Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons

There is need to develop a universally applicable end-to-end viral outbreak sample handling platforms to generate real-time epidemiological information that can be interpreted and applied by public health authorities. Highly sensitive and efficient whole-genome sequencing of the SARS-CoV-2 virus is critical for understanding viral transmission dynamics. Here, we developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 kb to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We here proposed an optimized protocol to synthesize cDNA using Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers, which has high yields of cDNA template for RNA and is capable of long-length cDNA synthesis from a wide range of RNA amounts and quality. The protocol proposed allows whole-genome sequencing of the SARS-CoV-2 virus with tiled amplicons up to 4.8 kb on low-titter virus samples and even where RNA degradation has occurred. This protocol reduces the time and cost from RNA to genome sequence compared to the Midnight multiplex PCR method for SARS-CoV-2 genome sequencing using the Oxford Nanopore.Peer reviewe