# Full-Genome Hepatitis B Virus Genotyping: A Juxtaposition of Next-Generation and Clone-Based Sequencing Approaches—Comparing Genotyping Methods of Hepatitis B Virus

## TL;DR

This study compares next-generation and clone-based sequencing for hepatitis B virus genotyping, finding that clone-based sequencing may detect more genetic diversity in some cases.

## Contribution

The study directly compares NGS and CBS for full-genome HBV genotyping, revealing unexpected advantages of CBS in detecting genetic diversity.

## Key findings

- Both NGS and CBS identified genotypes B, C, and I, as well as recombinant forms.
- CBS detected additional subgenotypes and mutations not found by NGS in two cases.
- Genotyping based on S ORF sequencing was shown to be unreliable and inconsistent with full-genome methods.

## Abstract

Background: The enhanced sensitivity of next-generation sequencing (NGS) for assessing hepatitis B virus (HBV) quasispecies heterogeneity over clone-based sequencing (CBS) is well documented. However, its comparative reliability for genotype determination remains an open question. Objective: This study aimed to directly compare the performance of NGS and CBS for genotyping HBV using the entire viral genome. Methods: We selected five challenging clinical samples that previously could not be subgenotyped or showed conflicting results when using direct sequencing of the S open reading frame (ORF). The full HBV genome from these subjects was amplified and then analyzed in parallel by both NGS and CBS. Phylogenetic analysis was subsequently used to assign genotypes. Results: Both methods identified a range of genotypes, including B, C, and I, as well as aberrant and recombinant forms. For three of the five subjects, genotyping results were identical between the two platforms. In the remaining two cases, however, CBS revealed greater complexity, identifying additional subgenotypes and recombinant/aberrant strains not detected by NGS. Notably, for three individuals, the genotypes determined by both modern methods contradicted earlier results from 2011 based on direct S ORF sequencing. Furthermore, the specific mutations detected were incongruent between the platforms, with CBS identifying a higher number of variants than NGS. Conclusions: Our findings indicate that genotyping results from NGS and CBS can be discordant. Contrary to expectations, CBS may uncover more genetic diversity, including a greater number of subgenotypes and mutations, than NGS in certain contexts. The study also confirms that genotyping based solely on direct sequencing of the S ORF can be unreliable and lead to misclassification.