PONE-D-25-08885Evaluation of shotgun metagenomics as a diagnostic tool for infectious gastroenteritisPLOS 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

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

Reviewer #1: 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: 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

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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: In this manuscript, the author compared the applicability of shotgun metagenomic and PCR for the diagnosis of infectious gastroenteritis. This is very relevant and will help improve diagnostic protocols and routines. The experimental setup appears reasonable, and the authors present their findings effectively. There are some points that I would like to discuss before making any decision.

• In this work, misclassification is closely linked to mobile genetic elements, which may be one reason. However, it is not the only one. Another possible reason may be the microbial abundance in metagenomics. Regardless of the reason, there is insufficient evidence in this manuscript to support mobile genetic elements as a cause of misclassification, and I believe this assertion is too strong to be made.

• The authors emphasize that bioinformatics analyses could impact the metagenomic results. This is true. However, I believe the manuscript does not provide sufficient evidence to support this claim. For instance, both the assembler and the binner could have a significant impact. In this manuscript, the authors chose to test only Megahit and Metabat2. It has been shown that both assembler and binner behaviour differ when they are used for low and high coverage data. So, preferring one over the other is very challenging in metagenomics. Indeed, many publications have suggested a multi-tool approach. Additionally, there are many different taxonomy tools; however, only Kraken2 and its associated tools were tested. Thus, although the statement is correct, the evidence is insufficient.

• I believe the manuscript will benefit from constructing a confusion matrix that includes both the false positive and negative rates for sequencing as a potential diagnostic method compared to the traditional routine method.

• What is missing from the discussion is the applicability of long-read sequencing to overcome the limitations in the field of metagenomic assembly, especially when it comes to mobile genetic element and their repetitive regions.

Please provide a percentage of unclassified reads for Kraken.

Please provide the percentage of unique mapped reads, depth of coverage, and breadth of coverage after mapping the reads back to assemblies (some of these values are provided but not in percentages).

The authors declared that they mapped the sequencing reads against the host genome where any actual maps were removed. This sounds like a correct approach; however, the authors did not specify the percentage of the host genome contamination.

L 415 / L 421: final spiked concentration is contrasting; please double-check.

L 440: One of the discussed points in this manuscript was the DNA extraction technique and its effectiveness. While I agree with the authors, unfortunately, the current work only presents DNA concentration and A260/A280 ratios. It would be nice to show the A260/A230 as well.

L 493 and L502: any reason to only use Megahit and Metabat2? It is hard to prefer one over the other, specifically for assemblers. Some assemblers performed better for low coverage data.

L 495: Why are authors considered co-assembly?

L 503: At this stage, it is uncertain whether the authors considered all the MAGs for downstream analyses or only selected medium and high-quality MAGs. Please also provide statistics on the quality of the MAGs.

L 508: Why did the authors suddenly decide to use GTDb-Tk for the taxonomy classification of MAGs?

L 526: What was the justification to shift to SPAdes for assembly?

L 527: How is percentage coverage calculated? The method is not consistent across the literature, so please provide more details.

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Reviewer #1: No

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Evaluation of shotgun metagenomics as a diagnostic tool for infectious gastroenteritis

PONE-D-25-08885R1

Dear Dr. Haugum,

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,

Adriana Calderaro

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 #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 #2: Yes

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

Reviewer #2: Yes

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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 #2: Yes

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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 #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 #2: The authors have satisfactorily addressed all reviewer comments in the revised manuscript. I therefore recommend the manuscript for acceptance.

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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 #2: No

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