Development of an NGS‐Based Estimation of Integrated HBV DNA in Liver Biopsies and Detection in Liquid Biopsies

This study characterized a hepatitis B virus (HBV) hybridization-capture next-generation sequencing (HBV-NGS) assay and applied it to develop a model for estimating the integrated HBV DNA (iDNA) quantity and for HBV genetics liquid biopsy. Using HBV monomers and reconstituted cell line DNA (SNU398, Hep3B, and PLC/PRF/5), the HBV-NGS assay demonstrated high coverage uniformity, reproducibility across HBV genotypes A-D, and 0.1% sensitivity for detecting iDNA. The iDNA sequence and structures from SNU398 and Hep3B are reported. An iDNA estimation model was developed using tissue biopsies from patients with serum viral load < 4 log IU/mL and validated using SNU398 and Hep3B cell line DNA. The assay's utility for HBV genetic liquid biopsy was evaluated using matched plasma-urine samples with HBV DNA levels ranging from high to undetectable. In this pilot study, HBV-JS was detected in all body fluids, regardless of viral load. These findings indicate that the iDNA from patients with negligible or undetectable viral replication can be assessed for iDNA elimination efficacy in drug development. Moreover, a sensitive HBV genetics liquid biopsy can be feasible even for patients with undetectable serum viral load. This study underscores the potential of NGS-based methods to advance HBV management.