PONE-D-20-22918

Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: study protocol

PLOS ONE

Dear Dr. Alberto Meyer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.  The Reviewers found merit in your study and we encourage resubmisison.

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Gianfranco D. Alpini

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a "Registered Report Protocol", where the investigative team plans to conduct an observational, case-control study to understand the composition of the biliary microbiota in patients with hepatobiliopancreatic diseases, compared to health controls. The study design is appropriate, given that well-designed observational studies are often found to be effective, compared to RCTs (randomized controlled trials).

I do have some additional comments that may require attention:

1. Sample size/Power: The sample size/power statement should explicitly mention the "name" of the statistical test used, and the "response" that was used to calculate this. The authors' mentioned effect sizes, but never mentioned the name of the test, whether it was one-tailed, or two-tailed, etc. a two-tailed test. Better to be specific (what test?).

2. Writing style: Even before presenting the sample size/power, I found lot of words and sentences used in describing 16S rRNA method, but I never found "what is the response" they will be using? Microbiome data is usually compositional in nature; see article below:

https://www.frontiersin.org/articles/10.3389/fmicb.2017.02224/full

The writeup needs to clearly describe the type of responses to be used, specifically, are they using the compositional data, or some "summary" of the compositional outcomes to conduct the comparison between disease and controls.

There is a pretty robust literature on conducing microbiome data analysis using compositional data, using standard freeware (like R); the authors are suggested to consider that, versus doing some rudimentary/half-hearted analysis. I strongly suggest addition of a qualified statistician/bioinformatician within the research team to produce nice data analysis results, and not some point and click.

3. Abstract/Statistical Analysis Methods: That brings me to the abstract & statistical analysis proposed. I see names of tests, like Student's t, and Man-Whitney tests. Again, it reads weird, when no responses are mentioned corresponding to the proposed tests, i.e., on which quantities those tests will be used.

4. Longitudinal Design: From the current writeup, study doesn't look longitudinal (correct me if I am wrong!). Plans to consider a longitudinal study maybe crucial (can be part of the Discussion/Conclusion Section), given that those may reveal time-varying compositional changes between the two groups.

Reviewer #2: In the current manuscript, Nascimento et al. reported a registered study protocol aiming at evaluating the difference of microbiota in bile samples between healthy people and those with hepatobiliopancreatic diseases. The authors well described the study procedure in human samples and the processing of DNA preparation for sequencing. However, the authors did not well justify several factors including aging and gender which significantly contribute to the difference of microbiota profiles in their statistical analysis. Based on previous studies (Gut microbiota and aging, Science 04 Dec 2015:Vol. 350, Issue 6265, pp. 1214-1215; Intestinal Microbiota Is Influenced by Gender and Body Mass Index, PLoS One. 2016 May 26;11(5):e0154090.doi: 10.1371/journal.pone.0154090. eCollection 2016.), the proposed statistical analysis which did not consider age, body mass index, and gender as variance factors may not obtain significant outcomes.

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Reviewer #1: No

Reviewer #2: No

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Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: study protocol

PONE-D-20-22918R1

Dear Dr. Meyer,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Gianfranco D. Alpini

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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

**********

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 and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors addressed my previous comments to a greater degree of satisfaction. I have no further comments.

Reviewer #2: Thanks for your responses. All my concerns have been well addressed. I look forward to the outcomes of your study.

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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