Multi-viral target Direct Detection and Nanopore Sequencing (DDNS) v1

This protocol describes the target specific amplification for direct detection of multiple viral pathogens from wastewater extracted RNA. This enables sensitive identification of multiple different viral pathogens of interest for both surveillance and research purposes. Targets include Enterovirus D68, Enterovirus 71, Rotavirus, Hepatitis A and E, Norovirus GI and GII and SARS-CoV-2. Wastewater samples should be concentrated using an appropriate method and viral RNA extracted following the method; MagMAX Viral RNA Isolation Large volume extraction protocol using 1.2 mL of concentrate for each extraction (see Viral RNA extraction below). This method then utilises highly-sensitive nested PCR approaches where amplicons are subsequently pooled and purified ready for library preparation for Oxford Nanopore or Illumina sequencing. Barcoding the amplicons prior to nanopore sequencing allows multiplexing of many samples, reducing turn around time and cost per sample.