PONE-D-24-15414Fast and accurate imputation of genotypes from noisy low-coverage sequencing data in bi-parental populationsPLOS ONE

Dear Dr. Lorieux,

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infrastructure. The following programs supported parts of this initiative: the French ANR project "LANDSREC"

(ANR-21-CE20-0012-03), the French Government France Génomique program through its International RIce

Genome INitiative “IRIGIN” project, and the CGIAR Research Program “RICE”."

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Additional Editor Comments:

The reviewers' comments pointed out that a revision is required to improve the current version of the manuscript. One of the most critical concerns regards the filtering step. It also needs to be clarified if the input of the three tested tools is the same. Please refer to the reviewers’ reports and the Reviewer’s Responses to Questions section for detailed comments, which could help you improve your manuscript. Please carefully address (and reply to) all the comments raised by all reviewers (this is mandatory).

[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: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: The authors propose a method to genotype bi-parental populations with low-coverage NGS technology and compare their approach against FSFhap and LB-Impute. An MLE algorithm as compared to HMM makes sense with the assumptions of the bi-parental populations with inbred and exclusively homozygous parental lines and the low running time and memory consumption looks good. However, I have some comments/questions as given below:

Major Comments

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- There is a heavy filtering of SNPs before being used by the tool. Filtering multiallelic SNPs and small indels is common in genomic tools (but newer versions of tools are updating their pipelines to incorporate them now) but further filtering of SNPs based on HWE parameters, read counts, etc might be a bit excessive. Do you have any explanation about this? How many SNPs are removed through these filtering criteria (in terms of percentage of all SNPs)?

- Do you compare NOISYmputer to FSFhap and LB-Impute with the input of these filtered variants to all three tools or are the filtering criteria for the three tools different? If variants are filtered differently for the three methods, is it possible to run the three tools on one VCF and compare the results? Is it possible that the high accuracy, better map size estimation, and breakpoint precision of the tool are the outcome of the heavily filtered set of SNPs?

- Line 259: How do you define too many or too few? Is it based on the average read depth or a constant value of X?

Also it is possible that the SNPs with too many reads might be duplicated regions but this information seems to be lost when they are removed. Any comments on this?

- What is the reason that windows in this work are based on the number of variant positions around a central SNP and not base pair distance-based? More clearly, why is an m-window around SNPj defined as SNPj-m to SNPj+m and not all the variants whose base pair distance is less than m from SNPj. Most references have gaps in their sequences and the existing window-system in the manuscript will include variants which can be large distances away from the central SNP if such a gap exists between the two variants.

Why was such a window-system used instead of a base pair distance-based?

- Why was the comparison done with FSFhap and also not with FILLIN (as the main publication of FSFhap does)? FILLIN has imputation capabilities that can also be compared with the imputation done by NOISYmputer.

I could not find a direct link to the simulated and real dataset used in the experiments. Each dataset used in the experiments should be stated clearly with a proper link. For simulations, an open source repository (e.g., Zenodo) might be used.

Minor Comments

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- Figure 2 shows that the performance of the algorithm is poor for data with high error rates. So even if the correct error rates are estimated, it does not help in generating accurate results and the tool depends on the underlying error rates.

This is not a very big issue since the tool primarily deals with Illumina reads, which have very low error rates (as stated in lines 354-357). But future ideas related to extending these tools to error-prone long reads will be unsuccessful based on these results. Is it possible to generalize the model further to allow high error rates?

- I would expect to see some stats about the performance of the algorithm in the abstract.

- Line 48 states that “Reducing sequencing costs through minimized per-sample coverage has an important experimental downside: LC-NGS mechanically introduces a series of issues, the main ones being:”

However, I can't see any connection of issue 3 listed in line 60 with the data being low coverage. It's a problem with short-read sequencing.

- Line 117: Where does the equation for the proportion of the genome with no recombination come from? Reference?

- Line 123: “computation time, which increases linearly according to the diplotype size” - Is there any time complexity analysis given as part of the work to show this?

- Line 153-155: Are nAi and nBi numbers or is it a multiplication between n and Ai and Bi. If they are numbers, consider superscipting the A and B. It is not clear how they are being obtained. The binomial expansion has them as variables and their values can range from 0 to ni. But in Line 162 in the equation, they are no longer variables on the LHS of the equation while they exist on the RHS of the equation. Which value is chosen for nAi and nBi and how are they chosen?

- Line 210-225: If BB is the segment to the left of position j and AB is to the right, why is the BB window defined for j to j+m and vice versa for the AB window? Since BB is to the left, the window is for j-m to j. Further definition of PSI as a product of 1-probabilities for the two regions. There is no explanation on why this approach has been taken.

- Line 244-245: The formulation of homozygous to heterozygous transitions seems to be easily generalizable for double recombination events of transitioning from homozygous to homozygous. Why not allow such events as default and allow users to choose whether to consider the double recombination transitions?

- Line 320: Does PoPSimul also simulate gaps in the reference or is it a uniform distribution of SNPs?

- Line 338-342: It can also be considered to report the results using other metrics like precision-recall or Matthews Correlation Co-efficient etc. Maybe even with a confusion matrix.

- Line 316-317: Specs of the cluster are given in the link but which of these machines did you use for the experiments. This information can also be added as a single line regarding the specs of the machine used.

Typos

-----

- Line 102: “leaving unimputed the regions"

- Line 132: “once could set”

- Line 157-159: End bracket missing in the expression in the middle.

- Line 218: Should this be SNPj+m and SNPj-m respectively, where “m” is subscripted?

Reviewer #2: The authors propose a specific scheme for handling imputation of individuals where the original genetic stock come from two completely inbred lines. They propose to compute the total number of reads supporting the two generalized alleles (A and B) and make the primary imputation decisions based on these counts. They claim that this technique will be more tolerant to sequencing and mapping errors compared to other prevalent approaches.

The authors claim that this approach is more efficient than a Hidden Markov Model, but in my mind, what's proposed is similar to a HMM where the emitted symbols are the read counts in each window. Combining windowing with transition probabilities for long range haplotype consistency wouldn't be impossible.

I would also be somewhat weary against the argument that it makes sense to have a single global error rate per origin line, since mapping and sequencing errors are highly variant-specific. On the other hand, I can of course agree that two parameters would maybe be better than a single global epsilon.

The use of the reconstructed chromosome length as a metric for qaulity is ingenious, but it takes some time to wrap your head around it.

Overall, I find the contribution to be interesting, but there are several assumptions made on the type of dataset and I think the manuscript would benefit from more clearly spelling these out and also contrast to related scenarios where they would not be applicable.

Reviewer #3: The paper is written well, I understood the problem of imputation and authors' approach to the maximum likelihood estimation. The results provided are understandable. However, related work or literature review could be improved.

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Reviewer #1: No

Reviewer #2: Yes: Carl Nettelblad

Reviewer #3: No

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PONE-D-24-15414R1Fast and accurate imputation of genotypes from noisy low-coverage sequencing data in bi-parental populationsPLOS ONE

Dear Dr. Lorieux,

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.

The authors addressed the main Reviewers’ comments; however, a couple of concerns remain that must be considered. For instance, I agree with Reviewer 2 that the authors should improve the repository documentation. Please consider the two minor comments from Reviewer 1.

Please submit your revised manuscript by Dec 05 2024 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:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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,

Andrea Tangherloni

Academic Editor

PLOS ONE

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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.

Additional Editor Comments:

The authors addressed the main Reviewers’ comments; however, a couple of concerns remain that must be considered. For instance, I agree with Reviewer 2 that the authors should improve the repository documentation. Please consider the two minor comments from Reviewer 1.

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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

Reviewer #2: All comments have been addressed

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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: Yes

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: Yes

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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: - I think this sentence in the abstract has issues, it should be rephrased: "NOISYmputer is particularly convincing when comparing the genetic map obtained on real datasets as it estimates accurately the size of the map where the two other software can mistake from hundred to hundred thousands centimorgans."

- Link to data in Zenodo should be added under "Availability"

- The code repository will benefit from better documentation about the list of output files that are generated. Multiple output and intermediate files are generated but no explanation for what each file represents have been provided.

Reviewer #2: I still think there would be room for improvement in the presentation, and I can agree with the request from reviewer 3 for additional references. I think even tools such as R/QTL and the original Lander-Botstein model are relevant in the sense that inferring trait status and outright imputation are mirror problems, especially in the biparental case.

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Reviewer #1: No

Reviewer #2: Yes: Carl Nettelblad

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Fast and accurate imputation of genotypes from noisy low-coverage sequencing data in bi-parental populations

PONE-D-24-15414R2

Dear Dr. Lorieux,

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.

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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: 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: 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: (No Response)

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Reviewer #1: No

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