PONE-D-21-18748

easyFulcrum: An R package to process and analyze ecological sampling data generated using the Fulcrum mobile application

PLOS ONE

Dear Dr. Andersen,

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PLOS ONE

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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: No

Reviewer #2: No

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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: No

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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 paper by Bernardo et al. explains the data-collection platform Fulcrum and easyFulcrum applications to organize ecological sample data collection. These applications are easy to use using mobile devices, and helps with identification of microscopic organisms based on their location, photographs and genotyping. This paper focuses on easyFulcrum R package which can analyze exported data from Fulcrum and process the data by combining geographical and sequencing data. These applications are designed for nematodes but can be used for any other organism.

Review Points:

1. In line 53 authors explain about Fulcrum but no reference paper is added.

2. In line 126, authors mention Genotyping template (google sheet) to enter data for isolated samples. Author explain well how to duplicate and use the template but fail to explain columns like ssu_pcr_date, its2_pcr_date etc. Also, there is no mention of the column name to input sanger sequencing data.

3. Authors need to explain how to input genotyping data and blast results if any new species is identified.

4. In line 176, author is suggesting to see methods. It will be helpful if authors mention which subheading in the methods to look for.

5. Authors should mention that Figure 1 is also published on the following website:

6. Figure 3A image quality is not good. Labels in the image are not legible.

7. For Figure 3, authors can include the “genotyping template” made for C.elegans, C.briggsae and C. tropicalis.

Reviewer #2: The manuscript sounds technically poor, I have following concerns should be addressed before any decision.

1. There are some typos and grammatical errors in the manuscript. It is strongly suggested that the whole work to be carefully checked by someone has expertise in technical English writing.

2. Key contribution and novelty has not been detailed in manuscript. Please include it in the introduction section

3. What are the limitations of the related works

4. Are there any limitations of this carried out study?

5. How to select and optimize the user-defined parameters in the proposed model?

6. There are quite a few abbreviations are used in the manuscript. It is suggested to use a table to host all the frequently used abbreviations with their descriptions to improve the readability.

7. Some sentences are too long to follow, it is suggested that to break them down into short but meaningful ones to make the manuscript readable.

8. Explain the evaluation metrics and justify why those evaluation metrics are used?

9. It seems that the authors used images of equations, please use editable equation format.

10. The Related Works section is also fair, yet the criteria behind the selection of the works described should be explained.

11. The title is pretty deceptive and does not address the problem completely.

12. Every time a method/formula is used for something, it needs to be justified by either (a) prior work showing the superiority of this method, or (b) by your experiments showing its advantage over prior work methods - comparison is needed, or (c) formal proof of optimality. Please consider more prior works.

13. The data is not described. Proper data description should contain the number of data items, number of parameters, distribution analysis of parameters, and of the target parameter itself for classification.

14. The related works section is very short and no benefits from it. I suggest increasing the number of studies and add a new discussion there to show the advantage.

15. Method description is detailed and overall convincing, yet there is a big formatting problem with the end of this section and the beginning of the following one.

16. Figures 1-4 are low quality and very unprofessional figures please improve them.

17. Use Anova test to record the significant difference between performance of the proposed and existing methods.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-18748R1

easyFulcrum: An R package to process and analyze ecological sampling data generated using the Fulcrum mobile application

PLOS ONE

Dear Dr. Andersen,

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 Oct 08 2021 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: http://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,

Syed Ahmad Chan Bukhari

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Reviewer 1 is still concerned. Would you mind revising accordingly?

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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

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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

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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

**********

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

**********

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: In this study authors are explaining how to use the R-package apps Fulcrum and easyFulcrum which have been used by them in previous study published in eLife (https://elifesciences.org/articles/50465). In this paper authors introduce easyFulcrum to combine Fulcrum data with genotyping data to make sampling easy.

1. easyFulcrum installation in the methods can be more elaborated to make it user friendly. Authors can simplify how to install R from devtools::install_github ("AndersenLab/easyfulcrum”), Imager and other prerequisites.

2. Genotyping templates have the option of entering Blast results but authors do not explain how the actual sequences of the PCR products linked to the C- and S- label in fulcrum.

3. Authors should mention examples of Molecular barcodes that can be used to identify organisms like DNA or RNA sequencing and lipid profiling etc.

Reviewer #2: The manuscript is relatively improved. Moreover, the manuscript is in a better format in terms of technicality and other results. I suggest accepting it.

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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 #1: No

Reviewer #2: 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.

easyFulcrum: An R package to process and analyze ecological sampling data generated using the Fulcrum mobile application

PONE-D-21-18748R2

Dear Dr. Andersen,

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,

Syed Ahmad Chan Bukhari

Academic Editor

PLOS ONE