This review aims to assess and recommend approaches for targeted and agnostic High Throughput Sequencing of RNA viruses in a variety of sample matrices. HTS also referred to as deep sequencing, next generation sequencing and third generation sequencing; has much to offer to the field of environmental virology as its increased sequencing depth circumvents issues with cloning environmental isolates for Sanger sequencing. That said however, it is important to consider the challenges and biases that method choice can impart to sequencing results. Here, methodology choices from RNA extraction, reverse transcription to library preparation are compared based on their impact on the detection or characterization of RNA viruses.

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT-PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17–21 °C and after 4 days at 11–15 °C, compared to 44% reduction at 7–9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram (gcg−1; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant difference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg−1. These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry.

In benchmarking bioinformatic pipelines for analyzing high-throughput sequencing (HTS) data sets, we provide method standardization for bioinformatics broadly and specifically for norovirus in situations for which no officially endorsed methods exist at present. This study provides recommendations for the appropriate analysis and classification of norovirus amplicon HTS data and will be widely applicable during outbreak investigations.