Hybrid Illumina-Nanopore assembly improves identification of multilocus sequence types and antimicrobial resistance genes of Staphylococcus aureus isolated from Vermont dairy farms: comparison to Illumina-only and R9.4.1 nanopore-only assemblies
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Antimicrobial resistance (AMR) in Staphylococcus aureus is a pressing public health challenge with significant implications for the dairy industry, encompassing bovine mastitis concerns and potential zoonotic threats. To delve deeper into the resistance mechanisms of S. aureus , this study employed a hybrid whole genome assembly approach that synergized the precision of Illumina with the continuity of Oxford Nanopore. A total of 62 isolates, collected from multiple sources from Vermont dairy farms, were sequenced using the GridION Oxford Nanopore R9.4.1 platform and the Illumina platform, and subsequently processed through our long-read first bioinformatics pipeline.
Our analyses showcased the hybrid-assembled genome’s superior completeness compared to Oxford Nanopore (R9.4.1)-only or Illumina-only assembled genomes. Furthermore, the hybrid assembly accurately determined multilocus sequence typing (MLST) strain types across all isolates. The comprehensive probe for antibiotic resistance genes (ARGs) using databases like CARD, Resfinder, and MEGARES 2.0 characterized AMR in S. aureus isolates from Vermont dairy farms, and revealed the presence of notable resistance genes, including beta-lactam genes blaZ , blaI , and blaR . In conclusion, the hybrid assembly approach emerged as a tool for uncovering the genomic nuances of S. aureus isolates collected from multiple sources on dairy farms. Our findings offer a pathway for detecting AMR gene prevalence and shaping AMR management strategies crucial for safeguarding human and animal health.
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I'm satisfied that all reviewer comments have been address and/or explained and that this manuscript can now be accepted for publication. Thank you to both the reviewers and the authors for a positive and productive peer review.
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Both reviewers are positive about this manuscript and give some suggestions for minor edits to improve it and increase clarity. Please respond to each of their points. Please do amend the title to be more specific about which version of the Nanopore technology was used in this study as suggested by reviewer 1, and add the additional metadata as suggested by reviewer 2.
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Comments to Author
Chakrawarti et al. present a thorough demonstration of the use of hybrid (Nanopore and Illumina) sequencing to assemble Staphylococcus aureus genomes, with particular focus on 62 isolates collected from various sources from 23 Vermont dairy farms between 2003 and 2020. The authors use various characteristics (MLST, spa type, AMR presence/absence) to compare Nanopore-only, hybrid and Illumina-only assemblies, supported by phenotypic testing for antibiotic sensitivity and/or gold standard Sanger sequencing for MLST assignment. The manuscript is well-written, contributes to existing literature, and draws valid, well-supported conclusions. I only have a handful of minor revisions/suggestions, detailed below: 1. Methodological rigour, reproducibility and availability of underlying data The strategies …
Comments to Author
Chakrawarti et al. present a thorough demonstration of the use of hybrid (Nanopore and Illumina) sequencing to assemble Staphylococcus aureus genomes, with particular focus on 62 isolates collected from various sources from 23 Vermont dairy farms between 2003 and 2020. The authors use various characteristics (MLST, spa type, AMR presence/absence) to compare Nanopore-only, hybrid and Illumina-only assemblies, supported by phenotypic testing for antibiotic sensitivity and/or gold standard Sanger sequencing for MLST assignment. The manuscript is well-written, contributes to existing literature, and draws valid, well-supported conclusions. I only have a handful of minor revisions/suggestions, detailed below: 1. Methodological rigour, reproducibility and availability of underlying data The strategies chosen for data analysis are generally the gold-standards used by the sequencing community (e.g. Flye, Medaka for Nanopore assembly, ABRicate for identification of AMR genes), and the methods are described in good detail, including tool version numbers and specific commands where relevant. The supplementary tables provide useful additional data and metadata. Minor revision: The method states that Guppy v4.0.11 was used for Nanopore basecalling, but not which level of accuracy (fast, high accuracy or super accuracy) - this is important an detail, since the level used has a major influence on the resulting accuracy of the basecalled data Minor revision: Although the underlying sequencing data is available from NCBI, I couldn't find any metadata linking individual samples to their accession numbers, which is important information to have. Extra columns (Biosample accession, Illumina SRA accession and Nanopore SRA accession) should be added to Supplementary Table S1. without this, it is not easy to reproduce the results. Minor suggestion: As the samples came from 23 different farms, a column in the metadata (table S1) showing which farm (anonymised, if necessary) each sample came from could be useful 2. Presentation of results The results are well-presented and easy-to-follow. Minor suggestion: The manuscript could be further improved by including some consideration of any differences in findings between samples from different farms and/or different sample sources (e.g. human skin swabs versus milk). This extra analysis could take some time though, so this is only a suggestion, and could potentially be included in future work instead. 3. How the style and organization of the paper communicates and represents key findings Good, no suggestions. 4. Literature analysis or discussion The introduction is thorough and well-referenced. Likewise, the discussion is well supported by literature and well organised. Minor suggestion: The results present details about the average number of contigs assembled for each assembly strategy. This seems to assume that each S. aureus isolate's genome could have been assembled into a single contig, and therefore that even the long read strategy failed to assemble closed genomes in some cases. However, some of the isolates may have possessed plasmids, meaning the number of contigs assembled would always be higher than one - at least one contig for chromosome, and one per plasmid. Whilst this does not change the results of the analysis (which clearly shows that the long read strategy outperforms the short read), it might be worth mentioning in the discussion. 5. Any other relevant comments Nothing else to add!
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The article is a useful example of pathogen genomics for detecting MLST and AMR genes in Staphylococcus aureus within an agricultural setting. I think the methods are sound and the presentation is good. Unfortunately it is now outdated that the conclusion of hybrid assemblies improve upon nanopore-only assemblies for this application. The new R10.4.1 nanopore flowcells show nanopore-only assemblies are comparable to hybrid assemblies. Please see our recent pre-print (https://www.biorxiv.org/content/10.1101/2024.01.12.575342v1) which shows this. I still think the article is valid for publication, but attention must be drawn to this in the title and text. Please change the title to something similar to 'Hybrid Illumina-Nanopore assembly improves identification of multilocus sequence types and …
Comments to Author
The article is a useful example of pathogen genomics for detecting MLST and AMR genes in Staphylococcus aureus within an agricultural setting. I think the methods are sound and the presentation is good. Unfortunately it is now outdated that the conclusion of hybrid assemblies improve upon nanopore-only assemblies for this application. The new R10.4.1 nanopore flowcells show nanopore-only assemblies are comparable to hybrid assemblies. Please see our recent pre-print (https://www.biorxiv.org/content/10.1101/2024.01.12.575342v1) which shows this. I still think the article is valid for publication, but attention must be drawn to this in the title and text. Please change the title to something similar to 'Hybrid Illumina-Nanopore assembly improves identification of multilocus sequence types and antimicrobial resistance genes of Staphylococcus aureus isolated from Vermont dairy farms compared to R9.4.1 nanopore-only assemblies'. And preface the early mentions of ONT/Nanopore with R9.4.1 so readers understand the context. One further question is why wasn't unicycler used for the hybrid assemblies but was used for the Illumina only assemblies? Was your method of hybrid assemblies more accurate than unicycler? Please show evidence if the case.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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