In-house primer panel driven resource-efficient whole-genome sequencing of hepatitis B virus

Hepatitis B virus (HBV) genetic variability influences disease progression, immune escape, and treatment response. Owing to limited whole-genome sequencing (WGS) data from Central India and lack of standardized commercial assays, this study aimed to develop an in-house primer-based HBV WGS workflow to characterize the molecular epidemiology and mutational profile of HBV circulating in Chhattisgarh, India. A total of 102 HBV-positive clinical samples with viral loads ranging from 3.11 to 9.08 log₁₀ IU/mL were analysed. WGS was performed using three sets of in-house designed overlapping primers covering the complete HBV genome, employing a customized sequencing workflow adapted from the COVIDSeq (RUO) assay. Sequencing quality, genome coverage, phylogenetic relationships, and mutational patterns were evaluated. High-quality sequencing was achieved with a mean Q30 score of 92% and genome coverage exceeding 98.7% across samples. Phylogenetic analysis revealed exclusive predominance of HBV genotype D. Sub-genotype D3 was most prevalent (63.7%), followed by D5 (18.6%), D1 (9.8%), and D2 (7.8%), with ayw2 (63.4%) and ayw3 (36.6%) serotypes. Mutational analysis identified frequent surface gene mutations within the major hydrophilic region, including the "a" determinant (T113S, T114S/P, K122R, N131T, F134Y/N, A159G), along with mutations in transmembrane and core regions. The precore nucleotide mutation C1858T was universally detected in genotype D isolates. This first HBV WGS study from Chhattisgarh demonstrates a cost-effective in-house sequencing approach for molecular surveillance suggesting minimum viral load of 3.11 log₁₀ IU/mL required for adequate coverage of entire HBV genome. The predominance of genotype D and immune escape-associated mutations underscores the importance of genomic monitoring to enhance HBV diagnosis, vaccination strategies, and clinical management in resource-limited settings.