PONE-D-20-22465

A workflow with RStudio: phylogenetic analyses and visualizations using mitochondrial cytochrome b gene sequences

PLOS ONE

Dear Dr. BILGE,

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 address the comments and suggestions made by the reviewers, particularly those regarding format and readability. Additionally, as reviewer 2 points out, there is little in this work that is specific to RStudio, and the strengths of some approaches are not fully explained. After addressing these concerns, the manuscript could be much improved.   

Please submit your revised manuscript by Nov 28 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Michael Scott Brewer, Ph.D.

Academic Editor

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?

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

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

Reviewer #1: Yes

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 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

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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: No

Reviewer #2: Yes

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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 presents exciting and beneficial research for computational phylogenetics, and should be published.

There are grammatical issues throughout the manuscript which should be addressed, and it may be advisable to enlist the aid of a dedicated copyeditor.

While fairly straightforward, I do think that Figure 1 needs a descriptive caption to convey what you want the reader to take away from the figure.

Likewise, the caption for Figure 3 discusses which commands were used to construct the figure, and while the purpose of the figure here is to demonstrate the utility of these commands, it would still be advisable to provide some explanation of what information the figure itself is meant to convey. For example, what do the vertical hash marks indicate? Does the variation in branch length tell us anything? You convey these things in the caption for Figure 4, but if you are going to only explain them in one figure why not do so in the first

figure of this type which the reader will see?

For Figures 5 and 6, it would be good to put "site substitutions" after the 0.006 over the scale bar, for clarity.

For the most part, the walkthrough is well written and easy to follow. However, I do have some comments:

In the walkthrough, you have readers install the "stats" package twice.

It would be good to make sure that the instructions in the walkthrough are written in such a way so that people do not just stop reading when they get to, for example, "If your arrays are already aligned, please proceed from "READING AND PLOTTING OF ALIGNMENT-NJ-MSAPLOT" section" and miss important instructions in the same paragraph. The above quote is followed by a general warning of how long the alignment can take, which is then followed by an important instruction about calling the previously aligned file to the RStudio console. Make sure the walkthrough is written to be useable by people with little to no knowledge of R. Your code is helpful and you want lots of people to use it, so the lower you can make the bar to entry the better.

Along these same lines, the walkthrough is written such that even if I do not need to align my sequences (as an example), I still need to perform some of the commands in the sequence alignment section in order to complete tasks described further down in the walkthrough. This could be another barrier to use of your

code by those not well versed in R.

Unfortunately, I was unable to test your code using the, relatively, small .fas files which I use for my research. Your code worked fine when tested with your example fsa file, but my 124 taxa, 1016 bp .fsa file caused RStudio to crash when I used nbin<-fasta2DNAbin(fname). I have not had trouble importing these data into RStudio in the past, so I assume it has something to do with your code. If your code cannot handle sequences of over 1000 bp, it should be noted in the publication. I was able to test your code on a ~500 bp, 137 taxa .fsa file, however, so your code works for shorter sequences.

Reviewer #2: I found this manuscript to have a very helpful purpose: providing a worked example of population genetic and phylogenetic analysis in R. The manuscript was mostly clearly written. Below I outline a few key areas for improvement.

First a few more general suggestions for improvement:

1) There is nothing specific to RStudio in this workflow. I would encourage the authors to simple find and replace all instances of "RStudio" with "R". The section explaining that RStudio is a popular IDE could optionally remain. I suggest this because the authors do not discuss any RStudio-specific functionality, so continually referring to RStudio is misleading or distracting. I also make this suggestion because there remains a substantial group of R users who are uninterested in adopting RStudio for their work, but who would still find the contents of this manuscript useful.

2) The authors repeatedly highlight that some returned objects are of the S4 class system. However, the authors do not explain what is significant about S4 classes, nor how they compare to the S3 and R5 systems, nor any potential enter-operability concerns among different class systems. Thus I would suggest either including more information about S4 and why it matters to the analyses, or I would encourage the authors to simply remove any reference to S4.

3) The code examples in the main text need to be properly formatted. As they stand now, they are extremely hard to read. The main issue is a lack of proper line breaking and indentation. R Markdown or Sweave could be used to produce the proper formatting.

Finally, here are a few edits for increased clarity:

lines 58--60: remove the sentence about Sweave, starting "Sweave, a powerful..." and ending "...and calculation results [10]." Sweave is never mentioned again, and so does not need to be introduced here. It is probably also no longer the first choice of most R users for making dynamical documents as most people now seem to use R Markdown and knitr.

lines 62--66: remove the sentences beginning "R also can be used..." and ending "...and store data analysis [10]." The purpose of this section is unclear and the use of the term "environment" (a very specific term in R) is incorrect in the technical context of R programming.

line 93: the semicolon (;) should be a colon (:)

line 98: delete "array"

line 180: instead of saying "S4 class" saw what the actual class name is

line 207: "subtracted" should be "extracted"

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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.]

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PONE-D-20-22465R1

A workflow with R: phylogenetic analyses and visualizations using mitochondrial cytochrome b gene sequences

PLOS ONE

Dear Dr. BILGE,

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.

Thank you for your improved manuscript. One of the original reviewers was kind enough to assess the changes, and they are happy with your edits. I did my best to evaluate the responses to the other reviewer's comments and found them satisfactory as well. Please address the minor edits suggested by the reviewers.

Please submit your revised manuscript by Jan 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Michael Scott Brewer, Ph.D.

Academic Editor

PLOS ONE

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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

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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

**********

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

Reviewer #1: N/A

**********

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

**********

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

**********

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: This is a much improved manuscript and will be a wonderful contribution to the ability of researchers to efficiently and effectively analyze and visualize phylogenetic data.

I do have a few very minor grammatical points:

Line 433: "and" needed after "tree type,"

Line 435: This sentence is a bit clumsy, perhaps reword to "While input-output files are frequently needed in phylogenetic analysis software..."

Line 446: "was" should be "were"

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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

[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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

A workflow with R: phylogenetic analyses and visualizations using mitochondrial cytochrome b gene sequences

PONE-D-20-22465R2

Dear Dr. BILGE,

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,

Michael Scott Brewer, Ph.D.

Academic Editor

PLOS ONE

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