RBD Amplicon Sequencing and Evolutionary Analysis of SARS-CoV-2 Variants from Wastewater Samples.

The surveillance of SARS-CoV-2 is significantly limited by the presence of numerous variants with distinct behaviors driven by rapid evolution. Although the conventional clinical genome sequencing method provides comprehensive data, it has limitations in capturing the epidemiological dynamics of the variants circulating in the community. In this chapter, we describe a protocol using RBD amplicon sequencing of wastewater samples. We present the detailed experimental procedure for wastewater sample processing, robust nested-PCR-based amplification of the target RBD sequence, and library preparation. Moreover, we describe a general pipeline of sequencing data processing and interpretation, showing the epidemiological and evolutionary trends of SARS-CoV-2 variants in the community.