Dear Mr. Guo,

Thank you very much for submitting your manuscript "Identification of potential biomarkers in dengue via integrated bioinformatic analysis" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ahmed Mostafa

Associate Editor

PLOS Neglected Tropical Diseases

A. Desiree LaBeaud

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: ALL CRITERIA ARE MET FOR METHODS

Reviewer #2: Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

- The objective of the study is to identify dengue infection biomarkers. In order to do so the authors analyze public data with a standard methodology.

Is the study design appropriate to address the stated objectives?

- Yes, it is. By analyzing available transcriptomes, filtering DEGs, proceeding with pathway enrichment and then looking for miRNAs, the authors are able to find potential biomarkers for dengue infection. However, without at least an in vitro experiment in KO or of some sort targeted pathways in cell line DENV-infected x control in order to validate what was found, the findings in this publication are susceptible to criticism.

Is the population clearly described and appropriate for the hypothesis being tested?

- In fact, no. All three of the datasets chosen for this study have data from different timepoints of infection (post symptoms onset). During infections and diseases progression, patients’ transcriptional profile is in constant change and dengue infection is no exception. In fact, dengue infection is known to exhaust T and B lymphocytes (present in PBMCs, the samples from these studies) during initial stages of the infection but this changes in the convalescent stage. Since the transcriptional profile of each stage is so different, which of the samples were used and compared? There are no mentions to this point in the manuscript.

Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

- Yes, the datasets combined have enough samples to ensure adequate power if all the samples are used. However, I do not believe a transcriptional profile from a convalescent patient would be equal from a patient in the initial stages of the infection. Since the study is trying to find biomarkers, by analyzing all of this data together the statistical tests should be removing potential DEGs from the initial stages just because they are not differentially expressed two weeks later. That being the case, should the authors take this into consideration, I cannot guarantee there are enough samples in these three datasets.

Were correct statistical analysis used to support conclusions?

- Yes, both limma and GEO2R are bioinformatics’ tools that are already published and are highly accepted by scientists.

Are there concerns about ethical or regulatory requirements being met?

- No, there are not.

Reviewer #3: Yes the objectives are clearly stated, still few things are lacking in the manuscript.

Yes the sample size is good enough to address the hypothesis.

1) As all the three gene expression data come from same disease condition but other factors are highly varying. The authors should explain in detail the significance and preselection condition for these datasets to consider in the study.

2) The author should state which method were used for data normalization.

3) What is the rational for selection of fold change value?

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: all criteria are met

Reviewer #2: Does the analysis presented match the analysis plan?

- Yes, it does.

Are the results clearly and completely presented?

- Yes, they are.

Are the figures (Tables, Images) of sufficient quality for clarity?

- Yes, they are.

Reviewer #3: 1) Yes, The analysis is presented matches the analysis plan

2) In results section

Figure 5 is missing

Figure 5c is missing

Figure 6 an 7 description is missing in the text.

3) In discussion part references are missing.

The author either missed or overlooked some parts in the results and discussion, it should be carefully checked and edited.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are ok except for the microRNA part , which is not done correctly to predict microRNAs as biomarkers for deng.

Reviewer #2: Are the conclusions supported by the data presented?

- Yes

Are the limitations of analysis clearly described?

- No

Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

- Yes, both in the introduction and in the conclusion

Is public health relevance addressed?

- Yes, identifying potential biomarkers for disease treatment (and in this case, infection control) are of extreme importance. Even more when talking about dengue, its relevance goes without saying given the number of infected people yearly.

Reviewer #3: 1) Yes the conclusion is well supported by the data presented, limitation of the study is clearly stated.

2) The author should add few points relating to future use of outcome in the conclusion section.

3) The author should corelate the study with public health importance in the conclusion section

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: it is ok

Reviewer #2: I strongly suggest an English revisor in case the paper is accepted due to some English mistakes. Text without numerated lines so it is hard to reference, but some examples in the introduction are:

"There is a few dengue vaccine but no specific antiviral treatment" -> are … vaccines

"A DENV vaccine can not elicits protection" -> cannot elicit

"mainstream view is that immunity leads to cytokine storm cytokine storm" -> repetition

"many patients with DENV infection without developing plasma leakage" -> do not develop

"which is meaningful to predict sever dengue" -> severe

Also, there is an inconsistency (probably a typo in the abstract?):

"Through overlapping, a total of 66 differentially expressed genes (DEGs) were identified, of which 53 were upregulated and 24 were downregulated." (results 3.1 show 13 downregulated, 53+24 do not add up to 66)

Reviewer #3: Major revision is needed.

(As above stated points are significant to the study design)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study is ok; but with no new findings for pathogenesis of deng discovered except cell cycle which is already known, and the microRNA sectis not done rightlyion

Reviewer #2: As stated before, a differentiation in samples from different time (days) since symptoms onset is needed. Since transcriptional profiles are distinct, in order to find better biomarkers it would be required to at least not group convalescent blood samples with initial infection samples. Also, an in vitro experiment in cell lineage (jukarT, B-LCL, ...) in which the identified miRNA were targeted (either via KO by CRISPR/Cas) or maybe the mRNAs were silenced by siRNA would also be interesting. But i also understand that since no funding is linked to this research, the in vitro experiment may be out of question.

Reviewer #3: The study is a good and scientifically sound. Biomarkers are an important biological components in diagnosis and identification of disease and its severity. The DENV infection in tropical and sub-tropical region are big challenges to the health department. Identification of potential biomarkers will help us in diagnosis and identification of drug targets.

The current approach is insilico based analysis hence a clinical validation is needed.

--------------------

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Reviewer #1: Yes: Mahmoud ElHefnawi

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Dear Mr. Guo,

Thank you very much for submitting your manuscript "Identification of potential biomarkers in dengue via integrated bioinformatic analysis" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ahmed Mostafa

Associate Editor

PLOS Neglected Tropical Diseases

A. Desiree LaBeaud

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes. The objective of the study is to identify dengue infection biomarkers. In order to do so the authors analyze public data with a standard methodology.

-Is the study design appropriate to address the stated objectives?

Yes, it is. By analyzing available transcriptomes, filtering DEGs, proceeding with pathway enrichment, protein-protein interaction and then looking for miRNAs, the authors are able to find potential biomarkers for dengue infection.

-Is the population clearly described and appropriate for the hypothesis being tested?

Yes, the authors specify all of it in the manuscript.

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

Yes, there is enough data from these datasets to account for any statistical analysis.

-Were correct statistical analysis used to support conclusions?

Yes, the packages and tools the group used are standard and widely accepted in the bioinformatics' field.

-Are there concerns about ethical or regulatory requirements being met?

No, as they are using public data there is nothing to worry about.

Reviewer #3: As per the authors reply over the reviewer comments, they are acceptable

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: -Does the analysis presented match the analysis plan?

Yes, it does.

-Are the results clearly and completely presented?

Yes, they are.

-Are the figures (Tables, Images) of sufficient quality for clarity?

Yes, yes. The .tiff quality is excellent and the figures are disposed in an organized and clear way.

Reviewer #3: Most the author reply is acceptable, except figure 1 explanation

The threshold was set as followed: P<0.05 and |log2FC|≥2. FC: fold change

Review comment by author:

However, considering that it can ensure significant

differences and screen out more differential genes for further analysis and comparability

before data sets, this study took p < 0.05 and FC ≥ 1.5 when analyzing each dataset.

Because when fold change took 2, the number of DEGs obtained from individual data

sets are relatively small.

This both statements contradicts please clarify

Figure 4 and 5 quality is poor

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: -Are the conclusions supported by the data presented?

Yes, they proposed to find biomarkers for dengue infection and so they did.

-Are the limitations of analysis clearly described?

Yes, the authors perfectly describe it in the manuscript. Even though there are no in vitro nor in vivo experiments, and this may limit their findings, this is discussed and accounted for. Nonetheless, these findings are a great contribution for future studies and may be helpful for researches in dengue therapy.

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Yes, they state it clearly to the readers.

-Is public health relevance addressed?

- Yes, identifying potential biomarkers for disease treatment (and in this case, infection control) are of extreme importance. Even more when talking about dengue, its relevance goes without saying given the number of infected people yearly.

Reviewer #3: The authors reply is acceptable

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: (No Response)

Reviewer #3: Minor revision

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: The authors did a great work with the revision. This sort of bioinformatics analysis is of utmost importance for identifying therapeutic targets and controlling diseases. There is one point I would like to address, though. In your first submission we discussed about different infection's stages and how it may affect the patients' transcriptional profile. I don't know about limma, but when analyzing RNA-seq with DESeq2 there is a test called Likelihood Ratio Test (LRT) that may be used (instead of the default wald test) to identify genes whose expression change overtime. There may be a similar or corresponding test that can be used on limma and may prove to be useful for your future analysis.

Reviewer #3: The authors reply is acceptable

--------------------

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 #2: Yes: Igor Salerno Filgueiras

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Mr. Guo,

We are pleased to inform you that your manuscript 'Identification of potential biomarkers in dengue via integrated bioinformatic analysis' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Ahmed Mostafa

Associate Editor

PLOS Neglected Tropical Diseases

A. Desiree LaBeaud

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

The quality of the figures must be improved "higher resolutions" in the published version of the manuscript