SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis

Abstract

During the course of the SARS-CoV-2 pandemic reports of mutations with effects on spreading and vaccine effectiveness emerged. Large scale mutation analysis using rapid SARS-CoV-2 Whole Genome Sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring transmission and rising levels of mutant strains. Here we report a novel WGS technique for SARS-CoV-2, the EasySeq™ RC-PCR SARS-CoV-2 WGS kit. By applying a reverse complement polymerase chain reaction (RC-PCR), an Illumina library preparation is obtained in a single PCR, thereby saving time, resources and facilitating high-throughput screening. Using this WGS technique, we evaluated SARS-CoV-2 diversity and possible transmission within a group of 173 patients and healthcare workers (HCW) of the Radboud university medical center during 2020. Due to the emergence of variants of concern, we screened SARS-CoV-2 positive samples in 2021 for identification of mutations and lineages. With use of EasySeq™ RC-PCR SARS-CoV-2 WGS kit we were able to obtain reliable results to confirm outbreak clusters and additionally identify new previously unassociated links in a considerably easier workaround compared to current methods. Furthermore, various SARS-CoV-2 variants of interest were detected among samples and validated against an Oxford Nanopore sequencing amplicon strategy which illustrates this technique is suitable for surveillance and monitoring current circulating variants.

Keywords: COVID-19; Lineage; Mutation; RC-PCR; SARS-CoV-2; WGS.