Dear Dr van der Hooft,

Thank you very much for submitting your manuscript "iPRESTO: automated discovery of biosynthetic sub-clusters linked to specific natural product substructures" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Our sincere apologies for the delays in the review process. We had many problems finding available reviewers.

Based the on the two reports received and my own general review, I agree on the usefulness of the tool and I do not think major changes are necessary in the paper.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jaime Huerta Cepas

Guest Editor

PLOS Computational Biology

Kiran Patil

Section Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

Dear Dr. van der Hooft,

First of all, our sincere apologies for the delays in the review process. We had many problems finding available reviewers.

Based the on the two reports received and my own general review, I agree on the usefulness of the tool and I do not think major changes are necessary in the paper.

Please address the minor changes suggested by the two reviewers or explain why they cannot be addressed.

best wishes,

-jaime

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The identification of subclusters within predicted BGCs is highly useful for both attempting to link a cryptic BGC to a proposed molecule or for attempts to predict encoded structure in the process of discovery. I believe iPRESTO will make an invaluable contribution to the Antismash framework. The manuscript is well written and I believe it should be accepted for publication. I only have two small comments that I believe will improve the manuscript and the ability of readers to better utilize this study.

- Only 45 subclusters were annotated but I believe the remaining unannotated subclusters should be provided in the excel spreadsheet, perhaps in a different tab. Information on these remaining subclusters may help other researchers with their predictions or may even be annotated by other studies in the future.

- Whilst the authors sometimes say that only inferences are made – I would like to urge the authors to remember that these are in fact only bioinformatic predictions and that direct evidence is lacking, particularly in the earlier parts of the manuscript. Whilst it is stated sometimes, I would like to ask the authors to go through the manuscript and check their language to ensure this aspect is clear.

Reviewer #2: Louwen et al. present iPRESTO, a tool which can explore a database of biosynthetic gene clusters (BGCs) to extract common subcluters. They adapt LDA (latent Dirichlet allocation) for this problem, which improves over the previous state-of-the-art. They show that they can reproduce some known findings and find some suggestive novel assignments for certain metabolites.

Overall, the work is well executed and the value of the approach is well demonstrated. I have only minor comments on how the work is presented.

Minor Comments

#1 The paper is framed as a tool that analyses a collection of BGCs, but to me it seems that most of the value is in the database that the authors explore. On the git repository, it seems that it is possible for a user to map their BCGs into the motifs that the authors have discovered ("Querying existing sub-cluster models"). I estimate that this is much more useful than running the subcluster discovery from scratch and I suggest mentioning it more promeniently in the manuscript. The authors do note that this will be included in antiSMASH in the future, but if the functionality is already available on the command line tool, it could be mentioned in the manuscript.

#2 PRESTO-STAT should be introduced in a way that is understandable even for readers that are not familiar with the prior method on which it is based.

#3 The motivation for why it is a positive thing that many nested sub-clusters are avoided by PRESTO-STAT/PRESTO-TOP should be explained better. IIUC, the problem with previous methods is that it reports too many overlapping/redundant nested sub-clusters. This should be made explicit early.

#4 Line 164: This is unclear to me. If I understand it correctly, the problem is to query a BCG for whether it contains (or approximately contains) any known sub-clusters. Even a fairly naive index would probably work because the number of subclusters that each Pfam domain only participates in is not that large. Perhaps I have misunderstood what was meant.

Line 469: I do not understand the sentence "For a sub-cluster to be considered it needs to consist of more than one gene, for which we set a cut-off of 1.1 on the summed feature probabilities." Which feature probability are being summed and how can their sum exceed 1.0?

Line 183: Figure reference is incomplete, should probably be 2C.

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Figure 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. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

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.

Dear Dr van der Hooft,

We are pleased to inform you that your manuscript 'iPRESTO: automated discovery of biosynthetic sub-clusters linked to specific natural product substructures' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Jaime Huerta Cepas

Guest Editor

PLOS Computational Biology

Kiran Patil

Section Editor

PLOS Computational Biology

***********************************************************

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have made fantastic efforts to address the reviewer comments and I think the manuscript is now ready for publication

Reviewer #2: The reviewers have addressed all my concerns.

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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