Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters

Abstract

Norovirus is a highly diverse RNA virus often implicated in foodborne outbreaks, particularly those associated with shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbor various pathogens, including human-pathogenic viruses. The application of Sanger or amplicon-based high-throughput sequencing (HTS) technologies to identify human pathogens in shellfish faces two main challenges: (i) distinguishing multiple genotypes/ variants in a single sample and (ii) low concentrations of norovirus RNA. Here, we assessed the performance of a novel norovirus capsid amplicon HTS method. We generated a panel of spiked oysters containing various norovirus concentrations with different genotypic compositions. Several DNA polymerases and reverse transcriptases (RTs) were compared, and performance was evaluated based on (i) the number of reads passing quality filters per sample, (ii) the number of correct genotypes identified, and (iii) the sequence identity of outputs compared to Sanger-derived sequences. A combination of the reverse transcriptase LunaScript and the DNA polymerase AmpliTaq Gold provided the best results. The method was then employed, and compared with Sanger sequencing, to characterize norovirus populations in naturally contaminated oysters.