Phylogeny analysis of whole protein-coding genes in metagenomic data detected an environmental gradient for the microbiota

Soichirou Satoh; Rei Tanaka; Makio Yokono; Daiji Endoh; Tetsuo Yabuki; Ayumi Tanaka

doi:10.1371/journal.pone.0281288

Peer Review History

Original SubmissionJuly 22, 2022
13 Oct 2022 Decision Letter - Alfonso Esposito, Editor PONE-D-22-20665Phylogeny analysis of whole protein-coding genes in metagenomic data detected an environmental gradient for the microbiotaPLOS ONE Dear Dr. Satoh, 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. ==============================As you can see the two reviewers gave completely opposite answers, I found your paper potentially interesting, however, the important technical flaws highlighted by one of the reviewers should be addressed.============================== Please submit your revised manuscript by Nov 27 2022 11:59PM. 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We will update your Data Availability statement to reflect the information you provide in your cover letter. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide [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: No ******** 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: No ****** 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: Clear and well-written manuscript. The introduction is relevant and theory based. The methods are generally appropriate. The results are clear. The authors make a systematic contribution to the research literature in this area of investigation. Reviewer #2: This paper describes a new method called MPASS to compare whole proteomes of shotgun metagenomic sequencing data. However, the methodology is not bioinformatically adequate and tool code is not provided. The reviewer doesn't think this paper is a scientifically sound bioinformatics paper. Representative points of the authors must be reconsidered are as follows. Line 383: When calculating const1 and const2, the reviewer was surprised that the authors do not conduct any correction for multiple substitutions in the same site. Since uncorrected methods tend to underestimate genetic distance, especially for the comparison between distantly related taxa (See molecular evolution textbook), the authors must correct multiple substitutions. Estimating const1 and const2 without correcting multiple substitutions is nonsense. Line 402: Ref. 26 does not say these taxa inhabit soils. Taxa choices are not adequate. To my knowledge, some of them do not inhabit soils. Sulfolobus islandicus: thermophilic archaea, habitat is hot springs Proteus mirabilis: pathogen, habitat is mammal gut but can be found in polluted soil Nitrosospira multiformis: habitat is soil. OK. Bacteroides fragilis: a very famous human gut microbe Acidobacterium capsulatum: habitat is soil. OK. Figure S1: This figure does not indicate substitution rates but just sequence identities. Provide the program code for MPASS. Otherwise other researchers cannot use MPASS. Maybe MPASS is a pipeline, so provide complete code is difficult. But at least the authors should provide some shell scripts that are merged some steps of MPASS processes in Fig. 1. These comments are only representative of methodologies. The reviewer has many other comments related to the metagenomic analysis results of this paper, but before the analysis results, using adequate methodology is necessary. ****** 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.0281288.r001
Revision 1
8 Jan 2023 Author Response Dr. Alfonso Esposito Academic Editor PLOS ONE Dear Dr. Alfonso Esposito, We would like to thank you and the reviewers for your handling our manuscript and also for the valuable comments. We agreed with most of the comments by you and all reviewers and extensively revised our manuscript. Responses to the “Journal requirements” and the reviewer’s comments are indicated below and in the attached MS word file “Response to Reviewers_SSatoh.docx”. The parts of the attached manuscript that have been revised are highlighted in red (RevisedMS_Track_Changes_SSatoh.docx). According to the reviewer’s comments, we improved our methodology by the application of a new calculation method, and provided the merged program code for almost all of the steps in MPASS processes and the additional Perl script for the automatic prefiltering of fastq files. We reconstructed all metagenomic trees and their related data with the improved program. We also corrected the sentences including the phrase “data not shown” in the manuscript, and provided the minimal data set for the results described in the manuscript according to the journal requirements of PLOS ONE. Although almost all reconstructed metagenomic trees and their related data were identical to those of the previous version of the manuscript, minor corrections associated with these reconstructed data in the manuscript are highlighted in red. We also highlighted the slight descriptive errors and correction of the format of the manuscript in red. I hope that these revisions are sufficient to make our manuscript suitable for publication in PLOS ONE. Sincerely yours, Soichirou Satoh [Journal requirements] First requirement; 2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Second requirement; 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Answer to the first requirement; Line 323 in Page 19 (Discussion): We deleted the phrase “data not shown” and added the following sentence; The topology of the metagenomic trees of simulated metagenomic datasets (Figs. 2 and 3), soil (Fig. 4), and aquatic samples (Fig.5) detected reliable group relationships, however, the integration of the coverage of each gene into metagenomic distances will require further theoretical consideration in future studies. Line 374 in Page 22 (Materials and Methods): We deleted the following sentence; The removal of short protein sequences contributed to the reconstruction of congruent trees (data not shown). Answer to the second requirement; We attached the original data for the metaphylogenomic trees and their related analyses as supporting information files (S1File.zip). [Answers to the comment of Reviewer 2] Thank you for your critical and careful reading. These comments are valuable for improving our manuscript and I greatly appreciate them. We have revised our manuscript according to your suggestions. I hope you will satisfy our revision. The followings are the answers to your comments. Comment; Line 383: When calculating const1 and const2, the reviewer was surprised that the authors do not conduct any correction for multiple substitutions in the same site. Since uncorrected methods tend to underestimate genetic distance, especially for the comparison between distantly related taxa (See molecular evolution textbook), the authors must correct multiple substitutions. Estimating const1 and const2 without correcting multiple substitutions is nonsense. Answer to the comment; We fully agreed with your comment that correction for multiple substitutions in the same site is very important. As this suggestion, we improved our methodology by adopting the Poisson-corrected substitution rate for the calculation of the metagenomic distances, and reconstructed all metagenomic trees and their related data with this new calculation method. Although almost all reconstructed metagenomic trees reproduced the same topology as that of trees constructed with the previous MPASS program, we think that our method is improved by your suggestions. Comment; Line 402: Ref. 26 does not say these taxa inhabit soils. Taxa choices are not adequate. To my knowledge, some of them do not inhabit soils. Sulfolobus islandicus: thermophilic archaea, habitat is hot springs Proteus mirabilis: pathogen, habitat is mammal gut but can be found in polluted soil Nitrosospira multiformis: habitat is soil. OK. Bacteroides fragilis: a very famous human gut microbe Acidobacterium capsulatum: habitat is soil. OK. Answer to the comment; We replaced the phrase “five soil bacteria species” with “five bacteria species”. Comment; Figure S1: This figure does not indicate substitution rates but just sequence identities. Answer to the comment; We calculated the Poisson-corrected substitution rates between 16S rRNA genes of various organisms, and showed the relationship of them to distances from the comparison of metagenomes in Fig. S1. Comment; Provide the program code for MPASS. Otherwise other researchers cannot use MPASS. Maybe MPASS is a pipeline, so provide complete code is difficult. But at least the authors should provide some shell scripts that are merged some steps of MPASS processes in Fig. 1. These comments are only representative of methodologies. The reviewer has many other comments related to the metagenomic analysis results of this paper, but before the analysis results, using adequate methodology is necessary. Answer to the comment; We fully agreed with your comment that the availability of the MPASS program by other researchers is also very important. I also hope that this program is used by many researchers to study various metagenomes. According to this suggestion, we provided two Perl scripts. First, we provided automatic program code for the down-sampling, assembling, gene prediction, and calculation of the metagenomic distances in the MPASS pipeline. Second, we also provided another Perl script for the automatic prefiltering of multiple pairs of fastq files by using the Trimmomatic software. We uploaded these two scripts (MPASS.pl, FilteringFastq_for_MPASS.pl) and a refined version of the previous script MPASS_core.pl (only for the metagenomic distance calculation) on the GitHub website (https://github.com/s0sat/MPASS), and wrote this URL in the manuscript. By using MPASS.pl and FilteringFastq_for_MPASS.pl, the researchers can construct the metagenomic distance matrices from their paired-end fastq files or those downloaded from public databases, automatically. Attachments Attachment Submitted filename: Response to Reviewers_SSatoh.docx https://doi.org/10.1371/journal.pone.0281288.r002
20 Jan 2023 Decision Letter - Alfonso Esposito, Editor Phylogeny analysis of whole protein-coding genes in metagenomic data detected an environmental gradient for the microbiota PONE-D-22-20665R1 Dear Dr. Satoh, 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. 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, Alfonso Esposito, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0281288.r003
Formally Accepted
24 Jan 2023 Acceptance Letter - Alfonso Esposito, Editor PONE-D-22-20665R1 Phylogeny analysis of whole protein-coding genes in metagenomic data detected an environmental gradient for the microbiota Dear Dr. Satoh: 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. Alfonso Esposito Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281288.r004

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