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The manuscript has been well-reviewed and revised accordingly, which is ready for publication now.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
I do thank the authors for integrating the suggested changes, and also especially for the better structured response in the last round of reviews.
In my opinion, this manuscript is ready for publication, and I do congratulate the author's to their work. It is very important that such methodological work is performed.
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Please follow the reviewers' comments and address the issues.
I thank and commend the authors for addressing all the concerns and suggestions raised during the first review round. I believe the manuscript is now ready for publication.
No further comments.
No further comments.
Review of «Impact of storage and extraction methods on peat soil microbiomes (#98263)»
General remarks:
In general, the reviewer’s questions were accurately addressed and the manuscript has developed in a positive way.
1. However, some issues remain, notably with the discussion section. Currently, the discussion section includes many results, and notably also results that were not presented in the result section. One example is the analysis on the Euryarchaeota. This has to be changed, as the discussion section is intended to treat only results that were previously presented, and to connect them with the literature. The paragraphs at lines 517-531, and L582-586 do for example not link the obtained results with the existing literature. These two paragraphs are given here as examples, but similar issues, which need to be addressed, exist also in other sections of the discussion.
2. Reviewer 2, minor comments 1: While I trust the authors that the samples come from three distinct habitats with different physico-chemical properties, little evidence is presented support this claim. The only change after the reviewer’s request was the addition of a remark specifying the general differences of C/N ratio and organic humification indices among the studied habitats (L142,143), as well as porewater pH, which is the main driver of soil microbiome structures. However, porewater pH did not change between bog and fen (L141,142), indicating that the habitats may not be chemically, and microbially as distinct as claimed.
3. Answer to reviewer 4, Experimental design, a): I do not see the changes made in the manuscript.The explanation, why 4.5g of peat were used for one experiment, while 8.9g were used for the other needs to be provided within the manuscript. Or do the 8.9g include the weight of the LG buffer? A change within the manuscript is needed to clarify this.
4. While I acknowledge the liberty of authors to choose the used terminology, I think it is not debatable to use defined terminologies in a scientific manuscript. Therefore, I do request the following adaptations to the manuscript:
- Inclusion of a definition of the term ‘humics’ within the manuscript; so far, only the term ‘humic acid’ is defined (L72-73). Given recent scientific debates questioning the nature of ‘humic acids’ (Lehmann and Kleber, 2015), it may be more accurate to avoid the term ‘humic acid’ and ’humics’ in certain contexts (e.g. L77,78 “Peat soils are […] high in humic acidcs”). Lehmann and Kleber (2015) explain that ‘humic acids’ do generally not occur naturally in soils, but correspond to analytical artifacts created during the alkali extraction. Of course, DNA extraction procedures could have similar consequences depending for example on the pH of the DNA extraction buffers, which may cause negative effects on downstream molecular analyses.
- Avoidance of terms that do not make much sense: use ‘Bray-Curtis dissimilarity’ or ‘Bray-Curtis distance’ but not ‘Bray-Curtis dissimilarity distance’ (legend Figure 2). If the authors insist on the used terminology a short justification is needed within the manuscript.
While this is a minor error, it is unpleasant from the reviewer perspective, that this error was not corrected, despite being mentioned in the first review.
- Use same spelling for the same terms: RNALater and RNAlater are used; Lifeguard and LifeGuard (this was also already pointed out in the first review).
- There are also some further inconsistencies, which should be avoided, e.g., sometimes kits are called ‘Mobio PowerMax’ (L173(, sometimes just ‘PowerMax’ (), sometimes “Qiagen’s PowerMax” (L183,184). While this does not confound meaning, it does decrease the clarity of the manuscript, which is a pity. It is also such inconsistencies, which were criticized in the first review round, and which were apparently not corrected.
- Also regarding terminology: it is not clear to me why the abbreviation ‘RC’ was chosen. Probably, a short explanation would increase clarity.
5. Storage in LG is shown to reduce the ASV richness by 42% in average. Therefore, it is not clear to me, why the LG storage method was selected for the second experiment testing different extraction procedures. This needs to be justified within the manuscript. This choice and potential consequences, e.g., testing the different extraction procedures on a subset of the community, need also to be covered within the discussion.
Finally, I would like to note that the review process of this revision was unnecessarily complicated. Notably, it was in general difficult to precisely check, what the authors changed following specific requests. This was mainly due to wrong line numbers that were provided (shift by about 4 lines), or due to the lacking indications of line numbers in the responses (e.g., response to minor comment 2 of reviewer 3, most answers to reviewer 4). Ideally the changes should be traceable within the answers of the authors.
I would appreciate if the authors could provide more carefully checked documents in the future to facilitate the peer-review process.
Specific comments:
E.g. L168, L171, L593: check “-80”, “-80C” “-80 deg” throughout the manuscript. The same with “-20”.
Figure 1: I think the figure has improved in response to the reviewer’s requests. However, it is misleading to include “3 ways of preservation” under the second experiment.
References:
Lehmann, J., & Kleber, M. (2015). The contentious nature of soil organic matter. Nature, 528(7580), 60-68.
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Please revise the manuscript by following the reviewers' comments. A point-by-point response letter is required when re-submitting your revised manuscript.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
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First of all I want to thank you for doing the work assessing these methodological impacts and doing the effort to publish, as this is a great service for the research community. Secondly, I want to compliment you on the thoroughness of the work. It was clearly written, embedded in the relevant literature, and clearly structured, with clear and easy to interpret figures. I don't think I have ever reviewed a manuscript where I had essentially nothing to contribute as it was so thorough and high quality work. So, my apologies for not being able to make recommendations for improvement as I saw none. Normally I write a laundry list of suggests edits and reflections, but I've got nothing...
I am writing to provide my review of the manuscript titled "Impact of storage and extraction methods on peat soil Microbiomes" submitted by Cronin et al. to PeerJ. Overall, the study presents valuable insights into the effects of storage and extraction methods on peat soil microbiomes, offering potential resources for the scientific community. However, I have identified several major concerns that need to be addressed before publication.
- Clarity of Microbiome Target and Methodology: The manuscript lacks clarity regarding the primary microbiome target. It is crucial to specify whether the study primarily focuses on bacterial communities, archaea, fungi, or eukaryotes. Given the diverse impacts of extraction kits and preservation methods on different microbial communities, specificity in targeting is essential. Additionally, confirming the primer sets used for amplification (e.g., 16S rRNA for bacteria and archaea, 18S rRNA for eukaryotes, ITS for fungi) is necessary to ensure methodological accuracy.
- Functional Genomics Analysis: Further analysis using tools like PICRUSt or MG-rast to investigate functional genomics is recommended. While the manuscript acknowledges the potential impact of storage and extraction methods on data interpretation, additional analysis could provide deeper insights into functional genomics, aiding in understanding ecological variation and relating to expression data.
- Identification of Optimal Methods: As a technical paper, the manuscript should aim to identify the most effective method/s for future studies. While no significant differences between preservation methods were observed, clarity on which extraction method/kit is superior for DNA extraction is lacking. Providing recommendations for future research or practitioners utilizing these methods would enhance the manuscript's utility.
Minor Comments:
- Clarification on the software version used for analysis (e.g., Qiime2) is needed.
Explanation for trimming the data to 250bp (e.g., quality control, primer removal) should be included.
Adjusting the Y axis in Figure 4 to represent cumulative percentage of 100% would improve clarity.
Confirmation if all phyla mentioned in the legend of Figure 4 belong to bacteria or other microbil communities is necessary.
Overall, while the manuscript addresses an important topic, addressing the aforementioned concerns is vital for enhancing its clarity and scientific rigor. I recommend revisions based on the provided feedback to improve the manuscript's quality and impact.
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Cronin et al. examine how storage and DNA extraction methods affect microbial profiles from climate-sensitive peatland ecosystems. Peatland microbiomes are crucial to climate, yet molecular extraction techniques are rarely standardized and benchmarked. The authors highlight biases in diversity metrics introduced by storing samples in Lifeguard buffer and extracting DNA using specific kits, which I believe would be useful to the peatland and soil microbiome science community. Overall, the manuscript is clear and well-written. I commend the authors for taking a systematic approach with best practices for addressing the research gap, testing reproducibility when required, and making solid arguments. I also appreciate them for considering the possibility of batch effects in a transparent manner. However, I suggest the following for improving the quality of the manuscript, prior to acceptance.
The research gap being addressed is meaningful and of interest to the community. The experiment design used is robust and sufficient to answer the research questions raised. The methods and discussion can benefit from additional details as stated below.
The findings presented in the article are valid, with caveats sufficiently discussed. I have provided some points to be addressed below, which I believe can help improve the quality of the manuscript.
Major comments:
1. The experiments would have benefitted from the use of community standards/spike-ins to objectively evaluate differences/biases introduced by preservation/extraction methods. It’s difficult to assess differences in microbial profiles without baselines. I suggest that the authors add text to discuss this caveat.
2. DNA yield is often affected by storage duration. Can the authors describe the time under storage prior to extraction? Can the authors also comment about randomization prior to extractions?
Minor comments:
1. The authors highlight in their abstract (lines 45-46) and later in their introduction that they use samples from habitats with distinct physiochemical conditions. Apart from Supplementary Table 1, I recommend adding additional text in the methods section to describe these differences.
2. I found it difficult to understand the difference between the kit stated in Line 167 and S kit. My understanding is that the former is a high humic acid protocol while the latter is not. Is this correct? If so, can this be rephrased for better clarity?
3. Can the authors comment on the specific chemical components of the buffers discussed in Lines 456-457 on their results? This will enable groups using other buffers to evaluate these results.
4. Can the authors in lines 440-441 comment on quality/yield being sufficient or otherwise for long read sequencing?
5. Please revise Figure 1. It's unclear here that the storage conditions were extracted only using the S method & that LG-preserved samples were extracted using all five different methods.
6. Can the authors consider increasing the symbol size for Figures 2, 3, and similar supplementary figures?
7. For Figure 4, please state in the legend for panel D what the rows and columns signify, distance/method used for row/column clustering and normalization used.
8. Can the authors consider re-plotting Figure 5. It was hard for me at least to examine the box plots embedded within the violin plots. Stating sample size here would also be helpful.
In general, the manuscript reads well and is relatively clearly structured. However, as pointed out below, several changes are required to improve the quality and clarity of analyses and corresponding deductions.
The length of the different sections seems disproportional to me: Introduction (1.5 pages), material and methods (6 pages), results (2 pages), discussions (2.5 pages); 5 figures and 0 tables.
The introduction of nucleic acid extraction from soil samples is shallow, as it only enumerates selected commercial kits. It would be better to introduce the basic principles of DNA extraction from soil, and which factors differentiate the different commercial kits. Furthermore, it is shortly mentioned in the method section (L149-151), that problems can arise when production of commercial kits are discontinued. This may pose a significant problem to long-term studies or long-term monitoring systems, and may merit to be mentioned more prominently than at present in the method section.
Besides a common R package, the newest references are from the year 2020. The introduction and discussion are therefore not up-to-date, which needs to be changed.
Not enough care was taken during the revision of the manuscript, some examples include:
- Inconsistent wording/lacking definitions of some terms, e.g., community structure (e.g. L54) vs community composition (L42) vs community profile vs microbiome profile (L102) vs community variation (L55); humic acids (L72) vs humics (L73); whole-community dissimilarity (L379) vs community composition differences (L381) vs Bray-Curtis dissimilarity distances (Figure 2). Personally, I like the terms community composition for presence-absence analyses, community structures for abundance-weighted analyses; and, I do also have a preference for pairwise community dissimilarity rather than pairwise community distance, due to the non-euclidean nature of the main dissimilarity metrics used for microbiome analyses.
- In my opinion, the word appreciable is used too many times. More precise statements would be preferable.
- L500, Figure 4: “the the”
- Two abbreviations are used for liquid nitrogen (LN and LN2)
- Lifeguard/LifeGuard is not always abbreviated to LG
- 4700xg (L182) vs. 4,700 X g (L185) vs 4,700 xg (L196)
- L516-L519: These are results not discussion.
The quality of figures is low; the resolution needs to be improved.
The research questions should be stated more clearly.
From what I understood, the experimental design has been well chosen. However, at times it is hard to follow the description in the method section. Points to clarify include, for instance:
a) L126: “~4 ml (equivalent to ~2g) of peat”, but L152: “4.5g peat […] or 8.9g of peat with Lifeguard”; how much was sampled?
b) I suggest to separate the two experiments, i.e., test of storage conditions and test of extraction methods, in the figure 1 as well as in the method section. Also, all DNA extraction methods should be mentioned in the same section of the methods. At present, method S (as well as the justification of method SR) are presented before (L144-157) the others (L162-L197).
c) L220-221: “Illumina specific adapter sequences”; are these the Nextera adapter sequences?
L224: Is the construct of the reverse primer correct? The first 15 bp are repeated, why?
d) L240-246 and L274-277: This is too much information; the experimental design should be described, but the naming of the samples is not relevant.
e) L249: which primers were used, with which adapter sequences (again Nextera?)?
f) L278: did the three RC samples yield PCR products?
g) Why were 6 and 9 replicates taken for LN and NAK storage conditions? Three of each were used to test different storage conditions, what happened with the rest?
h) The presentation of the statistical analysis section could be more concise.
i) L336: Which list of methanogenic genera was used to identify the relevant taxa?
In general, the findings are nicely presented and valid. However, some findings are over-emphasized or lack justification:
a) The significant impact of the extraction method is overemphasized, as it is mentioned in the first sentence of the paragraph (L388, L389). However, DNA extraction has a much lower impact on the community analyses as compared to the soil habitat (site and depth), and later in the paragraph it is even mentioned that DNA extraction is not significantly affecting community analyses (L395), if results from one DNA extraction method (RC), which represent “outliers” are not considered. This suggests, that the observation of the impact of DNA extraction method is largely caused by the difference of RC and other DNA extraction methods. This needs to be presented as such in the results but also in the discussion, and requires further analyses on the different impacts of the different extraction methods. For example, was there a difference between S and SR extractions?
b) L433: “Notably, for ASVs, only the …” This is also true for OTU-level analyses.
c) L456: “The most parsimonious explanation for these collective results is that Lifeguard does not work well on a subset of lineages, such that those cells degrade during storage and are absent from characterization.” Which taxa could be affected? What can be derived based on the presented data? Without substantiation by the presented data, this explanation reads more as a speculation.
The conclusions are not well justified:
L542-543: “can influence the interpreted ecology of a sample”. While I agree in general with this conclusion, I do not see, where this has been shown with the presented data. Habitat and soil depth were always the much stronger drivers of community metrics, which is also mentioned in the conclusion at L546-547.
L546-549: “impacts of storage or extraction […], but were appreciable enough that they could strongly impact data interpretation (e.g. network analyses, tracking of particular lineages of interest, evaluating finer-scale ecological variation, and relating to expression data).” Again, while I do in principle agree with this potential impact, none of the mentioned analyses have been performed. What is the basis of a potential “strong” impact? At the present state of the manuscript, this is not a conclusion, but a speculation.
It is not clear, if the first sentence of the abstract “[…] community composition is influenced by sample storage conditions and nucleic acid extraction methods, and the impact varies by sample type.” (L42-43) is a finding of this study, known from the literature, or a hypothesis.
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