Dear Dr. Soylev,

Thank you very much for submitting your manuscript "CONGA: Copy number variation genotyping in ancient genomes and low-coverage sequencing data" 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.

As you will see, all three reviewers appreciated your manuscript and the novelty and importance of your approach. In addition to the specific remarks that reviewers made, which I believe will improve your manuscript, two of the reviewers note that they were not able to compile and test CONGA. This is very important for a computational biology paper, and I ask you to please insure that the software can be compiled and used before resubmission, and address this question very clearly in the revision.

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,

Marc Robinson-Rechavi

Academic Editor

PLOS Computational Biology

Sushmita Roy

Section Editor

PLOS Computational Biology

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As you will see, all three reviewers appreciated your manuscript and the novelty and importance of your approach. In addition to the specific remarks that reviewers made, which I believe will improve your manuscript, two of the reviewers note that they were not able to compile and test CONGA. This is very important for a computational biology paper, and I ask you to please insure that the software can be compiled and used before resubmission, and address this question very clearly in the revision.

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: uploaded as an attachment

Reviewer #2: CONGA

Summary:

The authors propose CONGA, a specialized tool for the genotyping of copy-number variation in ancient genomes, which come with a plethora of challenges owing to low coverage, sample damage, and issues in library preparation. CONGA uses any given set of CNV calls as a truth set for genotyping using read-depth and split-read methodologies.

The authors evaluate the performance of their algorithm on simulated data, down-sampled real genomes, as well 71 ancient genomes, in a manner spanning a wide range of coverages and diverse ancestry.

The paper is written well - its question is clear, the goals of each section are appropriate, described effectively and concisely.

The authors do an excellent job of describing not only the strengths and applications of their algorithm, but also its faults, caveats, and areas of underperformance. The paper highlights the usability of CONGA while explaining clearly areas the tool is not suitable for analysis (such as genotyping ancient genomes in <5x coverage).

Additionally, the algorithm performs as expected. True-positive rate, as well as false-discovery rate largely scale with coverage. The algorithm tends to perform better for deletions than other types of CNVs, and CONGA is most performant for variants > 1kb as expected of short-read datasets.

The results are clear and the tool looks useful for those who are interested in genotyping CNVs in low-coverage ancient genomes. Overall, CONGA is deserving of publication in this journal without revision.

Major:

None

Minor:

Page 2, Line 83 - The CONGA algorithm is initially described in terms of read-depth and split-reads. However, a small line should be inserted here to quickly inform of the reader of how CONGA uses this information differently from already available read-depth and split-read callers.

Page 3, Line 119 - The authors title this section 'copy number predictions of CNVs' but note that CONGA does not evaluate >= 3 copies. This section should be renamed, as many working on CNVs may assume more capability here as opposed to simply detecting homozygous vs homozygous variation.

The authors mention that their input callset was determined across 4 datasets by choosing non-overlapping variants, but some polymorphic CNVs in the population may have overlapping breakpoints and thus such a heuristic may filter out genotype-able CNVs. It would be worth attempting to decide on a way to keep some of these CNVs in the input callset, perhaps based on some metric of reciprocal overlap.

Reviewer #3: In attached file.

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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

Reviewer #3: 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

Reviewer #3: 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

Attachments

Attachment

Submitted filename: Review.docx

Attachment

Submitted filename: CONGA_review1.docx

Dear Dr. Soylev,

Thank you very much for submitting your manuscript "CONGA: Copy number variation genotyping in ancient genomes and low-coverage sequencing data" 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.

Like the reviewer, I have not been able to compile your software. I checked with the reviewer, and they tried on Linux, as recommended in the instructions. While it is acceptable that the software compiles on Linux and not on other OSes, I cannot accept it for publication in PLOS Comp Biol if neither reviewers nor myself have been able to compile and run the code in at least one environment.

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,

Marc Robinson-Rechavi

Academic Editor

PLOS Computational Biology

Sushmita Roy

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:

Like the reviewer, I have not been able to compile your software. I checked with the reviewer, and they tried on Linux, as recommended in the instructions. While it is acceptable that the software compiles on Linux and not on other OSes, I cannot accept it for publication in PLOS Comp Biol if neither reviewers nor myself have been able to compile and run the code in at least one environment.

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The authors did a great job revising the manuscript incorporating the addressed points. The manuscript has gained in quality.

Unfortunately, the compilation of the code is still challenging, and I did not succeed with the given information. I am not sure if interested researchers will make this effort. Having a conda installation or a universal binary would be helpful. In the following I try to list the problems I encountered to compile the code:

System without root/sudo access:

Did not figure out how to get libbz2 and liblzma installed without root access.

System with root/sudo access (libcurl is missing):

$ sudo apt-get install libbz2-dev

$ sudo apt-get install liblzma-dev

$ make -C htslib/

…

gcc -g -Wall -O2 -I. -c -o hfile_libcurl.o hfile_libcurl.c

hfile_libcurl.c:46:10: fatal error: curl/curl.h: No such file or directory

46 | #include <curl curl.h="">

| ^~~~~~~~~~~~~

compilation terminated.

make: *** [Makefile:132: hfile_libcurl.o] Error 1

System with root/sudo access and cheating (disabling libcurl):

$ sudo apt-get install libbz2-dev

$ sudo apt-get install liblzma-dev

$ wget https://github.com/samtools/htslib/releases/download/1.16/htslib-1.16.tar.bz2

$ tar --bzip2 -xvf htslib-1.16.tar.bz2

$ cd htslib-1.16

$ ./configure ## libcurl is disabled

$ make

$ cd ../

$ git clone https://github.com/asylvz/CONGA --recursive

$ cd CONGA

$ mv htslib htslib2

$ ln -s ../htslib-1.16 htslib

$ make -C sonic

$ sed -i ‘s/-lcurl //g’ Makefile ## remove libcurl dependency

$ make</curl>

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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

**********

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

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. Soylev,

We are pleased to inform you that your manuscript 'CONGA: Copy number variation genotyping in ancient genomes and low-coverage sequencing data' 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,

Marc Robinson-Rechavi

Academic Editor

PLOS Computational Biology

Sushmita Roy

Section Editor

PLOS Computational Biology

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