<xhtml:span xmlns:xhtml="http://www.w3.org/1999/xhtml" xml:lang="en">Genomic analysis of the progenitor strains of <em>Staphylococcus aureus</em> RN6390 </xhtml:span>
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RN6390 is a commonly used laboratory strain of Staphylococcus aureus derived from NCTC8325. In this study, we sequenced the RN6390 genome and compared it to available genome sequences for NCTC8325. We confirmed that three prophages, F 11, F 12 and F 13, which are present in NCTC8325 are absent from the genome of RN6390, consistent with the successive curing events leading to the generation of this strain. However, we noted that a separate prophage is present in RN6390 that is not found in NCTC8325. Two separate genome sequences have been deposited for the parental strain, NCTC8325. Analysis revealed several differences between these sequences, in particular, between the copy number of esaG genes, which encode immunity proteins to the type VII secreted anti-bacterial toxin, EsaD. Single nucleotide polymorphisms were also detected in ribosomal RNA genes and genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM) between the two NCTC8325 sequences. Comparing each NCTC8325 sequence to other strains in the RN6390 lineage confirmed that sequence assembly errors in the earlier NCTC8325 sequence are the most likely explanation for most of the differences observed.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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The reviewers and I agree that your work comparing the genome sequence of Staphylococcus aureus RN6390 to available sequences for NCTC8325 is important given the widespread use of the former in laboratory work. The manuscript is well presented and will be of interest to readers of Access Microbiology and the wider microbiology community. The reviewers have suggestions for small amendments that will improve the readability of the manuscript that I would like you to consider, where appropriate, in a revision.
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Comments to Author
Overall Summary and Impressions: This paper aimed to show that the Staphylococcus aureus NCTC8325 that has been sequenced by the Wellcome Sanger Institute should be used as the reference genome for NCTC8325-derivative strains, such as the commonly used laboratory strain RN6390. Previous studies often reference the NCTC8325 genome that has been sequenced by the University of Oklahoma Health Sciences Center, but the authors were able to show genetic differences between this reference genome and RN6390. Although this paper is short, it highlights the importance of going back to the most frequently used laboratory strains and sequencing their genomes, to make sure that important information has not been missed out when these strains are being used for reference. However, I do have the following concerns …
Comments to Author
Overall Summary and Impressions: This paper aimed to show that the Staphylococcus aureus NCTC8325 that has been sequenced by the Wellcome Sanger Institute should be used as the reference genome for NCTC8325-derivative strains, such as the commonly used laboratory strain RN6390. Previous studies often reference the NCTC8325 genome that has been sequenced by the University of Oklahoma Health Sciences Center, but the authors were able to show genetic differences between this reference genome and RN6390. Although this paper is short, it highlights the importance of going back to the most frequently used laboratory strains and sequencing their genomes, to make sure that important information has not been missed out when these strains are being used for reference. However, I do have the following concerns (mostly minor): Line 59 - I don't think the ' in 'University of Oklahoma Health Sciences Center' is needed. Labels within figures. Adding labels to figures would greatly help for easy reading. For example, the genome alignments in Figures 2a and c can benefit from having labels (within the figure themselves) that designate that NCTC8325-Oklahoma is at the top and RN6390 is at the bottom. Lines 91-92 - It is unclear whether the large region being talked about is the Staphylococcus phage 6390, please revise the statement. Line 98 - Staphylococcus phage SAP40 doesn't seem to be in the phylogenetic tree (Fig2b), so I'm not sure what the authors are referring to in this line. Line 104 - Table S1 doesn't seem to have the ISP479c vs RN6390 table Discussion - it might be good to add a bit more on how the repetitive nature of the genes is due to sequence assembly error for short reads (other studies?)
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
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 authors have compared available genome sequences for the Staph aureus RN6390 progenitor strains and identified errors in publicly available genomes. This is important for researchers working with these isolates and basing their work on their available genomes. It is also a lesson to all researchers to consider potential errors in genomes. 1. Methodological rigour Line 74 - more detail on what kind of enhanced genome sequencing Line 79 - more information on parameters used for Snippy v4, and reference needed Line 82 - full stop needed 2. Presentation of results Fig 2a and 2c - more clarity regarding what these figures show as it is not clear for those unfamilar with progressiveMAUVE outputs Line 104 - 'overall very few' specify how many SNPs and highlight this on Fig. 2c Line 105 - 107 - where …
Comments to Author
The authors have compared available genome sequences for the Staph aureus RN6390 progenitor strains and identified errors in publicly available genomes. This is important for researchers working with these isolates and basing their work on their available genomes. It is also a lesson to all researchers to consider potential errors in genomes. 1. Methodological rigour Line 74 - more detail on what kind of enhanced genome sequencing Line 79 - more information on parameters used for Snippy v4, and reference needed Line 82 - full stop needed 2. Presentation of results Fig 2a and 2c - more clarity regarding what these figures show as it is not clear for those unfamilar with progressiveMAUVE outputs Line 104 - 'overall very few' specify how many SNPs and highlight this on Fig. 2c Line 105 - 107 - where 'it's plausible' is this written because information is not known? If this is the case, this should be referenced or made clearer, perhaps in the Introduction. Line 103 - authors reference Fig. 1a but this isn't present in the manuscript - it should just be Fig 1 Fig 2b - outer ring is labelled 'Firmicutes' but taxonomy needs updating to 'Bacillota' Line 214 - 'represents indicates' needs to be updated Fig 3 legend - no title given Line 120 - T7SS is in italics - please check this Line 120 - capital letter on 'indicated' 3. How the style and organization of the paper communicates and represents key findings 4. Literature analysis or discussion Line 147 - comment on the accuracy of long-read PacBio sequencing compared to short-read sequencing and enhanced sequencing done by authors Line 147 - 'C' missing on 'NCT8325' Line 154 - T7SS is in italics - please check this 5. Any other relevant comments
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
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
The following are the participants of the Microbiology Society's ECM Forum Summer Conference 2022 who collaboratively reviewed this article
Antonia Cutts, Karolina Pyrzanowska, Isabel Parreira, Raphael Galleh, Adrian Campey, Aoife Mulry, Emilene S. Morais, Eva Bernadett Benyei, Charlotte Platel, Charlotte Head, Jack Ferguson, Ella Rodwell, Imran Khan
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