Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater. Norwalk virus

Noroviruses are the leading cause of non-bacterial gastroenteritis across the globe costing US$60.3 billion per annum. Our project involved developing and optimising a nanopore-based method for typing genogroups I (GI) and II (GII) by sequencing partial RNA-dependent RNA polymerase and major structural protein genes from a single fragment of DNA amplified from wastewater. Reverse transcription, PCR and library pooling were optimised for the sample matrix and a consensus-based bioinformatics pipeline was developed. To confirm accuracy of consensus sequences derived from nanopore sequencing data three faecal samples from norovirus positive patients were sequenced using both nanopore and sanger sequencing and compared, to ensure both approaches yeilded equivilient results. After optimisation of experimental, sequencing and bioinformatic protocols the technique was applied to samples which were collected from hundreds of sites in England around October 2021, as part of the SARS-CoV-2 wastewater programme, and pooled into ten samples representing different geographical regions. Within the ten pooled samples 13 GI and 8 GII dual types were identified, highlighting the diversity of norovirus taxa present in wastewater in England at a single point in time.