Dear Dr. Maghembe,

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PLOS One

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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?

Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: No

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: This manuscript investigates consistence of host-response transcriptomic associated with bacterial and viral infections. The author proposes a conservative workflow based on modules and their transportability in an external dataset. Overall, the distinction between discovery and external projection is well maintained.

- The main limitation of the study is that the conclusions are based on only one external validation dataset (GSE73461). Include additional datasets or discuss this limitation more explicitly. For example: "These results support the external transportability of selected host-response modules across public infection transcriptomic cohorts." This statement could be slightly softened to avoid overgeneralization.

- The manuscript indicates that Gene Ontology terms were manually reviewed to define modules. More details (criteria) describing how these decisions were made would improve transparency and reproducibility. Are these modules optimal or most informative?

- The manuscript does not compare the proposed strategy with other commonly pathway scoring approaches (GSVA). A brief discussion explaining these limitations should be addressed. Only median differences and adjusted P-values are presented. Include effect size measures and confidence intervals.

Overall, the manuscript addresses a relevant methodological question regarding the reproducibility and transportability of host-response transcriptomic modules across independent datasets. The novelty is methodological rather than biological, regarding the reproducibility of biologically interpretable transcriptomic modules across independent cohorts.

Reviewer #2: SYNOPSIS AND TECHNICAL ISSUES

This manuscript describes a limited and largely sound piece of research seeking to identify characteristic patterns of gene expression that differentiate a viral infection from a bacterial infection in humans. The work explores transcript levels from two published compendiums of transcript information with known disease status. The premise of this work is that there exists collections of genes for which the change in expression reflects the infecting agent and that such collections can be applied more broadly. As I understood the report, one set of data was used as the training set to extract sets of characteristic genes. The expression of these sets of genes was analyzed in a second set of published data to evaluate the discriminatory power of the collection. The authors conclude that it is possible to identify such collections of genes that could be "transported" to other data sets.

The study was limited to these two data sets and did not aggressive explore the power of these particular findings. Specifically, the collections of genes were identified by computational means and with guidance provided to the investigator using information about biological processes (GO identifiers.) The premise was supported by the findings but not challenged. For example, how many of the identified genes were essential for each collection to provide discrimination? This would have been a valuable computational exercise (leave one out, leave two out, etc.) that could extend the utility of the genes identified in this single training set.

The singularly significant finding reported in the Discussion was that discrete programs of gene expression could be found with the power to discriminate among the infecting agent. Viral infections were characterized by antiviral pathways, interferon-stimulated pathways and the more vague anti-viral program of gene expression. Bacterial disease appeared to have a curious fingerprint in translational and ribosome metabolism along with mitochondrial respiration and oxidative phosphorylation.

This finding may prove to be of greater significance in future studies that violate the "directionally" found in these studies. For example, a novel viral disease that presents with the pattern of the bacterial fingerprint may provide unexpected insight into a novel nature of the viral disease and vice-versa. It is for this reason I find the study to be valuable. However, there are additional significant limitations in the manner in which the work was presented.

Technical limitations in this manuscript that need to be corrected include the following:

1. As noted above, the extent to which the gene sets can suffer the loss of specific members and still provide discrimination should be evaluated. Alternatively, if my understanding is so badly off-base, the manuscript should be re-written in a manner to make the objectives more clear to a naïve reader such as myself.

2. The gene sets analyzed were only identified by the accession number (GSE211567 and GSE73461). The nature of the samples needs to be described in substantial detail for independent evaluation by the reader. The content and nature of the gene sets matter much more than the accession number.

3. What was the logic of excluding control samples in the z-score reference set (lines 118-119). Specifically, what was the expected outcome of this exercise and how could it have affected the results?

4. The figure legends (captions) are entirely inadequate because they fail to explain what is depicted in each figure. These legends should state the finding, succinctly describe the method of analysis, and describe the elements in the figure needed to ascertain the finding.

5. What is the formal definition of concordance used here as shown in Fig. 1B?

6. What is the formal definition and mode of calculation for the "module score" from the mean z-score. What is this mean value derived from?

7. What is shown in Fig. 2C? Because the x-axis is not a continuum of values, the y-values have little reason to be joined and expressed in a scatter plot as shown. Moreover, I am a bit unsure of how one defines "projection significance" and what is meant by the "Main projection and "Primary-only z-score sensitivity?

STATISTICS

It is difficult to evaluate the statistical analyses because the methods were not adequately (or clearly) described. In so far as the primary query was a z-score, the results appear appropriate analyzed using corrections for multiple comparisons.

CLARITY

The manuscript is very difficult to understand because of the overly generous use of jargon and phrases that have minimal informative content. I would strongly encourage the authors to write in a simpler style. Even the word "transportable" leans toward jargon. This word, in the context of bioinformatic analyses, should be defined in plain English before being used so heavily.

Other phrases that are unnecessarily confusing include: "coherent expected-direction behavior," "site-aware discovery," "safeguards" (in the context of the analysis of gene expression), "strict discovery/projection firewall," and "frozen," to highlight phrases from the Introduction that could better be conveyed in simple English.

Of particular importance is the notion of "projection." The focus of this paper seems to be that clusters of related genes can be identified and applied to a new dataset to discriminate between viral and bacterial disease without redefining (or rediscovering) the clusters of related genes. This method of "fixed-module projection" should be defined in plain English to reach the broader audience that is typical for the PLoS journals.

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what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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External transportability of bacterial- and viral-associated host-response modules across public transcriptomic cohorts

PONE-D-26-30583R1

Dear Dr. Maghembe,

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,

Benjamin M. Liu, PhD, D(ABMM), MB(ASCP)

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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??>

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Main concerns have been satisfactorily addressed as the inclusion of GSE72810 and GSVA sensitivity analysis.

A few minor suggestion to interpretation of VIR_M2: consistency of VIR_M2 across cohorts was not supported maintained with GSVA.

Repetition: overstatement the absence of gene reselection, module redefinition, reweighting, or model training.

Reviewer #2: All concerns (of reviewer 2) have been well addressed. The reduction in jargon appears to have made the manuscript more accessible to the non-expert reader.

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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: Yes: Cezar Rangel Pestana

Reviewer #2: No

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