3D-PCR and NGS
CpG-rich regions of HBV cccDNA or secreted rcDNA were amplified using a
specific primer pair and TaqF polymerase. The resulting amplicons were
purified from the gel and extracted using the Qiagen gel extraction kit.
Equal amounts of the purified PCR products were then subjected to nested
PCR using TaqF polymerase at progressively decreasing temperatures
(95–82°C). The resulting PCR products were analyzed by gel
electrophoresis and subjected to next-generation sequencing (NGS).
Alternatively, for semi-quantitative analysis, 3D-PCR was performed with
SYBRGreen dye. Briefly, 3D-PCR amplicons generated at 87°C, 84°C, or
82°C were purified from the gel using the Qiagen gel extraction kit,
quantified using a Qubit 2.0 Fluorometer (Life Technologies), and pooled
in equimolar ratios. Adapters for Illumina sequencing were ligated, and
the libraries were sequenced using the MiSeq instrument (Illumina) with
250 paired-end reads. To analyze the sequences, FASTQC software and
Geneious software were employed for quality assessment, reference
alignment, removal of low-quality reads and nucleotides, and calculation
of indels. Custom Python codes (available upon request) were used for
mutation analysis and mutation context analysis. 3D-PCR primers are
listed in Table S3.