PONE-D-19-32855

A normalization method for next generation sequencing data using power transformation

PLOS ONE

Dear Dr. Wang,

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 author needs to study/review more on current popular normalization approaches and tools for RNA-seq data. More extensive comparisons are needed in the manuscript to show the performance of proposed method.

==============================

We would appreciate receiving your revised manuscript by Feb 21 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled '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.

We look forward to receiving your revised manuscript.

Kind regards,

Xiaofeng Wang

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was supported by a grant from the Simons Foundation (#422535, Bin Wang)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"This research was funded and terminated due job change for the corresponding author. Grant number provided in the manuscript."

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: This manuscript focuses on the proposal of a normalization method for RNA-seq data analysis. It is well written and reasonably sophisticated. Yet I have a couple of comments regarding its publication in PLOS ONE.

Major comment:

1. The first paragraph in Introduction Section mentions measurement errors. Maybe the author can add one or few sentences to explain what are the sources of measurement errors in next generation sequencing data? This added explanation may also help in making a connection between the first paragraph and the second paragraph.

2. Since this proposed normalization method assumes that all gene profile distributions have similar upper tail behaviors, can the author provide the frequency distribution in the upper tail area of the RNA-seq data for LCL to see if this assumption holds?

3. Math symbol convention: suggest using different math symbols/notations for different mathematical formulas:

Equation (1): uses j = 1 to n and uses j = 1 to i

Figure 1: x-axis label says Log(X), it should be Log(zi), right?

Line 91: suggest using a symbol other than x since x has been used in previous formulas.

4. Equation (2): it seems that the density function shown here is incorrect, assuming that the cumulative distribution function is correct. Please check.

5. Equation (3): it should be an approximate relationship, not an exact relationship (not exactly equal), right?

6. I am curious about the performance of the proposed method on gene profiles other than profile 47 and profile 107. Has the author done the data analysis?

7. Could the author provide the rationale that why the optimal estimates should be the pair that minimize the maximum distance between the two empirical distributions?

Minor comment:

1. The terminology ‘global profile’ was used in the abstract but not introduced in the main manuscript. Maybe the author can explain what does ‘global profile’ mean?

2. Line 21: what do loess and MA-plot stand for?

3. Line 73: m distinct values are the actual distinct values or all possible distinct values? If it is all possible distinct values, then the distinct value number m is the same across all n genes. If it is actual distinct values, the distinct value number m can vary from one gene to another, right?

4. The curves in Figure 2 are hard to see. Maybe the author can use other colors for the dotted lines and the dashed lines?

5. It is great that all the algorithms have been implemented in R. Yet I tried and found that the package bda is not available for download purpose. Maybe the author can provide further clarification for the R code implementation in the Conclusion Section.

Minor concern:

1. The second paragraph in Introduction Section lacks intention?

2. In Abstract: ‘the proposed normalization method use global profile information’ -> ‘the proposed normalization method uses global profile information’.

Reviewer #2: The author developed a Zipf plot based normalization method for RNA-seq data analysis by assuming similar upper tail distributions in all gene profiles. It takes the zero-inflated feature of RNA-seq data into account, and the results indicate that the proposed method improves data distributions. However, I have some major concerns:

1) The references in the manuscript are not state of the art. A lot of newly developed normalization methods for RNA-seq data in the past decade are not mentioned/discussed. Here are some papers: [1] Dillies. MA, et al. A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis. Briefings in bioinformatics, 2013. [2] Li, P, et al. Comparing the normalization methods for the differential analysis of Illumina high-throughput RNA-Seq data. BMC bioinformatics, 2015. [3] Zyprych-Walczak, J., et al. The impact of normalization methods on RNA-Seq data analysis. BioMed research international, 2015. [4] Abbas-Aghababazadeh, Farnoosh, et al. Comparison of normalization approaches for gene expression studies completed with high-throughput sequencing. PloS one, 2018.

2) The author should compare with those existing normalization methods (especially the one in DESeq2, which is the most popular tool for RNA-seq data analysis at the current stage) extensively in the manuscript to show the performance of the proposed method.

Minor comments:

The English writing is overall good, but there are still some grammatical mistakes. Please carefully proofread.

**********

6. 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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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. Registration is free. 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. Please note that Supporting Information files do not need this step.

PONE-D-19-32855R1

A Zipf-plot based normalization method for high-throughput RNA-Seq data

PLOS ONE

Dear Dr. Wang,

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.

Please make a minor revision based on the reviewers' comments.

We would appreciate receiving your revised manuscript by Apr 04 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled '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.

We look forward to receiving your revised manuscript.

Kind regards,

Xiaofeng Wang

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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

**********

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

**********

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: I appreciate the author for carefully addressing the reviewers' comments and significantly revising the manuscript. Yet I still have two comments regarding the presentation flow/the writing.

1. Thank the author for rewriting the introduction section to include the existing normalization methods for RNA-seq data. However I think these existing methods are not clearly described in the introduction section in terms of (a) the underlying assumptions (b) applicable scenarios (c) pros and cons (d) implementation and it is difficult to read paragraph 2, paragraph 3 and paragraph 4.

2. I agree that this manuscript needs further modifications in the presentation flow and in the language, especially in those newly added parts. For example, the author uses the phrases like 'we can see', 'we see', 'we want' a lot yet it can be better expressed. Also some phrases are not very formal, for example, Line 173 – Line 174 says that 'and manipulate the Zipf plot of profile Y' but the word 'manipulate' could perhaps be replaced by an alternative word suitable for this setting.

Reviewer #2: The author has carefully and comprehensively revised the manuscript in response to my comments. I do think that the paper has been improved considerably. I do not have any major comments on the revised manuscript, but a few minor concerns:

1. I appreciate the author's extensive discussion and comparison with other RNA-seq normalization methods. I think the message of how to select appropriate methods in the Conclusion section is important. I would suggest adding a brief sentence of this conclusion to the end of Abstract.

2. At the end of Introduction, the author says "This new normalization method helps to prepare high-quality data for further normalization or down-streaming analyses such as classification or clustering analysis." But no data of a further normalization, classification or clustering are shown. The author may reconsider this statement.

**********

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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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. Registration is free. 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. Please note that Supporting Information files do not need this step.

A Zipf-plot based normalization method for high-throughput RNA-Seq data

PONE-D-19-32855R2

Dear Dr. Wang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Xiaofeng Wang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: