Dear Dr. Guyeux,

Thank you very much for submitting your manuscript "CRISPRbuilder-TB: “CRISPR-Builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in Mycobacterium tuberculosis complex using SRA" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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

[1] A letter containing a detailed list of your responses to the 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.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Mihaela Pertea

Software Editor

PLOS Computational Biology

Mihaela Pertea

Software Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The work presented by Guyeux and co-authors in this manuscript focuses on reconstructing the CRISPR locus of Mycobacterium tuberculosis complex strains from short reads sequences directly. The authors developed a bioinformatics pipeline to achieve this goal and presented their results compared to those obtained from other similar tools. A major strength of their work is that the performance of their pipeline (named CRISPRbuilder-TB) is evaluated by reconstructing CRISPR loci of simulated and complete MTBC genomes, where the structure of CRISPR loci are known. They show that 1/3 of assembled genomes contain mis-assemblies and erroneous CRISPR loci, and thus advocate for the use of their pipeline to reconstruct CRISPR loci from short reads directly. They also show how existing tools do not capture the full diversity at the CRISPR locus, including duplications, spacer and direct repeat variants, and locations of IS6110 insertions.

Major comments:

- The authors should include documentation on their project GitHub page (https://github.com/cguyeux/pyMTC/) explaining how the pipeline can be downloaded and run locally by providing examples, in addition to any software requirements.

- The authors provide a comprehensive list of available CRISPR identification tools. However, they only compared their tool (CRISPRbuilder-TB) to two other independent tools (Table 1). It is not cleat to this reviewer why only Crass and CRISPRdetector were used as comparison tools, whereas other cited CRISPR predictor tools (CRT, CrisprFinder, PILER-CR, CRISPRCasFinder, CRTmeta or metaCRISPR) were not. As some of these tools only accept assembled genomes as input, simulated reads could be assembled first and then assemblies used to run the remaining CRISPR prediction tools.

- Both in Table 1 and Table 2 is not obvious from the text provided that reconstructed CRISPR loci by CRISPRbuilder-TB are identical to simulated loci (Table 1) or reference genome loci (Table 2). A simple visual representation of simulated and reference vs. reconstructed CRISPR loci is needed to easily identify and compare regions of homology, order of CRISPR features (DR, spacers) and variants (SNPs, duplications, IS6110) between simulated and reference compared to reconstructed loci.

- Although, the authors have published a separate study on the evolution of CRISPR locus on MTBC, they should discuss whether there is evolutionary evidence that the CRISPR locus is functional. According to https://www.biorxiv.org/content/10.1101/2019.12.13.875765v1 , the CRISPR locus “of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages.” Would the authors interpret these results as a clear indication of a non-functional CRISPR locus?

Minor comments:

Abstract:

In ‘compared to more generalist tools’, please indicate here what these tools are here in the abstract.

Authors summary:

Line 55: ‘using short read sequences’ not ‘Short Reads Archives’

Line 56: ‘current assembled genomes’

Line 60: list here what existing methods CRISPRbuilder-TB was compared to

Introduction:

Line 67: indicate in brackets and include citations of the first two Mtb sequenced isolates

Line 73: given that spoligotyping is not taxonomically precise or fully ‘correct’, please replace ‘allows a correct taxonomical assignment’ with ‘allows an approximate taxonomical assignment’

Line 89: in more than a hundred thousand samples. Also include citation at the end of this statement.

Line 97: they have the same limitations as in vitro spoligotyping techniques.

Line 99. Use WGS abbreviation instead of ‘whole genome sequencing’, here and elsewhere in the text.

Line 99. By traditional de novo assembly tools

Results:

a. Description of CRISPRbuilder-TB

Line 129. A known limitation

Figure 2. In Dataset #1 Validation sample, do the authors used 7 complete (sequenced to completion) reference genomes? If so, they may want to change ‘Assembled genomes

collection from NCBI’ to ‘Complete genomes collection from NCBI’

In lines 130 to 132. A more explicit description on how CRISPR locus associated reads are extracted is needed here. From Figure 1 step 1, DR sequences are used to fish out CRISPR locus associated sequences from short sequence reads.

Figure 1 illustrates very well CRISPRbuilder-TB pipeline but lacks a legend in the manuscript. Figure 1 would also benefit from including what bioinformatic tools were used at each step.

Line 138. It would be helpful to report how many SNP differences (mismatches) the authors find among variants of the same spacer. This would inform the maximum number of mismatches in silico spoligotyping tools use to search for spacer occurrences in SRA data.

Lines 148 – 149. What do the authors mean by ‘translate these contigs in strings of words’? Do they refer to annotating contigs with CRISPR locus features such as DR, spaces, etc. If so the authors may want to use the term ‘annotation’ and rename step4 in Figure 1 ‘Translation into explicit contigs’ to ‘CRISPR annotation of contigs’.

Line 151. Related to the point above, the authors may want to use the term ‘Unannotated regions’ of contigs instead of ‘untranslated parts’.

b. Reconstruction of simulated CRISPR

Table 1. It is not cleat to this reviewer why only Crass and CRISPRdetector were used as comparison tools, whereas other cited CRISPR predictor tools (CRT, CrisprFinder, PILER-CR, CRISPRCasFinder, CRTmeta or metaCRISPR) were not. As some of these tools only accept assembled genomes, simulated reads could be assembled first and then assemblies used to run the remaining CRISPR prediction tools.

Table 1. As pointed before, it is preferable to use the term ‘non-annotated contig sequences’ than ‘untranslated sequences’

Line 163. Indicate how short (in bp) the shortest reads used by CRISPRbuilder were.

Line 163. As pointed before, use ‘annotation’ instead of ‘translation’.

Evaluation of CRISPR locus reconstruction based on WGS data of MTC reference strains

Line 187. The authors should stress here that the seven reference strains had been sequenced to completion, that is, they had an ungapped and complete chromosome sequence resolved. This is somehow implied with the term ‘reference’ genome.

Line 203. Do the authors mean CRISPRbuilder-TB reconstructed sequences were identical to those of the complete reference genome? Or those assembled de novo from short reads? Same point in Line 212 ‘assembled genome’.

Line 207. Citation needed right after statement ‘as described previously’.

219. Do the authors mean ‘public reference genomes’ by ‘public assemblies’ here? As pointed before, it is important to differentiate between complete reference genomes (ungapped, resolved and annotated) vs. de novo assemblies (made up of contigs).

d. CRISPR region in other MTC assembled genomes.

Line 230. In the statement ‘Most sequences seemed of high quality.’, what quality control metrics did the authors used to assess the quality of these 187 MTC public genomes? What sampling strategy was used to select these 187 MTC genomes? Are these complete genomes or assembled?

e. CRISPR evolutionary events in MTC

As pointed before, for line 138. It would be helpful to report how many SNP differences (mismatches) the authors find among variants of the same spacer. This would inform the maximum number of mismatches in silico spoligotyping tools use to search for spacer occurrences in SRA data.

f. Computational speed

Discussion.

Line 323. Use ‘complete reference genomes’ instead of ‘reference samples’.

Line 325. Use ‘concordant with complete genome’ instead of ‘concordant with assembled ones’

4. Material and methods

a. Sequence data

Line 480 to 481, replace ‘both assembled genomes and short reads sequencing’ with ‘both complete genomes and short reads sequences”.

Lines 486 and 487. What selection criteria did the authors followed to select these assembled genomes from public repositories?

Line 489. Do the authors mean European Nucleotide Archive (ENA) by EMBL-SRA?

e. Locus reconstruction

Contiguage

It is not clear to this reviewer why the authors did not make use of an established de novo assembler (such as Velvet or Spades) to assembly selected reads and, instead, came up with their own algorithm.

f. MTC CRISPR simulation

Lines 708 – 712. Can the authors specify what bioinformatics tool they used to simulate short reads?

Reviewer #2: This is a well written paper that provides important novel insight in the CRISPR genetic diversity and organisation of the locus and will help refine the MTC global population structure. It presents a detailed and comprehensive rational, approach and data for the problem. In my opinion, this is an important and worth while tool that should complement the current apps used for refining MTC phylogeny.

I have a few minor points for the authors to address.

Line 39: put comma after pipeline

Line 43: replace generalist with generalised

Line 89: replace hundred with one hundred

Line 90: replace to explore with exploration of

Line 124-126: this does not make sense. Please re-write

Line 159: replace Tool's with Tool

Line 180: Why is the data not shown? Please explain and provide data in suppl figs.

Line 286: replace Go with Gb

Line 362: Could it be easily adapted to other species? Re-write...not appropriate

Figures:

Largely fine, but would recommend modifying/edit text and font styles to look clearer and optimum.

Typos:

The manuscript is generally well written, but a thorough read-through is needed as there are a number of typos, formatting and grammatical errors. Some of which I have highlighted above.

Discussion:

I would like to see a little more about how this software has resolved the frequency and nature of IS6110 insertions in the CRISPR locus. Also, how does the software cope with large scale deletions of the locus due to IS6110?

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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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.

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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: Yes: Francesc Coll

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, PLOS recommends that you deposit 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. For instructions, please see http://journals.plos.org/compbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Guyeux,

Thank you very much for submitting your manuscript "CRISPRbuilder-TB: “CRISPR-Builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in Mycobacterium tuberculosis complex using SRA" 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 first reviewer's recommendations.

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,

Mihaela Pertea

Software Editor

PLOS Computational Biology

Mihaela Pertea

Software 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:

[LINK]

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 successfully addressed the following major comments:

- Detailed documentation on how to download, install and run their tool on GitHub has been now provided.

- They argued well why the used short reads as the tool’s input, and not assemblies, and thus the choice of CRISPR predictor tools they used to compare their tool’s predictions against.

- Added some discussion on the functionality of CRISPR locus in MTC.

However, the authors haven’t addressed the following point:

The newly added Supplementary_File3.odt, Supplementary_File4.odt and Supplementary_File5.odt do not provided “A simple visual representation of simulated and reference vs. reconstructed CRISPR loci is needed to easily identify and compare regions of homology, order of CRISPR features (DR, spacers) and variants (SNPs, duplications, IS6110) between simulated and reference compared to reconstructed loci.”

All minor comments were successfully addressed.

Reviewer #2: None

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

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: Yes: Francesc Coll

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, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Guyeux,

We are pleased to inform you that your manuscript 'CRISPRbuilder-TB: “CRISPR-Builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in Mycobacterium tuberculosis complex using SRA' 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,

Mihaela Pertea

Software Editor

PLOS Computational Biology

Mihaela Pertea

Software Editor

PLOS Computational Biology

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