A SARS-CoV-2 Surveillance Sequencing Protocol Optimized for Oxford Nanopore PromethION v1

To identify and monitor SARS-CoV-2 variant evolution, UNC Charlotte has introduced a surveillance sequencing program for the campus and surrounding county area. We have generated a step by step protocol which has been optimized from the ”High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples” (Pater et al 2021, https://doi.org/10.1101/2021.02.09.430478) protocol for preparing SARS-CoV 2 viral genome libraries for next generation sequencing using the Oxford Nanopore PromethION instrument. The protocol is designed to work in a 96-well format throughout. It is intended to produce sufficient sequence for genome assembly to meet standards required by GISAID and NCBI for SARS-CoV-2 sequence submission. The protocol has been modified to achieve maximum yield and includes modifications to address isolate sequencing failures arising from low viral titer in some clinical samples (Cq values below 30). The protocol provides detailed guidelines and steps for working with high Cq value clinical samples, as well as a protocol variation for working in half-reaction volumes throughout to reduce sequencing costs.