Detection of SARS-CoV‑2 Variants of Concern with Tiling Amplicon Sequencing from Wastewater

The emerging variants of concern (VOCs) of SARS-CoV-2, e.g., Alpha, Beta, Gamma, Delta, and Omicron, have constrained the global response to the COVID-19 pandemic. They challenge our current capability to identify and distinguish variants from wastewater, due to the high likelihood of viral RNA degradation and the prior knowledge required for primer design. This study focused on the detection of multiple VOCs of SARS-CoV-2 using a high-throughput, multiplexed, amplicon-based sequencing technology, namely, ATOPlex. We first demonstrated that this method can discern multiple variants from artificial samples consisting of four synthetic strains of SARS-CoV-2. The ability of ATOPlex to identify VOCs was further validated using real wastewater samples collected from both an international passenger flight and local wastewater treatment plants. On the basis of phylogenetic analysis and the identification of single-nucleotide polymorphism on the genomes, the ATOPlex method was shown to be effective in detecting three VOCs, including two Beta variants and one Delta variant from either local wastewater or flight sewage samples, which were phylogenetically close to the variants that originated from France, Philippines, and the United States. We found this method is mutation-independent, rendering it a tool for proactive detection of SARS-CoV-2 VOCs in wastewater for the application of wastewater-based epidemiology.