PONE-D-25-23235Spatiotemporal structure of SARS-CoV-2 mutational frequencies in wastewater samples from OntarioPLOS ONE

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PLOS ONE

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Additional Editor Comments:

Dear authors,

I join the reviewers in thanking them for their clear manuscript and its significant contribution to the study not only of SARS-CoV-2, but also to the epidemiology of emerging diseases. Please review and attend the minor changes, which I believe will make your manuscript a more interesting work.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Dear authors,

Congratulations to this very nice (and interesting) manuscript. It is clearly written, nicely explained and easy to follow. In the following, please find more concrete comments:

INTRODUCTION

- p. 3, lines 3-11: Suggest to move to methods section as study area sub-section.

METHODS

- p. 4, lines 20-23: May this have an impact on the number of mutations detected?

- p. 5, Figure 1: Please add the number to the legend to show the magnitude of the number of samples, e.g. is a small circle one sample or ten or hundred?

- p. 7, line 1: "This normalization also conferred some robustness to variation in read coverage among samples...", Please discuss this further as it has an impact on your analysis, i.e. deriving spatial or temporal differences when in reality read coverages were different, i.e. not the same mutations were covered.

RESULTS

- General observation: Figures often placed before their call-outs in the text.

DISCUSSION

- p. 20, lines 6-7: Have you considered examining defining mutations for a variant to see whether your approach would be comparable to other data sources in terms of the time component?

- p. 20, lines 14-15: How do you ensure that this is really due to temporal differences in the occurrence rather than differences in coverages?

- p. 20, lines 16-17: Please consider: statistically significant yes, but also practically relevant?

- p. 21, lines 9-10: How did you derive this lower limit of detection for spatial structure, as there are studies showing differences between geographical units on a much smaller spatial scale?

- p. 21, lines 16-17: Your study focused on single mutations instead of lineage-assignment, for which you highlighted the shortcomings, so why bring back this argument in the discussion?

- p. 21, lines 17-21: Please provide an example for residential areas, such as Schmiege, Kraiseburd et al.:

https://doi.org/10.1016/j.scitotenv.2023.165458

- p. 21, lines 21-22: Please provide an example for this statement, such as Rios et al.: https://doi.org/10.1016/j.lanepe.2021.100202

Reviewer #2: The authors present a large study of SARS-CoV-2 sequencing of wastewater samples from the Ontario region of Canada. They focus on the nucleotide mutations rather than lineage deconvolution. I found the paper to be interesting, well formatted, and ready for publication. I have no suggestions or questions.

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Reviewer #1: No

Reviewer #2: No

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Spatiotemporal structure of SARS-CoV-2 mutational frequencies in wastewater samples from Ontario

PONE-D-25-23235R1

Dear Dr. Poon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Pablo Colunga-Salas

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear authors,

I recognize that the work has improved with the reviewers' comments. On the other hand, I am convinced that your manuscript is ready to continue the process and be published.

Reviewers' comments: