PONE-D-24-11148Reference Sequence Browser: An R application with a User-Friendly GUI to rapidly query sequence databasesPLOS ONE

Dear Dr. Tapias Gomez,

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Please adjust the manuscript according to the comments of the two reviewers.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

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4. If the manuscript contains new data, have the authors made this data 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: N/A

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5. Is the article 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 highlight any specific errors that need correcting in the box below.

Reviewer #1: No: There are misalignments between text and figures that need correction. These are detailed below

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 #1: This manuscript describes an R Shiny web application for supporting searches of sequence data repositories for the purpose of identifying reference sequence availability or discovery of gaps or missing sequences for target organisms. The app can be run online or the code downloaded to run locally. The Shiny app is designed to support searching 3 repositories for reference sequences for one to many organisms and standard barcode primers. The authors provide a reasonable justification for the application based on their experience and interviews with other scientists. The tool serves a niche for users who may not be savvy in accessing online data repositories to search for sequences. A key strength of the tool is that it provides a quick way to compare results across more than one data repository within the same platform rather than having to visit each data repository individually.

That said, there are some issues that could be improved. One is to review potentially related/similar work (e.g. crabs: Jeunen, G.-J., Dowle, E., Edgecombe, J., von Ammon, U., Gemmell, N. J., & Cross, H. (2023). CRABS—A software program to generate curated reference databases for metabarcoding sequencing data. Molecular Ecology Resources, 23, 725–738. https://doi.org/10.1111/1755-0998.13741

taxalogue: Noll NW, Scherber C, Schäffler L. taxalogue: a toolkit to create comprehensive CO1 reference databases. PeerJ. 2023 Dec 4;11:e16253. doi: 10.7717/peerj.16253. PMID: 38077427; PMCID: PMC10702336.) noting overlaps and differences and the degree to which the work fills a gap.

There are also some sections that could use some clarifications and corrections. In the section Case 1.1 under the third paragraph, the text states, ‘To get more information, the user would then inspect the CM like the Fig. 3.’ Figure 3 appears as a summary table rather than a coverage matrix (CM) as stated.

A little more careful alignment between text and figures would alleviate some potential confusions. For example, the text says,

‘As shown in the summary table, of the 11 invasive species searched, six organisms had at least one sequence.’ It is not clear how this information was derived from the summary table since it is not organized by organism. The text further says, ‘The vast majority of the sequences were found in the CO1 barcode and includes five organisms but the 16S barcode captures all six detected 148 organisms.’ That the majority are found in the CO1 barcode is clear from the summary table but one needs to refer to the coverage matrix (CM) to determine that there were 5 organisms detected under CO1 and six detected under 16S.

Similar additional clarification would help with the next paragraph. The text states, ‘one can see examples of the three different ways in which the RSB highlights missing information in the CRUX databases. Firstly, Lithobates catesbeianus, Dreissena polymorpha, and Euwallacea kuroshio are not in the database.

It would help to point out to readers the pattern that depicts this. From the Figure 2 example one can see that there are no sequences indicated for Lithobates catesbeianus (no numbers other than zero), but the text family and phylum appear so this makes sense.

Dreissena polymorpha does have sequences appearing for 18S and 16s so the above statement that there are no sequences for this organism appears incorrect. Perhaps this was meant to be Dreissena bugensis which does show no sequences appearing across the set of barcodes?

With respect to Figure 4. The text might be amended from this: ‘Here, there are CO1 sequences for species that were not present in CRUX. To ‘The NCBI search results show that there are CO1 sequences for species (Lithobates catesbeianus, Dreissena polymorpha, Dreissena bugensis and Euwallacea Kuroshio) that were not present in CRUX.

With respect to Figure 5, the caption is,’ Example of NCBI Summary Data table for the genus Xenopus’ However the figure title is ‘BOLD coverage matrix’. It appears that Figures 5 and 6 are inverted with respect to the captions /associated png files.

In summary the work has benefit for supporting search for and comparison of sequence availability across a set of data repositories. The authors however could provide some further discussion of related work (as pertinent) and provide more specifics and clarifications to assist readers in interpreting the tabular results.

Reviewer #2: In this paper, authors summarize an R application which allows users to access and download reference sequences from multiple genomic databases in order to assess reference sequence availability. It is clear that this application has a wide range of potential uses in metabarcoding studies, and that researchers of various levels of experience could benefit from using it. The compatibility with the taxize package and the ability to uncover gaps in information (e.g., species within genera of interest that possess or lack barcode data, underrepresented barcodes) are particularly useful features of the RSB that the metabarcoding and barcoding communities can greatly benefit from. I have very minor feedback for the authors:

1. Line 16: BOLD stands for Barcode of Life Data System; authors should make sure to use its correct name.

2. Lines 35-38: Example for the UC system is interesting but difficult to put into context. Is this expected to be high or low in comparison to other schools? Average? I realize that this is merely to illustrate that few students may have opportunities to gain experience in programming, but I wonder if the high-powered UC system actually represents an overestimate of how many students are exposed to this training. Maybe something like that could be mentioned.

3. Line 131: This paragraph is useful; I just worry that an inexperienced reader could interpret its message to say that a barcode should be chosen based on how many organisms it covers when a barcode should probably be chosen based on numerous factors (certainly inclusive of coverage, but also factors like species-level genetic variability). Maybe authors could mention something like that users can evaluate this factor “in addition to other factors specific to their study.”

4. Figures 1-3, 6: I was curious why trnL is included in these tables when it is a cpDNA marker and would not be used in exploring barcodes of amphibians. Upon further inspection, it seems that in the RSB itself the “barcodes” rows/columns appearing in these figures are just the standard list included in outputs. Does this mean, then, that coverage of only one cpDNA barcode can be explored for plants? If so, it may be worth a more detailed discussion of the utility of this tool for various types of organisms, or stating that this is not an exhaustive list of markers and researchers might be interested to use others not in the RSB (e.g., trnL has become the new standard for plants but those interested in a multi-marker approach or who might find utility for a more widely used marker like rbcL might benefit from knowing that they have other options). Since this tool is introduced as one that is beginner-friendly, additional information on why that set list of markers is used in the app and how individuals might consider alternatives (or expand on this framework to compare a standard barcode to lesser-used barcodes) could be helpful.

5. Line 200: Creating local reference libraries for study systems of interest is crucial to high-resolution and reliable metabarcoding data. I wonder if their importance could be more explicitly stated in this paragraph (the development of local reference libraries is also synergistic with the goals of RSB including identifying taxonomic and geographic gaps in barcode data availability).

6. Line 241: Could the authors briefly explain how users might deal with or anticipate homynyms in the framework of the RSB?

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Reviewer #1: No

Reviewer #2: No

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Reference Sequence Browser: An R application with a User-Friendly GUI to rapidly query sequence databases

PONE-D-24-11148R1

Dear Dr. Tapias Gomez,

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,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: No

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: No

Reviewer #2: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: No

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data 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 article 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 highlight any specific errors that need correcting in the box below.

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: The issues identified in the first review have been satisfactorially addressed. There is an issue with the protocol. The link generates an error so that the protocol can not be accessed

Reviewer #2: The authors have addressed comments appropriately and I think the manuscript is suitable for publication.

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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

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