Dynamics of SARS-CoV-2 Mutations in Wastewater Provide Insights into the Circulation of Virus Variants in the Population

SARS-CoV-2 high transmission and genomic mutations result in the emergence of new variants that impact COVID-19 vaccine efficacy and virus transmission by evading the host immune system. Wastewater-based epidemiology is an effective approach to monitor SARS-CoV-2 variants circulation in the population but is a challenge due to the presence of reaction inhibitors and the low concentrations of SARS-CoV-2 in this environment. Here, we aimed to improve SARS-CoV-2 variant detection in wastewater by employing nested PCR followed by next-generation sequencing (NGS) of small amplicons of the S gene. Eight SARS-CoV-2 wastewater samples from Alegria Wastewater Treatment Plant, in Rio de Janeiro, Brazil, were collected monthly from February to September 2021. Samples were submitted to virus concentration, RNA extraction and nested PCR followed by NGS. The small amplicons were used to prepare libraries for sequencing without the need to perform any fragmentation step. We identified and calculated the frequencies of 29 mutations matching the Alpha, Beta, Gamma, Delta, Omicron and P.2 variants. Omicron matching-mutations were detected before the lineage was classified as a variant of concern. SARS-CoV-2 wastewater sequences clustered with SARS-CoV-2 variants detected in clinical samples that circulated in 2021 in Rio de Janeiro. We show that sequencing of selected small amplicons of SARS-CoV-2 S gene allows the identification of SARS-CoV-2 variants matching-mutations and their frequencies calculation. This approach may be expanded using customizing primers for additional genomic regions, in order to differentiate current variants. Approaches that allow us to learn how variants emerge and how they relate to clinical outcomes are crucial for our understanding of the dynamics of virus variants circulation, providing valuable data for public health management.