-->PONE-D-25-50266-->-->GermVarX: A Robust Workflow for Joint Germline Variant Exploration in Whole-Exome Sequencing Cohorts-->-->PLOS ONE

Dear Dr. Nguyen,

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->4. If the manuscript contains new data, have the authors made this data 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.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.-->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.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: N/A

Reviewer #2: Yes

Reviewer #3: N/A

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-->5. Is the article 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 highlight any specific errors that need correcting in the box below. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: My main concern is that the described pipeline appears to be primarily a combination of existing software tools, which raises questions about its novelty and added value. The authors are encouraged to provide a stronger justification for the development of this pipeline—specifically, to clarify what unique features, integrations, or improvements it offers compared to existing solutions, and to articulate how it will advance or benefit the research community.

The authors state in the introduction: “While a few pipelines such as MagicPipeline [5] and star protocols wes [6] support multi-sample processing, they are predominantly script-based and command-line dependent, lacking key features such as automated orchestration, modular parallel processing, and scalability across diverse computing infrastructures.” However, it remains unclear how the proposed pipeline substantively improves upon these existing tools. Furthermore, many researchers working with WES data already employ well-established, in-house pipelines tailored to their specific workflows. As such, the practical advantages and broader relevance of the proposed pipeline to the wider research community are not evident.

Below are several additional comments and questions regarding the manuscript.

1) Authors use a 3-sample WES benchmark dataset for validation, but it would be more informative if they could conduct validations on additional data sets.

2) On page 6, they state: "the highest 185 F1-score (0.9649), balancing exceptional precision (0.9994) with strong recall (0.9327). Please clearly define these 3 metrics.

3) Figure 2 needs a legend.

4) In the subsection "Variant Filtering and Quality Control" on page 5, are users able to modify the filtering thresholds? For example, instead of DP < 10, can the users change it to DP < 20? This was not very clear.

5) Can authors also explain if their pipeline can handle if the users input the intermediate files? For example, if the users already have gVCF files, can the pipeline bypass the previous steps and start with the gVCF files?

6) A major improvement to the current manuscript would be to demonstrate that the pipeline can handle two or more cohort-level WES datasets originating from different sources and generate a merged, harmonized dataset. This is a technically challenging task, and successfully implementing such functionality would significantly enhance the novelty and impact of the work. However, it is acknowledged that this capability may be beyond the current scope of the study.

Reviewer #2: The manuscript submitted by Nguyen et al. describes a workflow for variant calling of population WES data. Notably, the workflow includes a step for joint variant calling to produce a single cohort VCF. The manuscript is well written and very clear. However, I have some comments:

1) The authors state that existing workflows are designed for single-sample analysis, while their workflow is optimized for multi-sample analysis (L13). However, one of the existing workflows they cite, Sarek, states in their documentation that it is very easy to run multiple samples via a CSV samplesheet. It seems to me that the authors should not emphasize single vs multi sample design, but rather their implementation of joint calling.

2) Related to above, I believe that the authors could expand more on how their workflow is different than the existing workflows they listed, and why those differences are important. As it is written currently, the only real distinction the authors make is the difference in design for single or multi sample analysis, which is insufficient. The authors could consider making a table to list the differences to make it easier for readers (i.e. potential users) to determine if this workflow is right for their application.

3) The workflow Github repo looks well documented, for which I commend the authors. I would also like to see the inclusion of a test dataset that users can run through the workflow to make sure the workflow runs correctly on their system.

Reviewer #3: The narrative is well-written, clear, and linear. Each section transitions smoothly. The authors have combined dual-caller consensus and an automated joint calling workflow that addresses the gap between single-sample and multi-sample pipelines. However, I would like to give a few suggestions:

1. Add runtime comparisons with other public workflows. A qualitative comparison will strengthen the claim of scalability.

2. Future work could include the integration of structural variant and copy number variation (CNV) detection modules, along with extending the workflow to accommodate whole-genome sequencing (WGS) and long-read data for comprehensive variant discovery. This would enhance its applicability to diverse genomic analyses.

3. Figure captions could be expanded to interpret the figure rather than reviewing the section text.

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-->7. 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 published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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GermVarX: A Robust Workflow for Joint Germline Variant Exploration in Whole-Exome Sequencing Cohorts

PONE-D-25-50266R1

Dear Dr. Thao Nguyen,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mainak Bardhan, MD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?-->

Reviewer #1: Yes

Reviewer #3: Yes

**********

-->2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.-->

Reviewer #1: Yes

Reviewer #3: Yes

**********

-->3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.-->

Reviewer #1: Yes

Reviewer #3: Yes

**********

-->4. If the manuscript contains new data, have the authors made this data 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.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.-->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.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: N/A

Reviewer #3: N/A

**********

-->5. Is the article 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 highlight any specific errors that need correcting in the box below. -->

Reviewer #1: Yes

Reviewer #3: 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: The authors have addressed the previous comments adequately, and the current reviewer does not have any additional comments.

Reviewer #3: The authors have addressed most of the comments, including those related to accuracy and computational performance.

They have also included an additional dataset of 120 samples of WES cohort to demonstrate the workflow’s reliability and accuracy.

The authors have also addressed the key differences between GermVarX and existing WES workflows.

Overall, the manuscript is significantly improved.

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-->7. 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 published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->..-->

Reviewer #1: Yes: Harold BaeHarold BaeHarold BaeHarold Bae

Reviewer #3: No

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