Phylogenetic trees of closely related bacterial species and subspecies based on frequencies of short nucleotide sequences

Yoshio Nakano; Yusaku Domon; Kenji Yamagishi

doi:10.1371/journal.pone.0268847

Peer Review History

Original SubmissionMay 6, 2022
27 Oct 2022 Decision Letter - Frédérique Pasquali, Editor PONE-D-22-13256Phylogenetic trees of closely related bacterial species and subspecies based on frequencies of short nucleotide sequencesPLOS ONE Dear Dr. Nakano, 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. In particular, in order to improve the reader perception on the merit of the new method of phylogenetic recostruction, authors need to provide : 1) a comprehensive comparison with other methods, 2) complete documentation on the R package "Phy5", 3) clear discussion on potential applications Please submit your revised manuscript by Dec 11 2022 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Frédérique Pasquali 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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: In this manuscript, author presented a useful alternative for phylogenetic analysis of closely related bacterial species. Escherichia coli, Shigella, Yersinia, Klebsiella, and Neisseria spp. or serotypes of Listeria monocytogenes were analyzed, and good results were observed. I think that this method is worth exploring further. This manuscript can be accepted after minor revision. Minor comment: (i) The paragraph (lines 153-161) is a repetition of the previous one (lines 144-152). (ii) Supplementary Data 2-4 is missing from the manuscript. (iii) Line 162: stain→strain. (iv) Line 174: E. coli should be in italics. (v) Line 210: Please revise ‘textitI.’ Reviewer #2: Review of the manuscript PONE-D-22-13256, titled "Phylogenetic trees of closely related bacterial species and subspecies based on frequencies of short nucleotide sequences". The article describes a method of phylogenetic reconstruction based on pentanucleotide frequencies. The method could be potentially useful. Unfortunately the current manuscript does not provide a comprehensive comparison with other methods, which makes it difficult to judge the merit of the new method. Also, the implementation of the method needs a documentation and a command line interface to be useful in practical applications. I think, this could be a useful contribution, if these points are addressed. Main comments: 1. Fig.2 compares trees based on pentanucleotide frequencies and 16S rRNA sequences. However, as you noted in the introduction, MLST methods have been already proposed for distinguishing Yersinia species. Such MLST-based tree, based on alignment of concatenated multiple gene sequences, should be included in this comparison. You should compare your method to available state-of-the art alternatives, not just to the weakest alternative method. 2. Figs 3, 4, 5 and 6 show trees based on pentanucleotide frequencies. Best known alignment-based methods should be shown in comparison. This would allow judging the accuracy of the proposed new method. 3. The manuscript needs a clear discussion about scenarios where the new method based on pentanucleotide frequencies should be preferred to other methods, and in which cases it should not be used. 4. The new method is implemented in an R package "Phy5". The package lacks documentation. There should be a step-by-step manual about how to install it, and apply it to a FASTA-formatted dataset. Ideally two scenarios should be supported: (1) Operating on a set of FASTA-formatted files, treating each file as a sample. (2) Operating on a single FASTA-formatted file, treating each sequence as a sample. For this method to be useful in practice, there should be a non-interactive way to generate distance matrix (e.g., in PHYLIP format) and a tree (in newick format), from command line. 5. I think the introduction part does not adequately introduce the topic of alignment-free sequence comparison, and the previous related works. It should include more examples of similar work, because a lot of studies have been done on this topic. See, e.g., these resources for some, but there are many more: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1319-7 https://en.wikipedia.org/wiki/Alignment-free_sequence_analysis 6. It's not clear what is the unique or novel point of this work. Sequence comparison based on nucleotide frequencies have been studies a lot in the past. A possible contribution of this article could be, e.g., a comprehensive comparison of this method with other methods, and a guide about when it's applicable. Or, a convenient implementation, allowing easy reuse of this method in practical bioinformatic pipelines. But currently the manuscript is also lacking in both of these regards. Minor comments. 7. Text, Line 28: "Yersinia is a gram-negative bacterium consisting of 19 species and includes 3 prominent human pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica [13]." ¬- I think more than 19 species are currently recognized, e.g., see the NCBI Taxonomy Database: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=629&lvl=2&lin=f&keep=1&srchmode=1&unlock 8. Text, Line 153: This part repeats the previous paragraph. ****** 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.] 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. https://doi.org/10.1371/journal.pone.0268847.r001
Revision 1
8 Mar 2023 Author Response Reviewer 1: Reviewer: Minor comment: (i) The paragraph (lines 153-161) is a repetition of the previous one (lines 144-152). (ii) Supplementary Data 2-4 is missing from the manuscript. (iii) Line 162: stain→strain. (iv) Line 174: E. coli should be in italics. (v) Line 210: Please revise ‘textitI.’ Authors: (i) The duplicated sentences have been removed. (ii) Supplementary Data 2-4 will be surely uploaded on the revised manuscript submission. (iii) The typo has been corrected. (iv) The word, E. coli, has been italicized. (v) The incomplete LaTeX command has been corrected. Reviewer 2: Main comments: Reviewer: 1. Fig.2 compares trees based on pentanucleotide frequencies and 16S rRNA sequences. However, as you noted in the introduction, MLST methods have been already proposed for distinguishing Yersinia species. Such MLST-based tree, based on alignment of concatenated multiple gene sequences, should be included in this comparison. You should compare your method to available state-of-the art alternatives, not just to the weakest alternative method. Authors: Figure 2B has been replaced with a phylogenetic tree based on the MLST method, and the 16S rRNA-based tree has been renamed as Supplementary Figure 1. The trees generated using pentanucleotide frequencies and MLST have very similar structures. Reviewer: 2. Figs 3, 4, 5 and 6 show trees based on pentanucleotide frequencies. Best known alignment-based methods should be shown in comparison. This would allow judging the accuracy of the proposed new method. Authors: The MLST method for Yersinia has been reported and we can analyze it according to the previously published papers. In contrast, effective MLST methods for Escherichia coli and Shigella have not been reported, so candidate genes for MLST analysis must be determined. If the reviewer believes that MLST analysis for E. coli and Shigella for Figure 4 and Figure 3 are indispensable for this paper, we will attempt such MLST analysis. However, this will likely take around 6 months to complete based on the timeline of the Japanese academic year. MLST or phylogenetic analyses for Ipomoe chloroplasts have also not been reported, so Figure 5 cannot be compared to other alignment-based methods. A phylogenetic tree built using 16S rRNA gene analysis has been added to Figure 6 according to the reviewer's suggestion. Reviewer: 3. The manuscript needs a clear discussion about scenarios where the new method based on pentanucleotide frequencies should be preferred to other methods, and in which cases it should not be used. Authors: This application is useful for analyzing bacterial species whose genome sequences have not yet been determined. Accumulation of short sequences can be used for phylogenetic tree analysis without identification of genes in those fragments. This method is suitable for use as a first candidate, as it can be analyzed without the need for prior knowledge of the genes. In addition, it would be difficult to use other methods in combination with SVM to distribute a large number of samples as draft sequence data. Furthermore, other methods would be difficult to identify species based on a large amount of draft sequence data in combination with SVM. These descriptions have been added at the end of the manuscript. Figure 6 demonstrates a case where this method should not be used. Reviewer: 4. The new method is implemented in an R package "Phy5". The package lacks documentation. There should be a step-by-step manual about how to install it, and apply it to a FASTA-formatted dataset. Ideally two scenarios should be supported: (1) Operating on a set of FASTA-formatted files, treating each file as a sample. (2) Operating on a single FASTA-formatted file, treating each sequence as a sample. For this method to be useful in practice, there should be a non-interactive way to generate distance matrix (e.g., in PHYLIP format) and a tree (in newick format), from command line. Authors: "Phy5" is not an R package. It is a web application based on the R package "Shiny". It can be used as a standalone application and its manual would be the same as that for Shiny. A command line version of "Phy5" has been constructed to use in bioinformatic pipelines. The command line version supports a set of FASTA-formatted files or a single FASTA-formatted file. In addition, a file of the resultant distance matrix and a newick format tree file are generated and saved. R scripts are not very useful for command-line use. In particular, it is not suitable for introducing choices by argv. Therefore, we have made the command line version more user-friendly by integrating it into a Python script. Reviewer: 5. I think the introduction part does not adequately introduce the topic of alignment-free sequence comparison, and the previous related works. It should include more examples of similar work, because a lot of studies have been done on this topic. See, e.g., these resources for some, but there are many more: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1319-7 https://en.wikipedia.org/wiki/Alignment-free_sequence_analysis Authors: Content from the review article by Zielezinski has been added to the introduction, and it has been cited. Reviewer: 6. It's not clear what is the unique or novel point of this work. Sequence comparison based on nucleotide frequencies have been studies a lot in the past. A possible contribution of this article could be, e.g., a comprehensive comparison of this method with other methods, and a guide about when it's applicable. Or, a convenient implementation, allowing easy reuse of this method in practical bioinformatic pipelines. But currently the manuscript is also lacking in both of these regards. Authors: We have submitted this alignment-free sequence comparison to various journals, and for the first time, we have received constructive comments (one of them demanded proof of the evolution from me). We acknowledge that this is no longer a novel idea, but phylogenetic analysis methods based on the frequencies of short sequences are still not widely used. We hope that another web or command line application may be useful for constructing phylogenetic trees based on genome wide sequences. We absolutely agree that command-line use would be a convenient implementation for practical bioinformatic pipelines, and the command line version of Phy5 has been built and embedded into a Python script. Minor comments. Reviewer: 7. Text, Line 28: "Yersinia is a gram-negative bacterium consisting of 19 species and includes 3 prominent human pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica [13]." ¬- I think more than 19 species are currently recognized, e.g., see the NCBI Taxonomy Database: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=629&lvl=2&lin=f&keep=1&srchmode=1&unlock Authors: "19 species" has been corrected "26 species". Reviewer: 8. Text, Line 153: This part repeats the previous paragraph. Authors: The duplicated sentences have been removed. Attachments Attachment Submitted filename: Response_to_reviewers.docx https://doi.org/10.1371/journal.pone.0268847.r002
3 Apr 2023 Decision Letter - Frédérique Pasquali, Editor Phylogenetic trees of closely related bacterial species and subspecies based on frequencies of short nucleotide sequences PONE-D-22-13256R1 Dear Dr. Nakano, 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. Additionally, please consider comment 2 of reviewer 2 at the bottom of this letter. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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. Kind regards, Frédérique Pasquali Academic Editor PLOS ONE Additional Editor Comments (optional): 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: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) 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: (No Response) 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: (No Response) 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: (No Response) Reviewer #2: I believe the authors addressed my concerns. This manuscript should be acceptable now. The only minor comment: The legend for Fig 6 should explain what is shown on panels A and B. ****** 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 ******** https://doi.org/10.1371/journal.pone.0268847.r003
Formally Accepted
12 Apr 2023 Acceptance Letter - Frédérique Pasquali, Editor PONE-D-22-13256R1 Phylogenetic trees of closely related bacterial species and subspecies based on frequencies of short nucleotide sequences Dear Dr. Nakano: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Frédérique Pasquali Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0268847.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .