Review Reports - The Use of Confidence Intervals in Differential Abundance Analysis of Microbiome Data

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The Introduction is too long and should be shortened to be more focused on the core objectives of the study.
2. Please clearly state the aim and specific objectives of the study at the end of the Introduction.
3. Add a clear heading before Section 1.1: Location parameters of microbial data.
4. The number of abbreviations is minimal, and all are already explained in the text; therefore, the Abbreviations section is not required and may be removed.
5. In line ~272, replace “comparing subsamples drawn form negative binomial” with “drawn from a negative binomial distribution.”
6. In lines ~65–91, scientific names (Porphyromonas gingivalis, Streptococcus mutans, Prevotella copri) are inconsistently italicized. Please ensure that all scientific names are italicized consistently throughout the manuscript.
7. Sentences in lines ~64–91 and ~101–109 exceed 50–60 words. Please split these into shorter sentences to improve clarity and readability.
8. In lines ~131 and ~230, there is inconsistency between sentence case and title case in headings. Please use one consistent heading style throughout the manuscript.
9. In lines ~180–182, “T-test's CI” should be corrected to “t-test CI” or “CI of the t-test.”
10. In line 287, remove the comma after “Similar” in the sentence:
  “Similar but more pronounced trends were observed.”
11. In lines ~274–276, replace “two-sampled hodges lehman estimation” with “two-sample Hodges–Lehmann estimation.”
12. In line ~316–317, the commented text “[M1]: Please check and confirm…” must be removed before submission.
13. In line ~274, replace “Welsh correction” with “Welch correction” throughout the manuscript.
14. In line ~276, replace “hodges lehman” with “Hodges–Lehmann” consistently throughout the manuscript.

Author Response

Comments 1: The Introduction is too long and should be shortened to be more focused on the core objectives of the study.

Response 1: Thank you for your comment. We agree that the introduction is relatively long. Unfortunately, the methodological background section common to such studies tends to be extensive, as it is necessary to properly contextualize the research and the specific issue we are trying to address. We structured it to gradually address potential questions, explain the current state of knowledge, and introduce our specific research problem.

To help readers follow the work and skip the extensive details presented at the beginning, we have restructured the Introduction with clearer sectioning and reduced the word count from 1930 to 1381. This was achieved by omitting the full text of the taxonomic examples (lines 65-95), deleting the concluding sentences on effect size uncertainty (lines 121-127), removing the geometric mean from the enumeration of central tendencies, as it is a relatively rarely discussed option (lines 157-159), and deleting the discussion of additional Hodges-Lehmann estimator properties (lines 189-198), as they are not central to the main discussion.

Comments 2: Please clearly state the aim and specific objectives of the study at the end of the Introduction.

Response 2: Thank you for your suggestion. We have added a standalone Section 1.2, "The Aim and Objectives of the Study," which clearly states the study’s aim and objectives. All changes are highlighted.

Comments 3: Add a clear heading before Section 1.1: Location parameters of microbial data.

Response 3: Thank you for your suggestion. We have added a "1.1. Background" section header before Section 1.2 and deleted the misleading original Section 1.2 header. The introduction now contains only the following 3 sections: 1.1. Background, 1.2. The aim and objectives of the study, and 1.3. Summary of the results. All changes are highlighted.

Comments 4: The number of abbreviations is minimal, and all are already explained in the text; therefore, the Abbreviations section is not required and may be removed.

Response 4: Thank you for your comment. We believe the abbreviations section is a required component.

Comments 5: In line ~272, replace “comparing subsamples drawn form negative binomial” with “drawn from a negative binomial distribution.”

Response 5: Thank you for your suggestion and for drawing our attention to this typo. The suggested correction has been implemented. All changes are highlighted.

Comments 6: In lines ~65–91, scientific names (Porphyromonas gingivalis, Streptococcus mutans, Prevotella copri) are inconsistently italicized. Please ensure that all scientific names are italicized consistently throughout the manuscript.

Response 6: Thank you for your comment. In our version of the manuscript, all species names are consistently italicized. No abbreviations were introduced for species’ names due to their infrequent mention.

Comments 7: Sentences in lines ~64–91 and ~101–109 exceed 50–60 words. Please split these into shorter sentences to improve clarity and readability.

Response 7: Thank you for your comment.

The longest sentence in these lines is 55 words:

“The challenge of detecting these changes in microbial composition is threefold: first, increases in pathobionts or decreases in key commensals are often minor; second, pathobionts and key commensals typically represent only a small fraction of the total microbial population; and third, the change in their relative abundance (which is already minimal) can itself be small.”

We have split this sentence into three parts (all changes are highlighted):

“The challenge of detecting these changes in microbial composition is threefold. First, increases in pathobionts or decreases in key commensals are often minor. Second, pathobionts and key commensals typically represent only a small fraction of the total microbial population. Third, the change in their relative abundance (which is already minimal) can itself be small.”

All other sentences in this paragraph contain 36, 45, 29, 38, 18, 32, 32, 47, 33, 19, and 31 words. We believe their consecutive placement contributes to the feeling that they are longer. This paragraph has been removed. This revision serves a dual purpose: shortening the overall Introduction and eliminating dense supplementary detail peripheral to the core argument. All changes are highlighted.

Comments 8: In lines ~131 and ~230, there is inconsistency between sentence case and title case in headings. Please use one consistent heading style throughout the manuscript.

Response 8: Thank you for pointing this out. We have changed all headings to the prevailing Title Case. All changes are highlighted.

Comments 9: In lines ~180–182, “T-test's CI” should be corrected to “t-test CI” or “CI of the t-test.”

Response 9: Thank you for your suggestion. The suggested correction has been implemented. All changes are highlighted.

Comments 10: In line 287, remove the comma after “Similar” in the sentence:
“Similar but more pronounced trends were observed.”

Response 10: Thank you for your suggestion. The suggested correction has been implemented. All changes are highlighted.

Comments 11: In lines ~274–276, replace “two-sampled hodges lehman estimation” with “two-sample Hodges–Lehmann estimation.”

Response 11: Thank you for your suggestion and for drawing our attention to this multiplied typo. The suggested correction has been implemented. All changes are highlighted.

Comments 12: In line ~316–317, the commented text “[M1]: Please check and confirm…” must be removed before submission.

Response 12: Thank you for highlighting this. The text in question is part of an editorial comment thread. We have submitted a reply, and the thread will be resolved after the revisions are processed.

Comments 13: In line ~274, replace “Welsh correction” with “Welch correction” throughout the manuscript.

Response 13: Thank you for pointing this out. The suggested correction has been implemented. All changes are highlighted.

Comments 14: In line ~276, replace “hodges lehman” with “Hodges–Lehmann” consistently throughout the manuscript.

Response 14: Thank you for pointing this out. The suggested correction has been implemented. All changes are highlighted.

Reviewer 2 Report

Comments and Suggestions for Authors

I was pleased to review this manuscript. The article is well written, informative, and represents a valuable and novel contribution to the field of metagenomic data analysis. I think you should accept the manuscript after addressing the minor suggested revisions.

Instead of referring to group diversity and individual diversity, it is more accurate to use beta diversity and alpha diversity, respectively.
The number of references is limited, and several statements lack proper citation. Additional relevant references should be included to support these points.

Author Response

The authors appreciate the reviewer's consideration of the work.

Comments 1: Instead of referring to group diversity and individual diversity, it is more accurate to use beta diversity and alpha diversity, respectively.

Response 1: Thank you for your suggestion. We agree that the terms "alpha and beta diversity" are more field-specific. We have implemented this change by using "alpha/beta diversity" as the primary terms, with our original phrasing ("within-sample/between-group biodiversity") retained in parentheses for clarity, as they might be more self-explanatory. All changes are highlighted.

Comments 2: The number of references is limited, and several statements lack proper citation. Additional relevant references should be included to support these points.

Response 2: The authors would like to thank the reviewer for the consideration of the work. Despite the vast body of accumulated data in metagenomic research, limited work is available on the specific topic discussed in this study.

The authors would also like to take this opportunity to refer the reader to ALDEx2, a tool for microbial analysis that was not mentioned earlier. Its omission from the list of popular tools presents an incomplete picture of the microbial differential analysis landscape.

Additionally, the authors would also refer the reader to the review by Lin and Peddada, which provides comprehensive research on FDR rates (including in methods utilizing multiple testing correction for hypothesis filtration) and a thorough description of modern microbiome marker discovery methods.