PONE-D-25-22924CoSMIC - A hybrid approach for large-scale, high-resolution microbial profiling of novel nichesPLOS ONE

Dear Dr. Shental,

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Academic Editor

PLOS ONE

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[Funding was provided by Sustainability and Energy Research Initiative by TI; The Yotam project and the Weizmann Institute sustainability and energy research initiative to DS; Combined Weizmann-Abroad Postdoctoral Grant for Advancing Women in Science and the Emergency Fellowships for Israeli Postdocs Affected by COVID-19 to TI. The Open University of Israel internal grant 10024 to NS.].

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Additional Editor Comments:

Reviewer #1:

Reviewer #2:

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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?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

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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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript describes COSMIC, a method that combines short and long reads to enhance 16S identification for uncharacterized microbiome niches.

The combination of short and long reads to benefit high-throughput sequencing is an interesting approach that has many implementations.

The research produces data, explores it, and presents it well.

Some minor issues need to be addressed in the current version. The most significant of them is the effect of sequencing errors on the system. This can be addressed by the two methodologies in the manuscript, PacBio and LoopSeq.

Reviewer #2: The paper "CoSMIC – A hybrid approach for large-scale, high-resolution microbial profiling of novel niches" introduces CoSMIC (Comprehensive Small Ribosomal Subunit Mapping and Identification of Communities), a methodological framework designed to enhance microbial community profiling. CoSMIC builds upon the previously developed SMURF approach. According to the authors, CoSMIC offers a practical solution for studying novel ecological niches.

The revisions made by the authors in response to the reviewers’ comments are adequate and satisfactory. However, some weaknesses remain that require further clarification and improvement.

1. The full and short titles are the same. Authors should correct this and write the short title.

2. The introduction remains too general and reiterates well-known limitations of standard 16S rRNA profiling (e.g., primer bias, short-read ambiguity) rather than emphasis the specific research problem CoSMIC addresses.

3. The final paragraph in Intruduction section presents partial results instead of clearly stating the research objectives and hypotheses. This should be restructured.

4. Additional citations are needed at the end of sentences in lines 60, 63, and 77 to support key statements.

5. I am not fully convinced by the preparation of the mock mixture. While the rationale is understandable, the analyses would be more reliable if a standardized mock community (e.g., commercially available reference mixtures) had been included.

6. Negative controls were not applied at any stage of the study. This omission makes it impossible to determine to what extent the observed microbial profiles might be influenced by contamination. Based on my own experience, even primer purification methods can significantly affect contamination levels and therefore should be considered.

7. The study does not provide an optimized, ready-to-use primer set. It appears that each user must test multiple primer combinations before identifying the most suitable ones. This lack of standardization reduces the reproducibility and usability of the approach.

8. The authors are reminded that, in accordance with the rules of biological nomenclature (ICZN/ICN), the first time a species is mentioned in the manuscript, the full species name must be provided together with the author citation. In all subsequent mentions of the same species, the genus name should be abbreviated, as required by taxonomic conventions.

9. The use of the Greengenes database is problematic, given that it has not been updated since 2013. Silva is a more appropriate choice.

10. The study does not explain why NCBI was not considered, nor does it compare results obtained with different reference databases. A comparative analysis would strengthen the conclusions and clarify which database is most suitable for CoSMIC.

11. Rarefaction curves for some samples did not reach a plateau, raising concerns about whether sequencing depth was sufficient. This calls into question the reliability of applying CoSMIC to novel and complex samples.

12. Relative abundance estimates are acknowledged as unreliable, but the discussion provides little evidence for how UMIs will solve this issue in practice. This limitation currently reduces the ecological interpretability of CoSMIC results.

13. Claims about harmonizing results across studies and niches are speculative and should be more cautiously stated, given unresolved issues with standardization, database completeness, and contamination controls.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-25-22924_reviewer_Comments.docx

Attachment

Submitted filename: CoSMIC_review.pdf

CoSMIC - A hybrid approach for large-scale, high-resolution microbial profiling of novel niches

PONE-D-25-22924R1

Dear Dr. Shental,

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,

Theodore Raymond Muth

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The revised version of the manuscript is now acceptable for publication. The authors have addressed all the issues raised in the review of the previous version. No further issues.

Reviewer #2: Dear Authors,

I am satisfied with your responses to my comments and therefore recommend accepting the article for publication in PLOS ONE.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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