A Sanger approach based on overlapping fragments to screen SARS-CoV-2 variants

Abstract

Since the beginning of the COVID-19 pandemic, the SARS-CoV-2 virus has undergone various genetic mutations which have led to the emergence of variants. The World Health Organization (WHO) defines Variants of Concern (VOCs) and Variants of Interest (VOIs) according to several criteria. These include significant changes in the transmissibility and pathogenicity of the virus characterized by mutations in the spike gene coding the spike glycoprotein. In this study, we designed ten Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) assays in order to identify mutations of SARS-CoV-2 in overlapping fragments. Each assay contained mutations on the fragments sequenced by a Sanger method. The genomic analysis of the fragments allowed to identify the variant according to the position of the mutations. The assembly of the 10 fragments refined the analysis, highlighting all the mutations present in the S gene. Finally, a comparison of methods using a Next-Generation Sequencing (NGS) approches for samples enabled the method to be validated. By this method we have highlighted a characteristic mutation of the lineage B of SARS-CoV-2. We showed the circulation of SARS-CoV-2 belonging to lineage A and B in the beginning of the pandemic in Gabon. We have identified the Alpha, Delta and Omicron variants. This method would allow laboratories with limited financial means or without NGS instrument to obtain sequences of the S gene. This method wase very effective to highlight the circulation of variants, in particular VOCs and VOIs, in this developing country, Gabon, during the COVID-19 pandemic.