Peer Review History

Original SubmissionApril 2, 2026
Decision Letter - Katherine Kokkinias, Editor

-->PONE-D-26-06953-->-->Study Protocol on Antimicrobial Resistance Burden, Transmission Dynamics, and Therapeutic Bacteriophages in Livestock and Exposed Farming Populations in Nagpur, India: An Integrated One Health Approach.-->-->PLOS One

Dear Dr. Kashyap,

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.

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Upon reviewing your manuscript, we have a few additional requests regarding the reporting for whole genome sequencing. Please include the following information:

  1. The facility that will be performing the whole genome sequencing, the type of flow cell, and the type of read pairs (i.e. 151-bp paired-end reads)
  2. The version of the tools that will be used and appropriate citations for those tools
  3. Details regarding phage genome assembly including the tools that will be used, version, flags

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Staff Editor

PLOS One

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Revision 1

Comment 1: The facility that will be performing the whole genome sequencing, the type of flow cell, and the type of read pairs

Response: We thank the reviewer for highlighting this important point. Whole-genome sequencing (WGS) will be performed at MedGenome Labs Pvt. Ltd., Bengaluru, India a certified high-throughput sequencing facility using the Illumina NovaSeq 6000 platform. Sequencing will be carried out using an S4 flow cell, generating paired-end reads of 150–151 bp (2 × 150/151 bp). A minimum sequencing depth of ≥30× will be targeted for bacterial genomes, while higher coverage (≥100×) will be used for bacteriophage genomes to ensure accurate assembly.

Comment 2: The version of the tools that will be used and appropriate citations for those tools

Response: We thank the reviewer for this valuable suggestion. Bioinformatics analyses will be performed using well-established and version-controlled tools. Raw sequencing reads will be quality assessed using FastQC (v0.11.9) and trimmed using fastp (v0.23.2). De novo genome assembly will be conducted using SPAdes (v3.15.5), and assembly quality will be evaluated using QUAST (v5.2.0). Genome annotation will be performed using Prokka (v1.14.6) and RASTtk. Antimicrobial resistance genes will be identified using ResFinder (v4.1), and sequence similarity searches will be conducted using BLAST+ (v2.13.0). Protein domain analysis will be performed using HMMER (v3.3.2). Phylogenetic analyses will be carried out using Snippy (v4.6.0), Gubbins (v3.2.1), and IQ-TREE (v2.2.0). Functional resistome profiling will be performed using the nf-core/funcscan pipeline (v3.0.0). All tools will be used with default parameters unless otherwise specified.

Appropriate citations for all tools have been included in the revised manuscript.

Comment 3: Details regarding phage genome assembly including the tools that will be used, version, flags

Response: We thank the reviewer for this insightful comment. Bacteriophage genome assembly will be performed using SPAdes (v3.15.5) in --careful mode to minimize assembly errors, with multiple k-mer sizes (e.g., 21, 33, 55, 77). For complex datasets, metaSPAdes may be used. Raw sequencing reads will be quality-checked using FastQC (v0.11.9) and trimmed using fastp (v0.23.2) prior to assembly. Assembly graphs will be visualized using Bandage (v0.8.1) to confirm genome completeness and potential circularization.

Phage identification and completeness assessment will be conducted using VirSorter2 and PHASTER. Genome annotation will be performed using Prokka (v1.14.6) and cross-validated using RASTtk, with further functional annotation using BLAST+ (v2.13.0) and HMMER (v3.3.2). To ensure suitability for therapeutic applications, phage genomes will be screened for the absence of lysogeny-associated genes, virulence factors, and antimicrobial resistance genes using ResFinder, CARD, and manual curation.

Attachments
Attachment
Submitted filename: Response to Reviewers.pdf
Decision Letter - Katherine Kokkinias, Editor

Study Protocol on Antimicrobial Resistance Burden, Transmission Dynamics, and Therapeutic Bacteriophages in Livestock and Exposed Farming Populations in Nagpur, India: An Integrated One Health Approach.

PONE-D-26-06953R1

Dear Dr. Kashyap,

As a Peer-Reviewed Funded Protocol, your submission is eligible for expedited review by in-house editors. Based on our evaluation, we are satisfied that your manuscript meets our publication criteria for Study Protocols, and is therefore considered to be suitable for publication subject to final journal requirements

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Kind regards,

Katherine Demi Kokkinias, Ph.D.

Staff Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Formally Accepted
Acceptance Letter - Katherine Kokkinias, Editor

PONE-D-26-06953R1

PLOS One

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PLOS ONE Editorial Office Staff

on behalf of

Dr. Katherine Demi Kokkinias

Staff Editor

PLOS One

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