Review 1: "RBD Amplicon Sequencing of Wastewater Reveals Patterns of Variant Emergence and Evolution"

RR\ID Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: The authors contextualize wastewater RBD amplicon sequencing of SARS-CoV-2 and highlight that although the targeted RBD region represents a small segment of the SARS-CoV-2 genome, the advantages of  RBD sequencing being faster, easier, and cheaper than whole genome sequencing are important characteristics that have resulted in an increase in the application of RBD amplicon sequencing in wastewaters and clinical samples. This research shows that SARS-CoV-2 wastewater sequencing is complimentary and provides additional information to the sequencing of clinical samples. Wastewater RBD amplicon sequencing show a more diverse compilation of variants that suggests the potential detection of variants circulating in cities that may otherwise go undetected. Their work also supports previous findings that show that wastewater sequencing has the ability to identify the circulation of new variants in communities prior to clinical sequencing. 

The impact of this work is found in the ecological and evolutionary perspectives that the authors derive from the generated wastewater sequencing datasets. Increases in alpha diversity into and during the winter period are highlighted by the authors to suggest that many variants emerge during these months, with discussion related to seasonal and colder month emergence and spread of new variants. The authors also point to low transmission period as periods of increased diversity of variant circulation. These discussions are substantive and an important contribution to the current literature on wastewater sequencing.