High-resolution HAV genotyping using amplicon-based NGS read mapping and quantification

High-resolution genotyping of Hepatitis A virus (HAV) is crucial for molecular surveillance and outbreak investigation. Traditional Sanger sequencing of the VP1/2A region is commonly used but struggles with mixed infections.

This study aims to develop and validate an amplicon-based next-generation sequencing (NGS) method for precise HAV genotyping and mixed infection detection, along with tracing the origin of clinical samples using HAV fasta sequences.

A VP1/2A amplicon-based NGS workflow was developed and tested on 24 Quality Control for Molecular Diagnostics (QCMD) samples and 25 clinical samples confirmed as HAV-positive by IgM serology and quantitative PCR (qPCR). The process involves mapping NGS reads to reference HAV sequences to quantify genotype composition. Results were compared with expected genotypes and some findings were validated via nanopore sequencing. The likely country of origin for the clinical samples was also determined.

The method achieved 100% concordance with expected results in all 24 QCMD samples and successfully genotyped all clinical samples. It identified a mixed infection of HAV subgenotypes IA and IB, which conventional Sanger sequencing could not resolve but was confirmed by nanopore sequencing. Additionally, a small outbreak of HAV subgenotype IA was detected among four family members in a northern Iceland town, and the probable origin of the 25 clinical samples was determined.

VP1/2A amplicon-based NGS offers a quick, high-resolution method for HAV genotyping, enhancing mixed infection detection over Sanger sequencing, making it suitable for routine diagnostics and epidemiological studies.