Dear Mr. Masutani,

Thank you very much for submitting your manuscript "Investigating the Landscape of Plant Mitochondrial Genomes by Long-read Sequencing" 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.

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. 

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[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).

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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,

Christos A. Ouzounis

Associate Editor

PLOS Computational Biology

William Noble

Deputy 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: Dear authors:

Please refer to the following suggestions.

Title:

To my liking, talking about ‘plants’ when you only study a particular group of plants, in this case only one species, it’s rather pretentious. I know it’s something that is used by many authors, but I do not agree with this. I would change to a more appropiate title that describes the group of plants that it’s used in the study.

Abstract

1. Edit ’is not easy.’ for ‘is not feasible.’

2. Edit: ‘Specifically, to construct a sensitive read classifier, we developed a method that

exploits two classic algorithms--partial order alignment (POA) and the hidden Markov

model (HMM).’ for ‘We developed a method that exploits two classic algorithms--partial order alignment (POA) and the hidden Markov model (HMM). to construct a sensitive read classifier.’

3. Edit ‘a 15\\% sequencing-error rate and produced highly correct clustering.’ for ‘a 15% sequencing-error rate and produced correct clustering.’ It is my believed that something is correct or not, you cannot state that something is highly correct.

Author summary

1. Edit: ‘For this prepose’

2. Edit: ‘unknown structure of plant mitochondrial genome’ for ‘unknown structures of plant mitochondrial genomes’

Line 12: Edit: ‘Illumina sequencers’ for ‘Illumina sequencing technology’

Lines 34-49: The last two paragraphs of this section must be exchanged for a better organization. Minor edits must be done to accomplish this.

Materials and Methods

Line 51: Eliminate ‘To begin’

Line 72 Edit ‘longest reads’ for ‘longest read’

Line 80 Edit: ‘shows the workflow so far’ for ‘shows the initial steps of the workflow’

Line 121: ‘POA graph to a yet another model’ for ‘POA graph to another model’

Line 124 Edit ‘introduce’ for ‘introduced’

Line 127: Edit ‘let us substitute’ for ‘we substitute’

Fig. 1. Legend

1. Edit: ‘The thik line’ for ‘The thick line’

2. ‘The thik line and thin lines represent a reference and long reads, respectively’ This is confusing in the figure. Please label which is the reference and which are long reads in the figure.

3. LU and U should be defined in the legend.

Results

Fig. 2. Legend

1. Edit ‘We sampled 40 reads and 160 reads from one template and the other, respectively’ for ‘ We sampled 40 reads from one template and 160 reads from the remaining template.’

Line 228: Edit ‘Results’ for ‘Performance’

Line 239: Delete ‘the’

Lines 240-241: Delete’As can be seen’

Line 246: Edit ‘clusters’ for ‘cluster’

Line 249; Edit ‘dedicated’ for ‘designed’

Line 254: Delete ‘Similarly,’

Line 262: Edit ‘more time’ for ‘longer’

Line 267: Edit ‘Results’ for ‘Performance’

Fig. 3. Legend

1. In (D), edit ‘other’ for ‘remaining’

Line 294: Delete ’For example’

Line 298: Delete ‘Specifically,’

Line 319: Edit ‘we speculate that the Kyoto stain’ for ‘it seems that this strain’

Line 320: Edit ‘and this structure is maintained within the strain’ for ‘and this structure has been maintained’

Line 326: Edit ‘speculate’ for ‘believe’

Line 325: Edit ’by means such’ for ‘by processes such’

Conclusion

Please discuss if your method would have any application for polyploid assemblies, specially autopolyploids.

Line 338: Edit ‘However, there is room for improvement.’ for ‘There is room to improve our suggested method.’

Line 342: Delete ‘Also,’

Line 347: Delete ‘Lastly,’

Reviewer #2: The manuscript addresses an interesting problem; different from the mitochondrial genomes of animals, those of plants exist as multipartite structures that are difficult to assemble and describe using only short-read sequencing methods. The manuscript addresses this problem by proposing a new method to cluster long reads using a K-means algorithm that uses a HMM and partial order alignments to assign each long read to a different cluster. In general, the algorithms are described with sufficient detail both in the text and by using pseudocode. The underlying data and code are available from Github. The authors might need to add some extra information to the legend of Figure 1, which is somewhat vague, and need to explain better how they choose a particular value for K (the pre-defined number of clusters), as the number of existing structural variants is a priori unknown and might be very high. The manuscript might also benefit from some additional details (e.g. details on the scoring scheme used in the POA dynamic programing algorithm, or information on the starting coordinates for the back-tracing in the same algorithm, considering that the authors follow a semiglobal approach to not considering erroneous trailing nodes). Overall, I think the manuscript contains some interesting new ideas and tools to deal with the problem of cataloguing the different structural components of mitochondrial genomes in plants, a problem that is attracting much attention in the recent literature and is still waiting for solutions.

I also came across some typographical and other minor errors that the authors may want to fix:

* In the Author summary:

- it contains -> they contain/their genomes contain

- prepose -> purpose

- Appling -> Applying

- the last sentence in this section needs to be fixed too.

* Main paper:

-line 7: "a gene" is a vague expression; the authors might want to be more precise here.

- l. 8: create -> produce

-l. 51: may useful -> may be useful

-l. 75: LU? Please clarify.

- legend of figure 1: thik -> thick

- l. 102: how is the threshold selected?

l. 124: introduced

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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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Reviewer #1: Yes: Marco Cristancho

Reviewer #2: No

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Dear Mr. Masutani,

We are pleased to inform you that your manuscript 'Investigating the Mitochondrial Genomic Landscape of Arabidopsis thaliana by Long-read Sequencing.' 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,

Christos A. Ouzounis

Associate Editor

PLOS Computational Biology

William Noble

Deputy 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: Nice job taking care of reviewers suggestions!

Reviewer #2: All comments have been addressed. Please note that there are two different symbols for shared authorship in the cover page, suggesting that some authors are missing from the author list.

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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.

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: Marco Cristancho

Reviewer #2: No