Wastewater Sequencing Using the EasySeq™ RC-PCR SARS CoV-2 (nimagen) V3.0 V3

This SOP describes the procedure for generating cDNA libraries from SARS-CoV-2 viral nucleic acid extracts from wastewater samples using an adaptation of the Nimagen EasySeq™ SARS-CoV-2 Protocol v4.01 (https://www.nimagen.com/gfx/Covid19/protcol_NimaGen_covid_wgs_v401.pdf). Version 2.0 of this protocol is an update to accommodate the change to using lyophilized indexes in the EasySeq™ RC-PCR SARS-CoV-2 (novel coronavirus) Whole Genome Sequencing Kit (NimaGen, SKU: RC-COV096). Updates to the SARS-CoV-2 WGS Panel probe mixes have also been made (currently v4.02) to improve coverage of new dominant variants of concern (VoCs), although these can be used with no change to this protocol. For the latest BED files containing primer details for analysis, check the download section of https://www.nimagen.com/covid19. Version 3.0 includes a minor update to the options for post PCR cleanup, following optimization of the composition of the AmpliCleanTM beads provided with the kit by NimaGen. Additionally, advice is provided for sequencing poor quality libraries resulting from a high proportion of SARS-CoV-2 negative samples. The first step of this protocol is an addition to the original method, which aims to remove any PCR inhibitors from extracted RNA samples and potentially concentrate the RNA further, before carrying out reverse transcription to cDNA. During the second step, which follows the original EasySeq™ SARS-CoV-2 Protocol v4.01, with some minor modifications, reverse complement PCR is performed using a panel of probes that will generate tiled amplicons covering the whole SARS-CoV-2 genome. The final step involves an improved library clean up that will remove unused/unincorporated PCR probes, which will be more abundant in samples that contain low viral RNA concentrations such as those from wastewater.