-->PONE-D-26-15640-->-->Accurate quantification of spliced and unspliced transcripts for single-cell RNA sequencing with tidesurf.-->-->PLOS One

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Reviewers' comments:

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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

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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

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 manuscript “Accurate quantification of spliced and unspliced transcripts for single-cell RNA sequencing with tidesurf” by Schleicher et al. describes the implementation of a new tool, tidesurf, for RNA velocity inference. The key advance of this tool is its ability to infer RNA velocity measures from both 3′- and 5′-based scRNA-seq protocols. The authors demonstrate clear advantages of tidesurf over velocyto, which was not designed to handle 5′-based scRNA-seq protocols but has nevertheless been used for this purpose, often leading to misleading results. They present a strong and defensible case for the superiority of tidesurf in analyzing 5′-based scRNA-seq data by identifying the underlying issues that affect velocyto’s performance.

The authors also compare tidesurf with alevin-fry and STARsolo, showing high concordance between these tools for 5′-based scRNA-seq protocols and demonstrating that all tools perform similarly on 3′-based scRNA-seq. The performance benchmarks provided in the supplementary materials are additionally helpful for understanding how these tools compare depending on what resources an investigator may have at their disposal.

One area where differences remain between tidesurf and alevin-fry is in datasets derived from human donors. The authors address this, noting: “The human genome contains more repetitive sequences than the murine genome [21], which could lead to differences in the (pseudo-) alignments of Cell Ranger and alevin-fry, especially for intronic sequences. Furthermore, the total unspliced counts were considerably lower on the two 5′ datasets, rendering small absolute differences relatively more important. Finally, alevin-fry’s quantification of total counts differed slightly more from the Cell Ranger output than tidesurf’s (Fig. 3B–D).”

I wonder whether there is a dataset that could serve as a ground truth (even a simulated dataset) to more directly evaluate the performance of these tools and clarify the sources of similarity or divergence between tidesurf and alevin-fry. One possible candidate is the dataset described in PMID: 38782298, which includes both long-read and short-read data generated from the same 10x Chromium samples. Although this dataset is not human, and the authors suggest the discrepancies may be human-specific, it could still provide informative insights for readers seeking to understand how these tools behave under conditions where transcript structures are more fully resolved.

Reviewer #2: 1. Is the manuscript technically sound, and do the data support the conclusions?

Yes. The study is technically solid and the comparisons across datasets and tools are well done. The data generally support the main conclusion that velocyto performs poorly on 5′ scRNA-seq data, while tidesurf and other tools give more consistent results. The conclusions are reasonable based on the presented evidence.

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

The analyses used (correlations, distribution comparisons, and velocity/PAGA comparisons) are appropriate for this type of benchmarking study. However, the statistical evaluation is mostly descriptive, and a bit more formal testing would improve clarity, although it is not critical.

3. Have the authors made all data underlying the findings fully available?

Yes. The datasets are clearly referenced with GEO/ArrayExpress accessions, and processed data and tools are described well enough to allow reproduction. Data availability is adequate.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Yes. The manuscript is clearly written and easy to follow. Only minor language polishing is needed in a few dense methodological sections.

5. Review comments to the author

The manuscript is well written and addresses an important issue in scRNA-seq velocity analysis. The findings are convincing and supported by multiple datasets and methods. T

A few minor points to improve the manuscript:

•The conclusions about “incorrect” assignments in velocyto could be slightly softened, since there is no direct ground truth.

•A small summary table comparing tool settings would improve readability.

Generally, this is a solid and useful methodological paper with clear practical relevance.

Reviewer #3: The manuscript talks about an interesting aspect of identifying spliced and unspliced RNA transcripts but I am little confused as to why the authors have downplayed Velocyto which looks like the crux of the entire manuscript. I understand that Velocyto has not been upgraded to include the pitfalls of 5’ chemistry but what if they do that upgrade soon and make Velocyto as powerful as 10X genomics softwares.

It would have been nice if the authors have introduced Tidesurf as the new command line software which addresses the pitfalls of existing softwares such as Velocyto in a sentence or two but not make it the crux of the entire manuscript.

Can the existing manuscript be rearranged and rewritten so as to put the focus completely on the USPs of Tidesurf highlighting the real time importance rather than downgrading an existing software which was introduced in 2018-2019. I would say few sentences about Velocyto are in harsh language which is not desired for the readers.

If that modification (major revision) can be done than the manuscript will be in a good shape and can be considered for publication.

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Reviewer #1: No

Reviewer #2: Yes: Addis Tekaw

Reviewer #3: Yes: Dr. Gauri Awasthi

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Attachments

Attachment

Submitted filename: PLOS One tidesurf - review.docx

Accurate quantification of spliced and unspliced transcripts for single-cell RNA sequencing with tidesurf.

PONE-D-26-15640R1

Dear Dr. Claassen,

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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Kind regards,

Andreas Neueder

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

Reviewer #2: All comments have been addressed

Reviewer #3: 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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

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: The authors went beyond what was expected from me and actually bring to light interesting differences between long-read and short-read sequencing in their response. I think this manuscript contributes to the understanding of 5' sequencing and brings a new tool to this assessment.

Reviewer #2: The authors have addressed my comments satisfactorily. The manuscript has been improved by adding formal statistical tests, clarifying the interpretation of the results, and softening conclusions where no direct ground truth is available. The addition of summary tables for tool settings also improves readability and reproducibility. Overall, this is a well-executed and practically relevant benchmarking study that provides useful guidance for RNA velocity analysis of 10x Genomics 5′ scRNA-seq data. I have no further major concerns and recommend the manuscript for publication after these revisions.

Reviewer #3: All comments have been addressed satisfactorily by the authors in the revised version. Recommended for acceptance.

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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.

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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: Philip J. Moos

Reviewer #2: No

Reviewer #3: Yes: Dr. Gauri Awasthi

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