Dear Dr. Guan,

Thank you very much for submitting your manuscript "Micro-dissection and integration of long and short reads to create a robust catalog of kidney compartment-specific isoforms" 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.

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Sincerely,

Katalin Susztak

Guest Editor

PLOS Computational Biology

Ilya Ioshikhes

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: In this paper, the authors used both PacBio Iso-Seq long reads and Illumina RNA-seq short reads’ information to analyze and identify expressed isoforms for defining microscopic structures. They introduced a pipeline, in which they used long-reads of kidney tissues from two compartments: glomerular and tubule-interstitial and validated the reads by RNA-seq reads of the same tissue. From their experiment, they identified a substantial number of transcription start sites (TSSs), transcription end sites (TESs) and Splice junctions from a filtered set of high quality transcripts.

The manuscript is well written and easy to understand. The figures and tables are also clear and elaborate. However, I have some concerns about the work:

Major:

1. The novelty of the study is limited. The authors use the existing methods to analyze the Iso-seq and RNA-seq data, and identify expressed annotated and unannotated isoforms. The contribution to the computational biology field is not clear. This study can be easy applied to other tissue samples with both matched RNA-seq and Iso-seq data.

2. hg19 annotation is quite old (2009). It would be better to run the experiments based on the latest hg38 annotation.

Minor:

1. Abstract line 2: insterstitial -> interstitial

2. Abstract line 5: microdissed -> microdissected?

Reviewer #2: The paper titled “Micro-dissection and integration of long and short reads to create a robust catalog of

kidney compartment-specific isoforms” describes an developed experimental and computational pipeline for identifying isoforms at microscopic structure-level. It applies Pacific Biosciences SMRT analysis software and Illumina reads approach to discover novel transcripts. Although it is a promising approach, current manuscript lacks details and interpretations.

1. The authors claimed that they developed the approach for isoform identification. However, as far as I know, there are some additional methods that have been developed recent years, such as Mandalorion (Byrne et al. Nat. Comm. 2017) FLAIR (Tang et al, Nat. Comm. 2020), TALON (Wyman et al. biorxiv). I do not mean that they need to compare to all existing methods, but it is important that a comparison is performed to demonstrate the performance of this developed approach.

2. In general, it would be better to describe with more details of read correction, transcript assembly, and transcript quantification in the results part of the manuscript to illustrate the power of this approach and the reason that this approach performs good, such as what are the advantages and what are the drawback of this approach.

3. To test the accuracy of this approach, it would be better to provide the rate of false discovered isoforms and illustrate the reason of these false discovered isoforms.

4. For enrichment results, it would be better to show P-values, gene/transcript count for each pathway, and top pathways in one Figure.

5. The Introduction and Discussion sections are not comprehensive and do not present readers a view of the field. The authors should expand it and describe what are already available and what are the specific features of existing methods for PacBio data. Some recently published methods on novel isoform discovery are not cited. While I understand that this paper focuses on application of PacBio data, it is still important to summarize state-of-the-art methods to present a comprehensive view of the current state of art.

6. The font size in Figures is too small to read

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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: No: Illumina RNA-seq and PacBio Iso-seq of the 25 samples are not provided.

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Dear Dr Yuanfang Guan,

We are pleased to inform you that your manuscript 'Micro-dissection and integration of long and short reads to create a robust catalog of kidney compartment-specific isoforms' 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,

Katalin Susztak

Guest Editor

PLOS Computational Biology

Ilya Ioshikhes

Deputy Editor

PLOS Computational Biology

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No further comments

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The authors have responded well to the previous critiques.

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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: None

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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: No