Peer Review History
Original SubmissionJuly 13, 2022 |
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Transfer Alert
This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.
PONE-D-22-19792The virtualome: a computational framework to evaluate microbiome analysesPLOS ONE Dear Dr. Arias, 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 submit your revised manuscript by 20/11/2022. 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:
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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. 4. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: "FB and CFA gratefully acknowledge support by the Roechling foundation. BS was partially supported by MINECO grant MTM2017-85020-P." 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: "FB and CFA gratefully acknowledge support by the Roechling foundation. BS was partially supported by MINECO grant MTM2017-85020-P." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Partly Reviewer #2: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: In this work, the authors use a statistical framework to generate virtual microbiomes that serve as benchmarks to compare the performance of amplicon and WGS-based taxonomic profiles. They find that the performance of taxonomic profiling is shckingly poor, especially if the reference datbases are incomplete, but also with complete databases. Moreover, the overlap observed between amplicon and WGS-based profiling is often due to false positives. The implications of this study, if true, are devastating. What I find most concerning is the poor performance of taxonomic profiling even if the reference database contains all the genomes from the microbiome. Given the strong implications of this result, I find it essential to verify that such poor performance is not just a limitation of the particular tool (GAIA) and parameters used for taxonomic profiling. To that purpose, it would possibly be enough to study what happens when using other popular tools for taxonomic profiling and the complete database (which is the best-case scenario). I understand that the authors may choose not to go that longer way, but then the article should explicitly state that its conclussions are valid for GAIA, but maybe not for other taxonomic profiling algorithms. From a technical perspective, the statistical properties and algorithms used to generate the virtual microbiome should be described in much greater detail. Given that generation of virtual microbiomes is central to (and possibly the main novelty of) the study, these details could even be presented in the Results section. If the authors prefer to focus the Results section on the comparison of taxonomic profiles, full details should still be provided in the Methods section. I am not fully aware of Plos ONE policies about code availability; however, following the FAIR principles for open and reproducible research, it would be good to share the code to generate virtual microbiomes in a public repository, such as github or zenodo. Personally, I think that the generation of virtual microbiomes that are compatible with the abundance distributions found in real-world microbiomes is the main scientific breakthrough of this paper and not making the method available substantially diminishes its interest. I appreciate that pseudo-code for the algorithm is included in the Methods, but it needs more detail (what does function "generateAbundances" do? what is "params"?). Related to the previous issues: - Discussion of Figures 2C-D require further clarification. Why are there unidentified species when using the complete databases? Do the figures include unsampled species? Are some species being classified as "unidentified" because their assignations are ambiguous? How is it possible that the incomplete database sometimes prudices fewer unidentified genera than the complete database? How is it possible that the complete database produces false positives (perhaps due to an inappropriate management of ambiguous hits)? - The fact that complete databases often lead to a large fraction of unidentified species and false positives suggests that the bioinformatic workflows used to simulate reads and assign taxonomy are intrinsically inaccurate or not properly optimized for the analysis of virtual microbiomes. Has this poor behavior been previously described for real or mock microbiomes (e.g., in the origial articles of Grinder or GAIA)? If not, how do you explain such poor behavior with virtual microbiomes? The fact that taxonomic profiles are fairly poor even when using the complete databse makes me wonder whether the results with incomplete databases would look better if a software different than GAIA had been used. Do you have any evidence that the findings reported here are qualitatively general and also valid for other taxonomy-assignment workflows? Minor issues and suggestions: - The portmanteau "virtualome" seems unncessary and not very intuitive. "Virtual microbiome", though longer, would be more informative and less cumbersome. - In Section 2.2, first paragraph, sentences following cite [36]: These sentences are somewhat confusing and counterintuitive. The problem is that they refer to two measures of diversity that need to be clearly disambiguated. Species richness (discussed in terms of "small" or "large" communities) and alpha-diversity (discussed just as "diversity") are both measures of diversity, but they affect the percentage of unsampled species in opposite ways: the smaller the species richness, the smaller the unsampled fraction, but the smaller the alpha-diversity, the greater the unsampled fraction. I think that the results would be much more intuitive if ambiguous references to diversity were replaced by more informative terms. Specifically the concept of species richness is broadly known and I see no reason to not use it (instead of talking about small/large communities). As for alpha-diversity, trends would be easier to understand if it was discussed in terms of species (un)evenness, because it is quite intuitive that higher unevenness implies more rare species that may be missed by the analysis. - Figure 2A: add quantitative legend (color bar) for the shades of gray that correspond to alpha-diversity. - Page 3: "the simultaneous detection of a species by both techniques does not ensure its positive identification, since the overlap between amplicon and WGS can be dominated by false positives". This requires a citation. If it is a new finding of this study, it should be specified. Reviewer #2: The manuscript of Serrano-Antón and collaborators entitled "The virtualome: a computational framework to evaluate microbiome analyses" reports the effect of database completeness on the metagenomics analysis outcomes, underlying the critical issues of the current bioinformatics approaches. The manuscript is well-presented, technically precise, and scientifically sound, with the experimental methodology used appropriated for the intended objectives. The presented framework remarked the discrepancies between the Target and the Whole Genome Sequencing approaches both in simulated and real data, taking into account the ecological structure of microbial communities. This study highlights the actual limitations as well as the known advantages of metagenomics investigations, underlying the need for an increased amount of genetics/genomics information in public databases to raise the overall accuracy of real-world data. I have a few questions for the authors: Page 5: Authors discussed the "ecological structure of microbial communities imposes a major intrinsic limitation to the capacity of microbiome analyses", but this part is not clear to me. Since the composition of the microbial community can widely vary in real-world data, often without a priori knowledge of the microbial composition under study, the small virtualomes approach tends to reduce the loss of species for technical purposes. In my opinion, the authors should highlight the different findings obtained by the use of the different alpha-diversity databases, as in the discussion the presented differences are not detailed as needed. Page 6: Regarding the sentence "Remarkably, the incompleteness of the databanks affected the analyses of virtualomes even when the 100% of their species were represented in the incomplete databases (the open dots do not lie in the diagonal in Fig. 2D)." how did the authors interpret these findings? The authors should add more details on their evaluation of the reported findings. Page 7: In the sentence "the number of true positives detected exclusively by WGS was normally greater than those obtained only by 16S" the authors highlight the difference between 16S and WGS results. Did the authors find any statistically significant difference between the number of real positives found by the use of Target compared to the use of WGS? What about the false positive? Page 9: "The discrepancy between 16S and WGS is pronounced for genera", so even by the use of Pearson Correlation, readers cannot properly get which method could be the best in the study of an unknown microbial community. In my opinion, authors should highlight the best and the worst of each technique, even by adding a summary table reporting the main findings in both real and simulated data, to help readers in understanding of the presented results. Page 13: is not clear to me if all reads that did not map to the G. mellonella reference genome were successfully classified by the use of the GAIA software, or if it was necessary to assemble and classify any Metagenome Assembled Genomes (MAGs). Can the authors clarify this point? ********** 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. 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Revision 1 |
PONE-D-22-19792R1The virtualome: a computational framework to evaluate microbiome analysesPLOS ONE Dear Dr. Arias, 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 submit your revised manuscript by Feb 05 2023 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:
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, Patrizia Falabella Academic Editor PLOS ONE Journal Requirements: 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. [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: (No Response) 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: N/A 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 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 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: In the revised version of the manuscript, the authors made a remarkable effort to show the generality of their results by reproducing some parts of the analysis with a different taxonomic profiling tool (Kraken). Some obscure claims regarding unidentified taxa have been removed and the origin of false positives is now clearly explained. Overall, these changes notably improved the quality of the manuscript and the robustness of its conclusions. I appreciate the authors' effort to better describe the algorithm that produces virtual microbiomes. I still do not understand why they did not release a public, functional implementation of their code, although I leave it to the Editor to decide whether that is a requisite for publication. Anyway, the pseudocode included in the methods section is still incomplete: there are 4 subroutines in EcologyGeneration that are not proprely described. Furthermore, I could not find the parameter values (mu, sigma, shape, scale) used to generate the abundance matrices. I understand that, from the authors' perspective, it is hard to let go of a fancy word like "virtualome", which is certainly catchy. However, I must insist that it is nonsense. There are already many "omes" in the recent biological literature, perhaps too many, but at least most of them are self-explanatory and semantically coherent. Genomes, proteomes, transcriptomes, interactomes, and microbiomes (to name a few) clearly refer to sets of genes, proteins, transcripts, interactions, and microbes, respectively. Accordingly, virtualomes would be sets of some kind of virtual entities. What does that mean? I could think of something like a set of virtual environments in a computing context, although that would be quite a stretch. However, the authors use the term to refer to a virtual microbiome (I guess that the rationale is to combine VIRTUAL with microbiOME). From a semantic perspective, that is nonsense. Following that rule, it could also mean virtual proteome, virtual interactome, virtual transcriptome, virtual metabolome... Or none of that. It is perhaps a funny word to describe the tool in an informal setting, among colleagues or perhaps as a joke in a conference if you want to get the attention of the audience. But I hardly see how it would be an acceptable (that is, unambiguous and semantically coherent) scientific term. Some typos: 239: "does to distinguish" -> "does NOT distinguish" 325: "These unevenness in the relative abundance implies". Remove "These" Reviewer #2: In this last version, the authors addressed all the concerns raised by the reviewers, extending certain sections and clarifying important aspects of the actual limitations of the metagenomics analysis. In particular, the new analysis performed using the Kraken2 well supported the results and conclusions of the manuscripts. ********** 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.] 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. |
Revision 2 |
The virtual microbiome: a computational framework to evaluate microbiome analyses PONE-D-22-19792R2 Dear Dr. Arias, 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, Patrizia Falabella 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 ********** 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: All my comments have been addressed. Therefore, I support publication of this work in Plos One. I would want to congratulate the authors for this fine piece of research, which will help guide future taxonomic profiling works based on metagenomics. ********** 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 ********** |
Formally Accepted |
PONE-D-22-19792R2 The virtual microbiome: a computational framework to evaluate microbiome analyses Dear Dr. Arias: 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 Prof. Patrizia Falabella Academic Editor PLOS ONE |
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