Sequencing Using a Two-Step Strategy Reveals High Genetic Diversity in the S Gene of SARS-CoV-2 after a High-Transmission Period in Tunis, Tunisia.

Recent efforts have reported numerous variants that influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral characteristics, including pathogenicity, transmission rate, and detectability by molecular tests. Whole-genome sequencing based on next-generation sequencing technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low- and middle-income countries. To decrease sequencing costs, we developed a primer set allowing partial sequences to be generated in the viral S gene, enabling rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole-genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred one nasopharyngeal specimens collected during the decreasing phase of a high-transmission COVID-19 wave in Tunisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allow the detection of first introductions in the country of already-known VOCs and VOIs, as well as other variants that have interesting genomic mutations and need to be kept under surveillance. The method of choice for SARS-CoV-2 variant detection is whole-genome sequencing using next-generation sequencing (NGS) technologies. Resources for this technology remain limited in many low- and middle-income countries, where it is not possible to perform whole-genome sequencing for representative numbers of SARS-CoV-2-positive cases. In the present work, we developed a novel strategy based on a first partial Sanger screening in the S gene, which includes key mutations of the already known VOCs and VOIs, for rapid identification of these VOCs and VOIs and to help better select specimens that need to be sequenced by NGS technologies. The second step consists of whole-genome sequencing to allow a holistic view of all variants within the selected viral strains and confirm the initial classification of the strains based on partial S gene sequencing.