PONE-D-22-03204Next generation biosecurity: Towards genome based identification to prevent spread of agronomic pests and pathogens using nanopore sequencingPLOS ONE

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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?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

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?

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Yes

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: ID PONE-D-22-03204: “Next generation biosecurity: Towards genome based identification to prevent spread of agronomic pests and pathogens using nanopore sequencing” by Frey et al.

The manuscript reports a protocol that can be useful to the research community interested in the identification of agronomic pests. The traditional methods are based on classical barcoding (or metabarcoding) that targets one or a few genomic regions of one or a small group of species. The barcoding approach requires some prior knowledge of the target organisms, which sometimes is lacking. In this manuscript, the authors take the metagenomic approach by sequencing all the DNA of the sample, so any genomic region of any organism can be sequenced and, if there is enough genomic information in the repositories, identified. In consequence, the proposal is more flexible than the classical one based on barcoding. I reckon that in the future (with richer genomic repositories) methods like the one proposed here based on metagenomics will overcome (meta)barcoding methods.

The protocol is described in sufficient detail in the main text and the supplementary material. The method is validated using more than 60 artificial single-species libraries of insects and a few more of bacteria, fungi, and nematodes.

Despite being very positive about the manuscript, I don’t like how results are presented. I have a couple of suggestions that may be useful to the authors in the preparation of a new version of the manuscript.

Perhaps Figure 1 is unnecessary. It is just to be expected to find a highly significant linear relationship between the number of mapping reads and the number of collected reads. This information can be included in the text.

On the contrary, I’ve found the description of sample identification too shallow (l. 231-237). The authors only provide summary statistics (Table 1 and 2) that show that the process of species identification is good, in general. However, this is not always the case, as there are 9 libraries in which the “assumed species” is different to the “species ID” (Supplementary Table 2: please clarify the meaning of these two columns). This criticism does not invalidate the method but is proof that the method is not perfect (as it happens with all DNA-based technologies for species identification). I encourage the authors to acknowledge the limitations of their method in the main text (in the results) and explain, if possible, the reason for the wrong or partial identification of some species (in the discussion).

A final comment about the application of the method to real samples. The manuscript uses artificial single species libraries and the bioinformatic pipeline is intended to provide one species per sample. But what would happen if the original sample contained more than one insect species? Ideally, the method should provide all of them, but would it? In addition, if the sample contained two phylogenetically close species (e.g. two species of Bactrocera) would their reads map in different contigs or would they be mixed? I understand that these questions can be difficult to handle at this stage, but the authors might consider adding their reflections to the discussion. Depending on their willingness, they could simulate two-species libraries by merging the fastq files of two single-species libraries, run the entire pipeline using the new fastq file, and see what happens.

Reviewer #2: This manuscript is introducing a WGA based protocol utilizing ON MinION sequencing to improve DNA-based species identification. the methods is described very well, sufficient details on all steps are provided. My major point of criticism is that the discussion section lacks several aspects mentioned in the results e.g., on the presence of genes of interest such as insecticide resistance genes, and the presence of arthropod-infecting

viruses and endosymbiotic bacteria. for a protocol description such as this it would be also very interesting to discuss the reference libraries used – where they sufficient? If not, how to improve that, where are the gaps, etc. ; or what about the pipelines you used? Where they useful, what could be alternatives also given that Geneious is not cheap?

Minor things:

L29 - 658 basepairs if you refer to COI

L58 - is needed not are needed

L64 - shown not proofed

L125 - add a 'the' between extractions and DNA

L128 - add the missing )

L214 – How did you compensate for retained sequences of prior runs? MinION flow cells are known to retain up to 15% of former runs even if their wash kits are used. Did you see a difference (or overlap) between runs of reused flowcells? This should also be briefly discussed.

L239 – table caption talks about Nematode data, but none is shown.

L309 – remove “should essentially enable to” and use ‘could’

L314 – Interest not interests

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Reviewer #1: No

Reviewer #2: No

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Next generation biosecurity: Towards genome based identification to prevent spread of agronomic pests and pathogens using nanopore sequencing

PONE-D-22-03204R1

Dear Dr. Buehlmann,

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,

Patrizia Falabella

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: Yes

**********

2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: 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: 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

**********

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

**********

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 is the second time that I review this manuscript. As in this first occasion, I am positive about its quality. The protocol is described in sufficient detail in the main text and the supplementary material, and the method is validated using artificial single-species libraries. The suggestions that I made in my previous review are satisfactorily considered in the new version, so I almost have nothing else to add.

Just one word of caution to the suggestion that Kraken is a good alternative to their bioinfomatic approach to identify species in DNA libraries. Kraken, like many other metagenomic classifiers, is rather prone to produce false positives (see, for example, Ye SH, Siddle KJ, Park DJ, Sabeti PC. Benchmarking metagenomics tools for taxonomic classification. Cell. 2019;178(4):779-794).

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Reviewer #1: No

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