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PONE-D-23-14875Kuura - An automated workflow for analyzing WES and WGS dataPLOS ONE

Dear Dr. Fey,

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.

In particular please address the reviewer comments about making your pipeline easy to install and run. Also please address the comment about the performance 

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Please address the reviewer comments below especially regarding the performance of the pipeline and its installation so that it can be easily installed, ran and tested.

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: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

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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: Yes

Reviewer #2: 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

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5. Review Comments to the Author

Reviewer #1: The manuscript provides a Nextflow-based pipeline that bioinformaticians can use in their analysis of genetic variants. The pipeline runs as described but the setting up requires some clarification.

Step 3 in line 154 of manuscript, the command used only works in the docker directory and the change of directory needs to be specified in the step.

Files in the GitHub repository are named differently (default.config to standard.config) and a better guide on how to set up the config files would greatly extend the user-friendliness of this pipeline. It would also be beneficial to add supplementary files the repository explaining what each file is or a README file. Users who have not run Nextflow before would appreciate these comments.

The analysis is limited since it does not compare the effectiveness of Kuura to other pipelines based on variants called, time taken for analysis, processing power, etc. Evidence of this comparison would further convince and guide the implementation of this pipeline to existing workflows.

Reviewer #2: This work decribed a whole genome/exome sequencing workflow (Kuura) that consumes fastq files and produces annotated short genetic variants with multiple standard QC reports. It is written in Nextflow, with Docker enabled. The authors are aware of other pipelines such as SeqMule, DNAp, SpeedSeq and GESALL, but it seems their codebases are no longer maintained (most recent change to them was two years ago to SeqMule). Another Nextflow DnaSeq pipeline is Sarek that is still actively maintained and has a relatively large userbase, however its codebase is heavy and its structure is complex.

In contrast, Kuura provides a light-weight solution that requires a minimal setup and is easy to expand functionalities. It can also serve as a backbone for more advanced workflows.

Major concerns:

1. The data presented in Table 3 are surprising. The precisions and recalls are worryingly low. These numbers should be much higher according to Zhao et al., 2020 (https://doi.org/10.1038/s41598-020-77218-4).

Minor concerns:

1. There is no Readme in the github repository. A Readme with a brief desription of the workflow, and instructions to setup and test is necessary.

2. The existing workflow exclusively supports Docker as a solution. However, Docker's incompatibility with many High Performance Computing (HPC) systems hampers its widespread utilization. The authors should consider to include Singularity. (For reference, https://depot.galaxyproject.org/singularity/ has a wide range of images)

3. There are hardcoded parameters in the workflow (e.g. https://github.com/dhanaprakashj/kuura_pipeline/blob/main/dnaseq/main.nf#L96, https://github.com/dhanaprakashj/kuura_pipeline/blob/main/dnaseq/main.nf#L128, https://github.com/dhanaprakashj/kuura_pipeline/blob/main/dnaseq/main.nf#L206). Please make them configurable.

4. The workflow will work best with a small number of exomes. I think it might struggle with large number of exome samples or whole genomes. The authors should discucss this.

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Reviewer #1: No

Reviewer #2: Yes: Jing Yu

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Kuura - An automated workflow for analyzing WES and WGS data

PONE-D-23-14875R1

Dear Dr. Fey,

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,

Alvaro Galli

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 #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 #2: Yes

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

Reviewer #2: N/A

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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 #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 #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 #2: The authors have addressed all my comments. However, as I ran through it again I think that having the option to include compensating set of variants, i.e. variants called by some variant callers, but not all, could be an important improvement, e.g. for finding rare variants underlying Mendelian disorders.

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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 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 #2: Yes: Jing Yu

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