Nested VP1 PCR and Nanopore Sequencing from Stool and ES Samples v1.11 v2

This protocol is updated 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. The protocol aims to amplify the VP1 region of poliovirus through a nested PCR using panEV primers followed by amplification of the VP1 sequence using the Q8/Y7 primer set. We advise the use of barcoded primers where possible as this greatly simplifies the subsequent library preparation process. Additional steps have however been included in the case that the second PCR step is performed either with standard Q8/Y7 primers or using Q8/Y7 with the barcode adaptors (BCA) attached. Primer sequences for the panEV primers, Q8/Y7 primers and modified Q8/Y7 primers are found in Dataset S1 of the publication. Sequencing of the panEV product is also possible through the removal of the VP1 nested PCR steps and the preparation of the panEV product for sequencing. Version updates: v1.1 – Updated to LSK-109 v1.2 – Minor formatting edits v1.3 – Streamlining and minor alterations v1.4 – Written inclusion of negative control in PCR v1.5 – Updated to dreamtaq and Y7R primers v1.6 – Updated MinKNOW instructions & minor edits v1.7 – Changed back to Y7 primers v1.10 – Updated to LSK-110, removed cleaning and quantification prior to pooling, updated taq volume in rtPCR, altered nested VP1 to use neat panEV product. V1.11 – Updated RNA extraction to include Proteinase K, removed DNA CS addition, added pooling of Y7/Q8 primers.