SMART-9N amplification enabling rapid nanopore sequencing of RNA viruses v1

This protocol describes a rapid Oxford Nanopore Technologies (ONT) workflow for untargeted sequencing of RNA viruses. It has been optimized for wastewater and nasal swabs processed using the Nucleic Acid Observatory (NAO) protocols Concentration and nucleic acid extraction of viruses from wastewater influent V.3and Concentration and total RNA extraction of viruses from nasal swabs V.1, respectively. It assumes that viral RNA extracts have undergone background depletion and does not detail those steps. In brief, RNA is reverse transcribed using SMART-9N amplification and prepared for sequencing with ONT's Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24). In our experience, this approach consistently yields sufficient material for ONT’s recommended inputs and recovers a diverse range of RNA viruses from both wastewater and nasal swabs, with long reads, high genome coverage, and reduced background noise.