PCR-Sequencing Approaches to Assess Informative Mutations in SARS-Cov-2 Spike (S) and ORF7, ORF8 and N Genes Characterizing Variants of Concern and Variants of Interest

Abstract

Background: The high infectivity rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the prolonged duration of coronavirus disease 2019 (COVID-19) pandemics have contributed to the emergence of viral variants endowed with evolutionary advantages, leading to enhanced infectivity. The tracking of these lineages is urgent. However, the need to sequence whole-viral genomes through next-generation sequencing (NGS) represents a barrier hampering the massive identification of these variants. Therefore, in the current study, we developed Sanger-sequencing approaches targeting regions of interest containing vast lineage-defining mutations in the SARS-CoV-2 S gene and ORF8 region, allowing for unambiguous identification of all SARS-CoV-2 variants of concern (VOCs) and of interest (VOIs).Methods and results: Primers were designed for polymerase chain reaction (PCR) and nested-PCR to amplify and sequence samples with a low-viral burden. The primers’ annealing sites conservancy were checked in a large group of sequences. Amplification protocols were standardized, and sequencing reactions were performed in a cohort of samples for validation. The primers were highly efficient and sequencing of the targeted regions matched those generated by NGS in the same samples. The sequencing results allowed for the unambiguous identification of B.1.1.7, P.1 and P.2 samples, and would also allow the identification of B.1.617.2, B.1.351 and B.1.427/B.1.429 lineages, which were absent in our cohort.Conclusion: Implementing Sanger-sequencing-based approaches to identify SARS-CoV-2 lineages may represent an alternative to tracking these variants by more laboratories around the world and providing valuable molecular and epidemiologic information to inform health systems.