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.