PONE-D-24-15864Effects of storage conditions on the microbiome of fecal samples collected from dairy cattlePLOS ONE

Dear Dr. Jaramillo-Jaramillo,

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PLOS 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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

Reviewer #2: No

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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

Reviewer #2: 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: Statements and declarations:

Ethics: Field research was performed, but no permit details or authorisation was given in the section provided. Please include a statement if such permits/approvals were not required in your setting, or provide the approvals for the connected study for which the farms were enrolled.

Title:

In the last decade, efforts have been made to standardise microbiome terminology. Following these efforts, it would be more appropriate to refer to the “microbiota” instead of “microbiome” in this study, as the entire system and environment were not considered, but rather just the effect on the microbes themselves.

Introduction:

Lines 54 – 55: “Ribosomal” is more commonly used. Please do not omit articles like “the”, e.g., “the resistome”, “the mobilome”.

Methods:

Line 90: Was this temperature tested and proven to be stable for the length of time samples hung out, especially those from NS? It also matters whether the samples were directly on the ice packs or not. Depending on the quality of the box, we have found that temperatures can actually vary substantially, and could, arguably, have “worse” outcomes than having them at a semi-constant RT.

Line 95: Could you make it easier for the readers by specifying that it is the first 11 sample “sets” that arrived from the enrolled farms, and not individual samples?

Lines 99 – 108: Freezing the 3 day fridge subsample and not the 7 day fridge subsample has the potential to introduce bias, because the samples were treated differently – by one set going through a freeze-thaw and the other not. Freeze-thaw should be avoided in general, which is why a lot of stool extraction kits recommend not thawing frozen samples before extraction. I understand this was for batching purposes, but you need to keep in mind when analysing the data.

Lines 109 – 111: Why weren’t the 260/230 values considered? High fiber stools can often present difficulties for extractions and result in “dirty” DNA. While Illumina is more robust to this than some other chemistries, it is still a good measure of whether sequencing would have worked or not.

Lines 125 – 128: As far as I’m aware, GreenGenes was last updated in 2013. While QIIME2 often uses GG in their tutorials, in part because it is smaller and easier to compute, it would be hard to motivate to use such an outdated database as reference, for a paper up for review in 2024. I’d recommend switching to SILVA (there should be classifiers available for your version of QIIME2) or RDP.

While it is not incorrect, I would be wary of using the term OTU to describe biological taxonomic assignments, due to the potential confusion with the clustering outputs OTU vs ASV.

Line 143: Had to jump to results to check, but since the sequencing kit would have allowed up to 100 000 reads per sample I find it strange that the reads were so low per sample. Was this expected given results from other studies? The rarefaction depth is not “low” in the general sense, just unexpected given the potential output that the kit could have given, and the generally high bacterial load in the sample type used.

General methods comments: From past experience, it is easy to fall into this trap of not describing the sequencing and bioinformatics parts of the methods well enough. The methods here (especially in terms of sequencing pre-processing and analysis) really need more description for it to truly be reproducible. A lot of this could go in the supplementary methods to avoid clogging up the main text.

Some examples of what is missing:

Preprocessing: did you demultiplex, did you do any pre-QIIME2 QC, how was trimming of adapters/primers done? Which parameters were passed to DADA2? Which data did you take from QIIME2 to import and use in STATA, R, etc.? It is not clear that the taxonomic assignment was also done in QIIME.

What were your sequencing controls: Contamination is a huge issue in microbiome studies, so this needs to be addressed – you always need a negative(s)! Did you test your bioinformatics pipeline with a mock community control to ensure that it works properly?

Results:

Line 150: What do you mean with could not be sequenced? Did it fail library preparation or sequencing QC? How many samples were left in each group? Perhaps this information could be better represented in a table with the read ranges and things.

Lines 161 – 162: Figure labels 2B and 2C should be 1B and 1C.

Beta diversity: Would the immediate freezing not be better suited as a “control” to do comparisons against?

Table 1: Where is the adjusted p after Bonferroni correction column ?

Line 199: Relative abundance is not a good measure of taxonomic change on it’s own. Differential abundance, as compared to the “standard” of freezing immediately or another sample assigned as control, should be performed. Also think about investigating deeper than just Phylum level. Diff abundance can be done in QIIME2 (ANCOM, ANCOM-BC, gneiss) or R (various packages).

Discussion:

Line 249: Check the autocorrect typo for OTU.

Line 273 on: If F/B ratio is in the discussion, it should be introduced in the results in the taxonomy section first.

The discussion does not touch enough on WHY variability in results due to storage is a problem and should be countered where possible. Just one example is the fact that knowing that if certain microbes are affected more than others, it could mask or skew data, like which functions or resistance profiles can be detected or predicted in downstream analysis,.

Figures: It’s potentially just the version I received, but all the figures are a bit fuzzy. Why are figures 1 and 2 greyscale, when all the rest are in colour? It is harder to discern between groups with the greyscale.

Reviewer #2: Manuscript: Effects of storage conditions on the microbiome of fecal samples collected from dairy cattle

This study addresses a crucial and underexplored issue in bovine research: the effect of different storage conditions on the microbiota of dairy cattle. The aim was to investigate the effects of refrigeration at 4°C for three and seven days, as well as ethanol preservation, on the microbiota diversity of pooled fecal samples, with immediate freezing at -80°C after collection serving as the control. Establishing practical protocols for storing and transporting fecal samples from farm animals is essential, as researchers often face challenges in deciding the best strategies for collecting samples from farms located far from research laboratories. This study provides valuable information on the impact of refrigeration and ethanol preservation on fecal samples from cattle for microbiota studies.

Title: Change microbiome for microbiota and throughout the manuscript.

Introduction:

Line 46 to 62. This review can be condensed into a single paragraph (most information is already well known), concentrating on the knowledge gap that this study aims to address would be of importance.

Several studies in human and veterinary medicine (including dogs, cats, and horses) have evaluated the effect of different storage conditions on fecal and ruminal microbiota (e.g., Moossavi et al., BMC Microbiology, 19(1), 145; Granja-Salcedo et al., PLOS ONE, 12(4), e0176701; Barko et al., PLOS ONE, 19(2), e0294730). Acknowledging this previous work is recommended; however, it is even more important to clearly state how the present study differs from previous research to help the reader understand its significance. For instance, a previous study (Song SJ, mSystems. 2016;1(3):e00021-16) strongly recommend against 75% ethanol for preservation of microbiota, so here in the introduction the authors can hypothesise whether in cattle 75% ethanol would perform similar or different.

Line 77: Additionally, the introduction of the manuscript fails to explain the importance of establishing a practical storage and transportation method for pooled sampling in bovine research. Also explain when pooled samples could be (or are) used in bovine microbiota research

Line 77: any hypothesis?

M&M

Line 124: Was the location of the farm considered in the statistical models for alpha- and beta-diversity? Samples from NS were kept refrigerated below 8°C for 24 hours, while those from PEI were kept refrigerated below 8°C for only 3 hours. Studies in horses and dogs have shown changes in fecal microbiota after 6 hours of temperature exposure. Could the storage duration before processing have affected the microbiota analysis?

Results:

Line 161: this sentence appears to be incomplete.

Line 172: What is the meaning of largest discrepancies with less variability? Restate please

Line 190: Please discuss why the effect of storage condition was different for each of the b-diversity measures used?

Line 199: Please explain why the taxonomy analysis was performed only at the phylum level and not at the genus level. In this study, the V6-V8 region of the 16S rRNA gene was amplified, yielding DNA fragments of approximately 400-500 bp. This length allows for genus-level analysis, which could determine which genera within each phylum are affected by the storage conditions.

Discussion

Line 225: any reference supporting this statement? Also, which bacteria are more likely do increase or decrease with the type of storage and preservation method used. do the results of the present study support previous findings?

Line 227: what about in species with similar diets and likely similar gut microbiota?

Line 235: “significant varied”? increase or decrease?

Line 236: any idea why?

Line 261: which species? All or in which species has been the effect of refrigeration being investigated?

Line 262 – 268: in practical terms and based on your general objective, is it Ethanol 70% recommended or no for preservation of bacterial DNA?

Line 269 – 272: is there any studies in calves or adult cattle showing the effect of farm on the fecal microbiota?

Any study limitations?

Figure 1 and 2: consider using color in the figure.

Are the dataset Publicly available? If so, provide information about where and project number.

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Reviewer #1: No

Reviewer #2: Yes: Diego Gomez

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PONE-D-24-15864R1Effects of storage conditions on the microbiota of fecal samples collected from dairy cattlePLOS ONE

Dear Dr. Jaramillo-Jaramillo,

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.

Please submit your revised manuscript by Aug 10 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Timothy Omara, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #1: (No Response)

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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

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 #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: I find it odd that a group of authors' who state that their expertise is not bioinformatics, would undertake to write a manuscript based almost entirely off of bioinformatics analyses, and not bring onboard an expert? It is not that the analysis was poorly done, it is just that you should not use not being an expert as an rebuttal to leave out details needed for reproducibility of the protocol.

For future studies, it is the responsibility of the authors to ensure that they are familiar with the workflow of the sequencing provider and that correct procedure is followed - from experience one cannot assume that providers would do the "proper" things. Surely it would have been possible to have reached out and asked the provider what kind of controls they had instead of rebutting saying you assume they did?

Lastly, while the focus of the study was not to present a robust microbiota profile, I would argue that in order to properly represent the changes happening as a result of the conditions the samples were exposed to, other experimental factors such as contamination (which could skew results) should have been been controlled for, and the data QC and taxonomy assignment be as robust as possible. Differential abundance is also not hard to do in QIIME2, and could have easily been applied to see which, if any, phyla are differentially abundant and therefore the main drivers of changes due tot the conditions.

As adding controls is clearly not possible at this stage, please at least add a statement indicating that extraction negative controls and positive controls were not included in the protocol and please consider performing more robust analyses in future. It would only serve to strengthen the results you see, regardless of the focus.

I accept the remainder of the changes and/or rebuttals to my comments.

Reviewer #2: Thanks to the authors for addressing the reviewer's concerns. This reviewer does not have additional comments.

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Reviewer #1: No

Reviewer #2: No

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Effects of storage conditions on the microbiota of fecal samples collected from dairy cattle

PONE-D-24-15864R2

Dear Dr. Jaramillo-Jaramillo,

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,

Timothy Omara, PhD

Academic Editor

PLOS ONE

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