Whole genome sequencing of hepatitis A virus: adapting Illumina protocols for foodborne investigation

Abstract

High-throughput sequencing-based whole-genome sequencing (WGS) is highly effective for identifying viral pathogens in microbial research. However, applying WGS directly to foodborne viruses remains challenging because food matrices contain PCR inhibitors and viral titers are typically much lower than those found in clinical specimens. This study aimed to develop a WGS method for analyzing the hepatitis A virus (HAV) genome in clams using the Illumina MiSeq platform. To enhance the HAV WGS method, we applied four approaches to HAV-positive clam field samples: size-exclusion chromatography for sample preparation, a specialized RNA extraction method, optimized cDNA synthesis, and the selection of DNA polymerase. Nine complete HAV genomes were obtained from clams. The obtained HAV genomes and their genetic characteristics were then compared based on phylogeny. Before optimization, only four clam samples yielded detectable amplification; however, following optimization, two additional samples became amplifiable, resulting in six samples suitable for downstream WGS analysis. The developed WGS method was able to sequence low contamination levels of 2.91-3.61 log10 genome copies/mL, achieving coverage of 97.5% and 92.6%. Notably, this study confirmed an average sequencing depth of up to 82.20x and a minimum depth of 25.19x. As a result of sequencing, one HAV-IA, and eight HAV-IB genotypes were identified from six clam samples including the multiple strains. The sequence identity between the strains from clams and serum was 97.80% for HAV-IA and 95.2-97.80% for HAV-IB. This method of viral WGS in food samples may contribute to rapid genotyping, understanding virus evolution, and enhancing epidemiological surveillance in foodborne virus outbreaks.