Single-molecule and long-read sequencing reveals structural diversity of hepatitis B virus haplotypes including...

Hepatitis B virus (HBV) exhibits diverse pathological states during chronic infection. While defective genomes with deletions and splicing have been sporadically reported, the quasispecies structure and their role in disease progression have remained unclear due to technical constraints of conventional short-read sequencing.

Using rolling circle amplification (RCA)-nanopore long-read sequencing, we analyzed near full-length HBV genomes (approximately 3.2kb) at the single-molecule level from 6 serum samples obtained from 5 patients. Haplotypes were reconstructed from reads with point mutations, and structural linkages with deletions and splicing were evaluated.

Diverse haplotypes (three to five per patient) involving mutations across the HBV genome, predominantly in putative immune-targeted regions (core, preS, and surface), were detected in all cases, along with variants containing different deletions and splicing patterns. Notably, in chronic hepatitis and hepatocellular carcinoma cases, a substantial portion (29.5-71.3%) of circulating HBV DNA consisted of deletion and splicing variants, which clustered significantly in specific haplotypes (p < 2.8 × 10⁻⁵). Longitudinal changes in haplotype composition involving point mutations and different deletion/splicing patterns were observed in one patient before and after HBeAg seroconversion.

HBV exists as a population comprising multiple haplotypes with mutations, deletions, and splicing interconnected and dynamically changing according to disease state. Monitoring circulating splicing variants and haplotype dynamics may serve as novel biomarkers for disease understanding. This single-molecule long-read analysis elucidates the molecular basis of HBV quasispecies evolution.