCR mix was prepared as follows: 5 ul of HBV DNA (1 ng/ul); 5 ul of 10*LA Taq uffer; 26.5 ul of H2O; 4 ul of 2.5 mM dNTPs (1/4 dNTPs biotin-labeled); 2.5 ul of 10 pmol P1 (TTTTTC ACC TCT GCC TAA TCA); 2.5 ul of 10 pmol P2 (AAAAAGTTG CAT GGT GCT GG); and 0.5 ul of LA Taq enzyme (TakaraBio, Inc.). PCR was subjected to the following cycling condition: initial denaturation for 3 min at 94 C; 32 cycles of denaturation for 30 s at 94 C, annealing for 50 s at 56 C, and extension for 2.5 min at 68 C; final extension for 10 min at 72 C. Amplicons were purified by Ampure beads (Beckman Coulter, Inc.) and were then fragmented by Covaris E-210 (Covaris, Inc., Woburn, MA) to 250 bp500 bp through the 2% agarose electrophoresis. Single-stranded HBV probes were generated by high temperature denaturation at 94 C for 5 min. Sequencing libraries of 170 bp insert size were constructed for following the instruction of Illumina. Genomic DNA was sheared to around 150 bp200 bp DNA fragments by Covaris E-210 (Covaris, Inc., Woburn,MA). These fragmentswere purified, end blunted, A tailed, and adaptor ligated. 10 cycles of PCRwere performed after size selection in 2% agarose gel. The concentration of libraries was quantified by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Libraries were hybridized with HBV probes at 47 C for 24 hours and then washed to remove un-captured fragments. The eluted fragments were amplified by 16 cycles of PCR to generate libraries for sequencing. Libraries were quantified and proceeded to 101 cycles paired-end index sequencing in the Illumina HiSeq 2000 sequencer according to manufacturer's instructions (Illumina Inc., San Diego, CA).