PONE-D-23-08120Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae)PLOS ONE

Dear Dr. Shimura,

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.

Please submit your revised manuscript by May 21 2023 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,

Matthew Cserhati, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Additional Editor Comments:

Besides the comments made by the reviewer, I also have the following observations regarding th manuscript:

1. Several typos occur in the text; the English could use a little bit of improvement.

2. Please define basic terms, such as protocorm.

3. Lines 21-23: “The family Orchidaceae comprises the most species of any monocotyledonous family and has interesting characteristics such as seed germination induced by mycorrhizal fungi and flower morphology that co-evolved with pollinators.” Is incorrect, it is more accurate to say that the fungi and the flower morphology co-adapted with pollinators. We are talking about already existing structures that underwent adaptation.

4. Line 190: table 1: incomplete value: 1048,92

5. Why did the VL assembler have such a low mapping rate in data sets 1 and 3? Does this nit skew the results? Shouldn’t this assembler be excluded? Would the results change that much?

[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: Partly

**********

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

Reviewer #1: Yes

**********

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

**********

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

**********

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: In this paper, Kambara et al. reported the establishment of a de novo assembly pipeline for transcriptome data from the wild orchid Cypripedium. Using the contig set obtained by the established method, analyses of differentially expressed genes (DEGs) regarding mycorrhizal formation and maintenance were conducted. I agree with the authors’ idea that the de novo assembly pipeline established in this paper is effective and useful for the species for which genome sequences are not available such as Cypripedium to conduct RNA-seq. However, I have serious concerns about the statements of results and discussion based on the afterward transcriptome analysis because no biological replicates of samples were available.

For example, the authors showed TPM value based on one sample data, but at least three biological replicates are generally demanded to demonstrate the gene expression levels (e.g., qRT-PCR). Generally, DEGs should be obtained based on reliable statistical analysis. Therefore, I am regret to say that GO enrichment analysis of the DEGs obtained from no biological replicate work has no biological meaning. I understand that the authors would like to validate the established pipeline by the afterward transcriptome analysis. However, I think the statement should be more restricted, considering the results obtained from insufficient replicates. Hence, I would like to propose that Fig. 4-6 are moved to supplemental information, and the statement regarding this part should focus on the validation. In particular, the discussion part (Line 389-421) is overdiscussed. Hence, I recommend that the contents of this part should be reconsidered and shortened as far as possible.

In addition, I noticed that the abstract does not contain a conclusion at the end. Likewise, which content is the point the authors’ would like to claim to readers in this paper is not clear in the discussion part. Hence, please reconsider these contents and compositions.

On the other hand, I think the validation of the assemblies using the data of genome-sequenced orchids is not enough because the authors compared only the number of predicted genes. The authors suggested the accuracy of assembling only based on the number of predicted genes, but I think that the accuracy should be shown based on the sequence identity. Thus, please add such analyzed data. I think the top five list summarized in Table 4 is also not required. In addition, the assembled sequence data obtained in this study is not available, and only the unique gene IDs were shown in the manuscript. Considering that other researchers may verify or use the results obtained in this study, I strongly recommend that the representative assembled data should be deposited in public database database/website or submitted as supplemental data. I hope that the authors will revise the manuscript according to my comments.

Minor comments:

Line 45: ‘orchid mycorrhiza fungi’ should be ‘orchid mycorrhizal fungi’.

Line 64: ‘et al’ should be ‘et al.’ (same afterward)

Line 72: Words at the beginning of a sentence are not abbreviated. (same afterward)

Line 73: Considering the content of previous sentence, ‘However’ is not a suitable word for connecting.

Line 108: The expression of ‘12-14 weeks’ is not suitable to show the timing to harvest each sample because two weeks range is so different. Please reconsider and show it for each sample.

Line 109: Please state the details about the strains WO97 and FT061. The current statement is not enough to understand what the authors used.

Line 226: What is ‘mapping rate’? I could not get its meaning. (same afterward)

**********

6. 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 #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae)

PONE-D-23-08120R1

Dear Dr. Shimura,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Matthew Cserhati, Ph.D

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)

**********

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 #1: No

**********