Direct Detection of poliovirus and Nanopore Sequencing (DDNS) - Stool v2

This protocol is an update from the protocol described in the paper "Rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing" by Shaw et al in the Journal of Clinical Microbiology (2020), DOI: 10.1128/JCM.00920-20 and is commonly known as Direct Detection of Poliovirus by Nanopore Sequencing (DDNS). The protocol aims to amplify the VP1 region of poliovirus through a semi-nested PCR using a pan-Enterovirus primer and polio specific primers followed by amplification of the VP1 region using a polio specific primer set. We use barcoded primers as this greatly simplifies the subsequent library preparation process. Within the protocol steps quality control checks are included. Following the QC steps as you go ensures the validity of your results and provides easier troubleshooting when needed. This protocol is for use with Oxford Nanopore kit14 chemistry ligation sequencing reagents and can be used with the MinION Mk1B or GridION sequencer.