PONE-D-21-29551Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fishPLOS ONE

Dear Dr. Masao Adachi, 

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PLOS ONE

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Additional Editor Comments:

The manuscript have been reviewed by the three independent reviewers.

I thank you for considering the Plos One as potential vehicle of your research outcomes.

I am pleased to inform that all the three reviewers have recommended publication of the manuscript after minor revision. The comments and suggestions given by all the three reviewers are very pertinent . I would request you to revise the manuscript accordingly and resubmit for final decision. In addition to comments by three reviewers I think that the details of designing and selecting the primers are ambiguous , reviewers have also commented on this issue.

The manuscript is acceptable after incorporating all amendments suggested by the reviewers.

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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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 study aims to assess the efficiency of PNA clamp to suppress the amplification of fish DNA and compare it with the efficiency of blocking primers. 24 species of herbivorous fish are tested in the study. The authors design three blocking primers and one PNA clamp to determine the organisms that are a part of the diet of herbivorous fish. The results clearly indicate the dominance of PNA clamp over blocking primers in suppressing the fish DNA amplification by a huge margin.

The results from this study could be of a good application in studying aquatic food web and nutrient cycle while being species specific. The manuscript is well written and comprehensible. All my comments and questions are stated herewith.

The authors have stated the limitations of previously used methods, but the limitations of DNA barcoding accompanied with molecular cloning as a method could be elaborated a little more to consolidate their point of the need for a new method (inclusion of PNA clamp in this case during amplification) because the point that it is laborious and time consuming (line 44-47) could be applicable to most of the processes described and is general in my opinion. As DNA barcoding has been previously used for determining the prey organism of Siganus fuscescens and Scarus ovifrons, I think it would be better to specify the limitations from that experiment as well to strengthen the claims made by the author.

Regarding the table no. 4. For langsdorfii, no observations are included in the table and the genus name has not been provided, please address to that as it has been mentioned in line no. 325-327 of the manuscript in relation to a 100% suppression by BlockFish_long7

Also, Since Naso vlamingii does not seem to produce amplicons without blockers at 65 and at 60 degrees Celsius, and produces only 0-50% of relative amount of amplicons, was it tested at lower annealing temperatures than 60 degrees to see if more amplicons are being produced for Naso vlamingii without the blocker. Was there any limitation that did not allow more observations for the said species such as the sample size or availability.

Please include the page numbers and make sure to review the submission guidelines for the journal to check the formatting of the manuscript.

A general question for someone not a specialist in this field, This is more of a personal curiosity regarding the use of V9 and V8f region, Is there a possibility to use any more regions on 18S rDNA or an inclusion of more regions of 18S rDNA as biomarker, if yes would that be beneficial or the results would be similar in preparing the blockers and PNA clamp?

Reviewer #2: The manuscript shows how efficiently the gut microbiology of herbivorous fish can be investigated by focusing on 18S rDNA of only prey organisms and blocking the DNA content of fish. The study was novel as the prey content has not been identified in herbivorous fish. The manuscript is well written. Would suggest minor revision. The changes required are:

1. The details of designing and selecting the primers were not clear in the manuscript. If added in supplementary, I would suggest to add that in the materials and methods section where the primer designing has been mentioned. Also mention from which organism/s each primers has been designed.

2. I feel metabarcoding results should also be properly mentioned. The prey organisms mentioned in Table 2 were only used for conducting mock tests? Incorporate the metabarcode analysis of prey organisms (the list of organisms or classes of organisms that are observed in fish by PCR blocking technique).

The manuscript can be accepted after incorporating these minor corrections.

Reviewer #3: Few corrections:

1. BlockFish_long7 has only 6 nucleotide overlap with the 18SV9R which does match with the text in line 119.

2. In the Table 2 labels of columns Mock 1, Mock 2 and Mock 3 does not give the idea about what the numbers given in the columns represent (in line 236 mentioned but it should be clearly labeled in table also)

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Reviewer #1: Yes: Malayaj Rai

Reviewer #2: No

Reviewer #3: No

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Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish

PONE-D-21-29551R1

Dear Dr. adachi,

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.

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Kind regards,

Ram Kumar, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Prof Masao Adachi

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.

Thank you ones again for considering the PLOSE ONE as potential vehicle of disseminating your research outcomes.

Prof. Ram Kumar

Handling Editor

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: Yes

Reviewer #2: 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

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: The study effectively shows the potential for use of blocking primer and 18S metabarcoding as a means of dietary analysis of herbivorous fish by blocking the expression of fish DNA during amplification. The study is novel and has potential for future works related to the accurate identification of prey using metabarcoding as well which would provide information that could be experimented in aquaculture industry. All my questions and suggestions have been addressed to.

Reviewer #2: All the questions have been addressed. In the acknowledgement section, pls remove 'the' mentioned in the line 502-503 (for "the" revising). Can be accepted.

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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: Yes: Rai, Malayaj

Reviewer #2: No