Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SEAGRASS STRUCTURAL AND ELEMENTAL INDICATORS REVEAL HIGH NUTRIENT AVAILABILITY WITHIN A TROPICAL LAGOON IN PANAMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear, unambiguous, professional English language used throughout. Intro & background to show context. Literature is redundant. Structure conforms to PeerJ standards, discipline norm. Some figures are inappropriately described and labeled. Raw data supplied.
Experimental design: Original primary research within Scope of the journal. Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap. Rigorous investigation performed to a technical & ethical standard. Methods described with sufficient detail & information to replicate.
Validity of the findings: All underlying data have been provided; they are robust and controlled. Conclusions are not well stated, are not well linked to original research question & are not limited to supporting results.
Additional comments: In the manuscript “Seagrass structural and elemental indicators reveal high nutrient availability within a tropical lagoon in Panama” the authors presented a comprehensive dataset on seagrass coverage and composition across 24 sites in Bahía Almirante Bay, using a single survey. They focused on spatial variation in leaf morphology (length and width), elemental content (% nitrogen and phosphorus) and stable isotopic signatures (δ13C and δ15N) in the dominant turtlegrass Thalassia testudinum. The sensitivity of seagrasses in Bahía Almirante to light and depth was revealed. The authors documented the eutrophic status of this bay. Additionally, they conducted a literature review to compare N and P content in the leaf to values reported for T. testudinum in other regions across the Caribbean Sea.

     It is good work; however, I see some inconsistencies here.
   1. It was “single time point”, the authors did not include monitoring of the benthic community of the bay in the goals of the work, but they devoted two paragraphs to it in the Introduction, made assumptions in the Discussion and Conclusion. If they wanted to focus on monitoring of the benthic community in the bay, then it would be correct to make a comparative analysis of their results with data on seagrass temporal variability of Caribbean Coastal Marine Productivity Program (1998) for two sites of Bahía Almirante, as well as with the study described T. testudinum nutrient content for three sites of the Bocas archipelago (Carruthers et al. 2005b). At the same time, it is necessary to make appropriate additions to the goals of the work and in M&M. If monitoring will be included in the goals of the work, then Fig. 6 should indicate the average values obtained by the authors only for points close to the sites that were studied earlier (Carruthers et al. 2005). Without this analysis, the monitoring text must be removed from the manuscript or minimized upon before acceptance.
   2. There is a weakness in the statistical analysis. Before a statistical comparison, the proportional data must be transformed (square root or arcsin), as they are percentages. I think that untransformed data set may be the reason of unsatisfactory results of the cluster analysis of the benthic community composition between sites that used % cover per group. If the benthos is primarily composed of bare sediment or covered practically only by dominant T. testudinum, corresponding % data gives too heavy weight in calculations, crushing data on other components of the community. If the authors still will not get an interesting result, then the cluster analysis could be excluded from the results of the work. The authors' comparative analysis of the composition of communities living near the open ocean, the city and other areas is quite good and relevant. And it will not be necessary to keep silent about the places that did not fall into the cluster, or for some reason fell into one large (medium) cluster.
   3. I think that the calculation of the mean value, and especially SE and CV, for biomass is incorrect since the distribution is far from the normal distribution. It is advisable to enter the data in the text what is the fundamental difference between places with a biomass of 20-40 and 120-140 g per m2. There may be different stages of succession, or/and different growth rates. May be they are young and old individuals?
   4. References are too mach.

     Some notes on the text, tables, and figures:
Line 61. Seagrasses are sensitive to light not only in Bahía Almirante. The authors did not study nutrient availability in the bay water or bottom sediment.
The sentences located on lines 64-66 and 127-128 should be removed.
Lines 62-64. I do not understand why “The high nutrient availability and depth-restricted distribution of seagrasses suggest that many of these meadows may be approaching or have already surpassed a ‘tipping point’, in which seagrasses retreat to shallower depth”.
The sentences located on lines 112-113, 193-195 and 280-282 should be supplemented with references.
Lines 172 and 308-309. What means “long-lived”?
Lines 172-175 and 182-195. The literature data and the justification for the need to study N, P, δ15N and δ13C contents should be included in the Introduction.
Lines 189 and 199. It is necessary to transcribe the abbreviations DIC and IRMS.
Lines 280-282. Need to move it to the Discussion.
Lines 388-389. Mathematical statistics allows you to remove extreme bouncing values from the dataset, however, if there is a lot of data.
Table 2. It is necessary to explain the reason for the appearance of a large SE value and why the median is much higher than the mean value.
Fig. 1 or Fig. 3. It is desirable to draw isobath.
Figs 2 and 6. It is necessary to draw (a), (b), (c), etc.
Fig. 4. It is necessary to enter in M&M an explanation of why the transformed data is shown on the Y-axis, and not the absolute values.
Fig. 4b. It is necessary to draw a regression line and specify the correlation coefficient.
Fig. 6. It is necessary to include in the table title, which is shown by the black and blue lines. Specify the year of the previous study.
Fig. 7. “Distance to the open ocean”. Pacific Ocean or Carribean Sea? Linear regression and coefficient of correlation must be indicated. “Depth” and “Distance to open ocean” are not “seagrass structural and elemental characteristics”.
Fig. 8. Poor quality. What does the color of the dots and lines mean?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROBUSTNESS ANALYSIS OF METABOLIC PREDICTIONS IN ALGAL MICROBIAL COMMUNITIES BASED ON DIFFERENT ANNOTATION PIPELINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors present a comparative analysis about the effect of the choice of gene annotation methods on metabolic reconstructions and on the inference of minimal bacterial communities. The paper is well written and provides the necessary sources and materials.
The topic of this analysis is very important. Indeed, we know that metabolic reconstruction is affected by the identification of enzymatic functions from the genome. But this effect is rarely measured. As shown here, its effect on analyzes performed from metabolic reconstructions can be also very significant. But unfortunately the article does not go very beyond this observation...

Here are my major comments and propositions in order of importance
- As mentioned in the discussion, this analysis does not give us any idea of the sensitivity and specificity of the reaction lists inferred by each analysis. An efficient way to measure their accuracy would have been to compare the result of these analyzes with a PGDB already cleaned manually. Unfortunately, none of the PGDBs in the list seem to have been manually curated by another team. Maybe, it would have been interesting to compare the result of these analyzes with a PGDB already manually cured such as E. coli even if it is not part of this bacterial community. Even without doing this comparative analysis which may seem a little outside the scope of the paper, it would have been interesting to have an idea of the quality of the networks reconstructed by indirect measures, such as the number of connected components in the reconstructed network (without the ubiquitous metabolites) or the number of holes in the pathways identified by the pathway tools.

- It would have been interesting to focus on the prediction quality of EC numbers. It does not seem to me that the authors indicate if they only take into account full EC numbers. It can also be a measure of quality. Also, are certain classes of EC numbers (up to the first 2 or 3 digits) over or under represented by certain pipelines?

- The Pathologic tool of the pathway-tools infers a first list of reactions from the enzymatic annotations, then deduces a list of possible metabolic pathways in the organism. The reactions returned by Pathologic contain this first list of reactions but also the other reactions composing the inferred metabolic pathways, commonly called "pathway holes". The authors do not specify it in the text but I imagine that they took all the reactions, i.e. those associated with a gene annotation and those corresponding to pathway holes. However, we know that these pathway holes can be numerous and that there are many false positives in these inferred metabolic pathways. It would therefore have been very interesting to compare the number of inferred metabolic reactions obtained only by the enzymatic annotations.

- The Miscoto algorithm selects minimal communities. But I imagine that like any result of optimizations, there could be several communities of minimum size, which could change the conclusions made from Table 1. This point is not discussed in the paper.

Minor comments
p8 l126. Authors mentioned 6 experiments but they mentioned above only 5 different analyses.
p8 l136. The github link provided by the authors does not contain "the detailed description of the code and data used...". This link claims that we can find the different files in the cluster but we don't have access to the cluster.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROBUSTNESS ANALYSIS OF METABOLIC PREDICTIONS IN ALGAL MICROBIAL COMMUNITIES BASED ON DIFFERENT ANNOTATION PIPELINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Karimi et al examined the impact on metabolic prediction when different genome annotation software was used. The manuscript is well-written with proper literature reference, sufficient background info. Tables and figures look good too.

Overall, this work by itself has a lot of value in that with the emerging number of new genome annotation tools, how the choice of annotation software impacts the downstream analysis such as metabolic network construction needs careful evaluation so that researchers could be well-informed and choose the proper tools for various research purposes.
Experimental design: This manuscript raised a very good research question and provided some observations.
It is understandable that the authors started with 81 genomes from isolated strains. However, questions like how this evaluation would perform on microbes which have not been isolated and only have incomplete/ near complete genome info, because this is very common in a lot of microbial communities, where most of the microbes could not be cultured. Adding discussions on such topic would greatly enhance the generality and application of the current findings.
In my opinion, it would also greatly strengthen the results if the author could compare the predicted metabolites with the actual measured metabolites in this microbial community, either from other papers’ results or measured in a synthetic community with the 81 isolated microbes and algae. Otherwise, one would not know which predicted metabolites are false positive, and the discussion can only limit to comparing the numbers of different metabolites from pipelines.
Also, it would be nice if the author could share the parameters used for each annotation pipeline for reproducibility.
Validity of the findings: In my opinion, how/whether this knowledge from the manuscript could be translated to other microbial communities is not well-stated. How the presented results could add any insight to the existing knowledge in the field is not clear to me.
Some questions to consider: Now that you see different performance of the annotation pipelines, what is your hypothesis on what may have caused it? Any further work to test the hypothesis? What do you suggest other researchers when they want to do metabolic prediction in a similar situation? Etc…
Additional comments: -	Of the 81 strains, how many percent do they cover the whole algal microbial community (e.g. based on culture-independent sequencing method)? Are these 81 strains representative?

-	When you do metabolic network reconstruction, how do you define the input (e.g. culture medium)? Did you take into consideration of the abundance info when building the network? As it is very likely these 81 strains do not exist in equal abundance in the community. Any inter-species interactions considered in the modeling?

-	Figure 1: any statistical difference among the pipelines?

-	Figure 2: It will also be interesting to see the ANOSIM stats for phylum.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROBUSTNESS ANALYSIS OF METABOLIC PREDICTIONS IN ALGAL MICROBIAL COMMUNITIES BASED ON DIFFERENT ANNOTATION PIPELINES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Please see general comments below.
Experimental design: Please see general comments below.
Validity of the findings: Please see general comments below.
Additional comments: In the manuscript titled “Robustness analysis of metabolic predictions in algal microbial communities based on different annotation pipelines” Karimi et. al. present a study that compares different genome annotation pipelines and their resulting impact on draft genome scale metabolic reconstructions. The aim of this study is relevant as the number of available microbial genomes is expanding rapidly and the quality of the resulting annotation impacts downstream analyses.

Overall the study design is appropriate and results valid. However, the authors could enhance the utility of the results if there was a more concerted effort to understand the source of the differences between the annotation pipelines.

Comment 1:
The primary weakness of the manuscript is the lack of analysis as to why the different pipelines result in different metrics. Without this assessment, a reader cannot deduce the strengths and weaknesses of each of the pipelines. As a result, the only conclusion from the study is that there are differences between the pipelines, which is largely self-evident since they leverage different databases and search algorithms. While a comprehensive assessment of the differences is out of scope for such a study, a cursory investigation into some of the differences would enhance the manuscript. Some suggested areas to explore:

- The annotation pipelines were run with default settings (Line: 99). Are the homology search cutoffs significantly different in the default settings for the different pipelines? Often homology search is able to assign the biochemistry of the reaction (the first 3 digits of the EC) but the substrate specificity of the 4th digit of the EC is less certain. If the homolog cutoff for one of the pipelines is lower it will call more EC numbers resulting in more reactions, but they will be low confidence. Running a more stringent version of the homology search might address some of the discrepancies in reaction count.

- The authors look at the unique pathways in the draft metabolic reconstructions (Line 183). Perhaps a manual annotation of a few of these pathways might elucidate the source of the different results in the different pipelines?

Comment 2:
The numbers for figure 3 are perplexing. The authors state the Prokka reconstruction had 2535 reactions shared with all the other tools and 390 that were unique to Prokka for a total of 2925 reactions. The Supplementary Table states there are over 133,000 reactions in the 81 microbe consortium. On initial look, this is an exceedingly high amount of redundancy considering the broad taxonomic distributions of the representative microbes. Since it is consistent between all the pipelines, this may be an issue with metabolic reconstruction tool. Since the follow-on analysis includes metabolite production capability, issues with the recon tool will be projected into this analysis and affect the authors’ conclusions about that analysis. This should be addressed by the authors.

Comment 3:
The initial analysis of different metrics between the tools is interesting.However, the statistical analysis needs more context. What is the goal of that analysis? How does it help understand the differences between the annotation pipelines? The Bray-Curtis Index is an interesting mechanism to create a uni-dimensional dissimilarity value from a genome annotation comparison. Unfortunately it isn’t described in the text. Additionally, it isn’t clear how NMDS contributes to an increased understanding of the differences between the different tools. While this analysis encompasses all of Figure 2, there are only 4 lines of text describing the value of this analysis.

Comment 4:
UpSet is the name of a tool, not the name of a known analytical/visualization method. It would be helpful, for almost all of the results section, to develop topic sentences that outline the gap/desired result of the analysis. Then follow up with the tool/method that would facilitate that analysis. An example (Line 173):

“To further explore the differences in the metabolic network reconstructions and to highlight the
specificity of each annotation pipeline we aimed to determine the conserved and unique content between the genome annotations from these different pipelines. An UpSet diagram enables this comparison by…”

As written, the reader doesn’t know why the authors are choosing a specific analytical tool or method nor what the intended outcome of that analysis should reveal and it’s connection with the overall study aims.

Comment 5:
The paragraph starting on line 183 is difficult to understand. It would be helped by use of a topic sentence. It is clear the authors are trying to summarize a very large, interacting set of results, which is difficult. However, the paragraph as written doesn’t clearly communicate the desired results (unique metabolic pathways elucidated in each of the pipelines).

Comment 6:
Line 151: This line is currently written presenting an average and range. Since it comes right after a comment about using the different annotation tools the reader may think the different tools generated this wide range of results. In fact, I believe the authors are saying the different species in the consortium have different genome sizes and they are reporting that distribution. Since this is expected, I would suggest not reporting an average but instead focus on the range of genome sizes in the community.

Comment 7:
Overall, the Discussion and Conclusions are well written. In particular, it was important to point out that a larger number of EC numbers doesn’t necessarily result in a more accurate network; it may in fact result in false positives when tested for metabolic capabilities. The final comment on manual annotation is important but untouched in this work. Similar to the primary weakness, it would enhance the manuscript if there were a few small examples where manual annotation resolved discrepancies between tools.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROBUSTNESS ANALYSIS OF METABOLIC PREDICTIONS IN ALGAL MICROBIAL COMMUNITIES BASED ON DIFFERENT ANNOTATION PIPELINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No new comment.
Experimental design: No new comment.
Validity of the findings: No new comment.
Additional comments: Thanks to the authors for addressing most of my comments. The manuscript is now clearer and richer. I have still one major comment and few minor comments.

Major comments:
- The authors evaluated the quality of the reconstructions by counting the number of orphan and dead end metabolites. They claim that "The lower these values the more connected a metabolic network is". I think that this statement is not always true. We can imagine a network with a lot of dead end (outputs of the network) and orphans (inputs) that contains a single connected component. On the contrary, a network with less dead ends and orphans can be divided into several connected components. I think that the number of connected components (and optimally, the distribution of their sizes and the size of the biggest component) should complete these indices. I think that these connected components should be computed by removing ubiquitous metabolites, such as ATP, water, etc... Furthermore, it's clear that the number of connected components will strongly affect the results of the Miscoto algorithm.

Minor comments:
l 111 : had -> had been (or has been ?)
l 129 : conversion to SBML already mentioned in the previous paragraph
l 190 : In terms of EC numbers, Prokka predicted more than the other pipelines -> I'm not sure that this sentence is grammatically correct.
Fig 2 : What do represent the grey polygones ? If the authors don't comment them, they sould remove them to make the figure clearer.
l 342 : With the new EC number comparative analysis, this statement is not completely true anymore.
l 355 : to reconstructed -> to reconstruct


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROBUSTNESS ANALYSIS OF METABOLIC PREDICTIONS IN ALGAL MICROBIAL COMMUNITIES BASED ON DIFFERENT ANNOTATION PIPELINES
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The revised manuscript addresses my comments and questions well and I think it is now ready to be published at Peer J.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ROBUSTNESS ANALYSIS OF METABOLIC PREDICTIONS IN ALGAL MICROBIAL COMMUNITIES BASED ON DIFFERENT ANNOTATION PIPELINES
Review round: 2
Reviewer: 3

Output:
Basic reporting: See General Comments below.
Experimental design: See General Comments below.
Validity of the findings: See General Comments below.
Additional comments: The changes to the manuscript made by the authors addressed most of my comments. I feel the revision is a much more approachable version of the manuscript and more effectively communicated the results.

There is some proofreading required, but it is minor.

The manual curation of select reactions was a welcome addition. I was hoping for a more clear root-cause for the differences in the pipelines but it seems that it is elusive (no fault of the authors!).

The only thing I would have liked to have seen would been an adjustment of the pipeline parameters to a more stringent version. It is possible the pipelines converge on a largely consistent "core" reconstruction for reactions with high homology but the low homology content is highly affected by the underlying databases used by the different pipelines. In general, the e-value cutoff of 10e-5 or 10e-6 is very forgiving (in my experience of performing manual reconstructions) and will lead to many ambiguous reactions being added. The authors stated in their response that they "deliberately chosen to take the perspective of a standard user" but I would argue the standard user who came across this manuscript would greatly appreciate finding a study that performed some perturbation of the standard parameters.

This also ties into the final comment in the discussion about manual curation and the cost-benefit trade-off of time and reconstruction accuracy. It is very helpful to have an automated process to establish the core reconstruction and flags the ambiguous content for manual curation. I feel like this study is the perfect venue to explore the stringency parameters and the impact on the core reconstruction.

The study is still sound, but I do feel this is a missed opportunity that would increase the scientific contribution of the work.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE IDENTIFICATION, CLASSIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSF GENE FAMILY IN HYPERICUM PERFORATUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Ms entitled ' Genome-wide identiﬁcation, classiﬁcation and expression proﬁle analysis of the HSF gene family in Hypericum perforatum (#52717) by Zhou et al. has been critically assessed all for the analytical details, finding of scientific novelty, and linguistic representations. Honestly, genetic factors of high-temperature stress in plant systems are of scientific interest keeping in view the rising concerns of global warming and its effect on agriculture and the ecosystem. Therefore, the appropriateness of the article may be considered. However, the authors could have made a better effort to bring significant findings from their analysis. Several issues in the article require serious attention before can be considered for publication in a prestigious journal like PeerJ.

Overall, the Manuscript needs a thorough check for clarity of sentences, rechecking of all typos, grammar, and usage of the English language. Several sentences are unclear due to ambiguous statements and may need recasting them to improve the clarity of statements. On several occasions, authors have left without the citation year in text, which indicates their very casual manuscript finalization before the submission. My strong recommendation is to get the manuscript checked from any Native English speaking person or take the help of a professional language editing service.

The 'Introduction section' may be highlighted with the need to analyze HSFs in the plant system with prior work or citations. Several minor suggestions were mentioned in the pdf article itself. Some of the References cited are old and maybe replaced with recent citations. May include article: Sci Rep. 9, 5581 (2019). https://doi.org/10.1038/s41598-019-41936-1 and cite accordingly.
Experimental design: In the M&M section, authors may use the 'Heatster' program to annotate their sequences and compare them with the Phylogenetic groupings. Also, authors may need to include more in silico analysis like the evolutionary significance of HSF gene expansion and protein modeling with active sites.

Authors may clearly mention the Phylogenetic analysis parameters, like the Alignment method, amino acid substitution Models used, basis of selecting the models, etc. Consider using MEGA-X in MUSCLE. May consult https://doi.org/10.1038/s41598-019-41936-1 for details.
Validity of the findings: The results section may be strengthened further with the suggested new analysis.

In the Discussion section, the authors fail to sufficiently explain any scientific contribution to the field. The authors may need to revise this section to bring out the salient and new findings from their study.

Similarly, authors may also strengthen the 'Concluding section' with highlights of their study and how it is beneficial or in what way it may contribute to the scientific prosperity of the plant per se.

The authors may check and add references as suggested for the manuscript.
Additional comments: Authors may recheck the Ms again and again to minimize all typographic and grammatical errors. Also, they may need to bring their best efforts to reveal any significant scientific findings from their study. With these major revisions, I am sure their manuscript will be improved to be considered for publication in the PeerJ journal.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE IDENTIFICATION, CLASSIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSF GENE FAMILY IN HYPERICUM PERFORATUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript the authors identified and characterized several heat shock transcription factors in H.perforatum using various bioinformatic analysis tools. Considering the important role of HSFs in a plant’s response to heat stress, it is important to perform bioinformatic analyses and expression profile studies of HSFs to better understand this response pathway. Given the importance of H.Perforatum extracts in pharmacological applications and the challenges in its production, it is important to identify HSF  genes in this plant species which could provide the foundation for exploring methods to understand and regulate these stress responses.  Studies such as the work in this manuscript contribute towards such efforts. This work is within the scope of PeerJ journal. The manuscript is written in professional English for the most part and the structure conforms to PeerJ standard. Most figures are of publication quality and appropriately annotated. The raw data relevant to the manuscript is available. A few minor points are noted below.
1.	Figure 1: HpHSF23 is missing in the figure.
2.	Figure 3: HpHSF08 is listed as a member of group A. However, according to the figure it should be HpHSF18
3.	Figure 3: A3 is missing while A9 is duplicated. Is one of the A9 duplicates actually A3? Please correct the group names.
4.	Reference figure 7 in paragraph on lines 218-229
5.	Figure 8: Mention what the * indicates in figure legend
6.	There are some minor grammatical errors in the manuscript such as line 41, 47,67,80, 114,173, 176 and 239 which upon correction will improve the quality of the manuscript.
Experimental design: The authors have defined the objective of the study well. The bioinformatic analyses and biochemical characterization are well structured and seem to be performed accurately following standard procedures in the field. However, the authors must clarify the following point to improve the overall clarity and transparency of the manuscript.
1.	Please elaborate on the statistical analyses performed for the RT-qPCR assay to distinguish between significant and insignificant changes in the HSF expression levels.
Validity of the findings: Overall, the results are detailed and presented in a clear manner for the most part. Appropriate analyses have been performed and reported in accordance with standards in the field. The discussion of results is thorough, and the conclusion answers the original research question. There are some points outlined below which must be revised prior to acceptance of the manuscript.
1.	Line 31: Please elaborate what “vegetarian attack” indicates.
2.	Line 163: Was the finding that all the HpHSF proteins were unstable, an expected result? What does this finding imply in terms of the function of the protein?
3.	Line 180: Please use the scientific names of the plants as that is what is used in the figure 3 title and mention the common names in parenthesis.
4.	Line 191: Please clarify what “Peu” stands for?
5.	Line 224-225: The text states “Moreover, the expression of HpHSF11, HpHSF18, HpHSF13 and HpHSF07 in leaf were higher than that in other tissues”. However, according to Figure 7, the expression of HpHSF12 appears to be higher than both HSF7 and HSF11. Is there a reason HpHSF12 was not included in the statement above?
6.	Lines 226-228: HpHSF03 and HpHSF22 are expressed at very low levels or not expressed at all. Please comment on this in the text.
7.	Lines 233-234: “As shown in Figure 8, the expression of HpHSF2, 11, 12, 21 had no significant change”. However, in figure 8, there are * on the bars and the levels do look different. Please clarify what the * denotes which would resolve this ambiguity.
8.	Figure 8: For HpHSF 21 and 22 plots, why is the 0h time-point not at 1 on the Y-axis considering this is relative expression? If this is in error, please rectify.
9.	Please clarify what plant tissues were used in the heat stress response experiments. If it was the entire seedling, please mention that.
10.	Have the authors compared how the expression levels of HSFs change in response to heat stress in mature plants versus young plants/seedlings? If not, the authors should perform a comparative study to answer understand if it’s the same or different HSFs that are affected in young and mature plants. This might help to identify how similar or different the heat stress signatures are in the plant depending on its stage in the life cycle.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE IDENTIFICATION, CLASSIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSF GENE FAMILY IN HYPERICUM PERFORATUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of the article by Zhou et al. has been carefully checked now. The improvements in the article are clearly visible compared to the previous version. The English language usage is clearer and references are made up to date now. The article may be accepted for publication.
Experimental design: The experimental designs suggested were mostly included and this fits well with the Aims and scope of the PeerJ. The research questions are now better stated to initiate a platform to work further in this direction.
Validity of the findings: The findings are now being discussed sufficiently towards acceptance.
Additional comments: Suggestions were mostly addressed by the authors. no new suggestions are made.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE IDENTIFICATION, CLASSIFICATION AND EXPRESSION PROFILE ANALYSIS OF THE HSF GENE FAMILY IN HYPERICUM PERFORATUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript and the rebuttal letter adequately address the previously raised issues.
Experimental design: The revised manuscript and the rebuttal letter adequately address the previously raised issues.
Validity of the findings: The revised manuscript and the rebuttal letter adequately address the previously raised issues.
Additional comments: The revised manuscript and the rebuttal letter adequately address the previously raised issues.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BACPHLIP: PREDICTING BACTERIOPHAGE LIFESTYLE FROM CONSERVED PROTEIN DOMAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript is very clear, the tool design is well explained and the benchmarks provided should enable any reader to rigorously use this tool and interpret its results.
Experimental design: Hockenberry & Wilke present a new tool (BacPhlip) to identify temperate from virulent phages base on their genome sequence. BacPhlip relies on the detection of marker genes tied to a Random Forest Classifier, which is a robust experimental design.
Validity of the findings: BacPhlip seems to work as well as could be hoped/expected. It is also available on github, and easy to download and run. I thus only have a few minor comments about the text itself.
Additional comments: Minor comments:

l. 10: “Bacteriophage species” should be probably be “Bacteriophages”, as infection cycles are often determined for individual phages, not necessarily a whole species.

l. 10: “temperate (lysogenic) and virulent (lytic)”. Since temperate phages are defined by their ability to enter both lysogenic and lytic cycles, I would suggest moving the “lysogenic” and “lytic” parenthesis to the next sentence, i.e.: “temperate and virulent. Temperate phages are capable of a latent phase of infection within a host cell (lysogenic cycle), whereas virulent phages directly replicate and lyse host cells upon infection (lytic cycle).” (the same is true for l 31-32).

l. 29: “meta-genomics” should be “metagenomic”

l. 68: “errors” may be more clear as “erroneous annotation” (if I understood the intended meaning correctly).

l. 79: “that was” should be “that were”

l. 139: for non-specialist, “lysogeny-associated domains” may be more clear as “lysogeny-associated protein domains”

l. 194: “to detect” would be more clear as “to be detected”


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BACPHLIP: PREDICTING BACTERIOPHAGE LIFESTYLE FROM CONSERVED PROTEIN DOMAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written, and describes the problem, previous solutions, and the gap in knowledge that they aim to fill.

Figure 1 is clear and appropriate, although the figure is the inverse of the text describing it: the paragrap discuss classification accuracy (e.g. BACPHLIP is 98.3% accurate) while the figure shows inaccuracy (% incorrect predictions).

Figure 2 is less informative, and it does not relate to the information in the text describing the figure. For example, the text (lines 202-209) state the "Top 50 protein domains ... accounted for 85% of the model weights" but it is impossible to see that from Fig. 2.

The manuscript demonstrates the utility of BACPHLIP in predicting the lifestyle of temperate and virulent phages, an area of increasing concern.
Experimental design: The manuscript carefully defines the problem and identifies the data set and approach that will be used for the solution. The manuscript combines data from Mavrich and Hatfull with the Random Forests from McNair et al to idenitfy the lifestyle of phages.

The main difference between this approach and McNair is the use of a selected set of domains that are likely to be invovled in lysogeny. McNair used all the proteins in the genome, and thus discovered proteins that may be associated with lysogeny. However, as noted in the manuscript, the approach applied here has identified phages whose mechanism of lysogeny is apparently unknown and novel.
Validity of the findings: The authors took every effort to crouch their results in the appropriate caveats that may affect their findings. They are careful both in the manuscript and in their git repository to remind users that this tool has not beeen designed to work with fragmented/incomplete genomes, and to acknowlege the inherent bias in the data set that they used to train their models.

Given those caveats, BACPHLIP shows remarkable progress in classifying the lifestyle of phages over previous work.

In my experience, I was able to run BACPHLIP and generate meaningful results.
Additional comments: This is the next step advancement over the previous work, that integrates many more genomes to result in higher accuracy.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BACPHLIP: PREDICTING BACTERIOPHAGE LIFESTYLE FROM CONSERVED PROTEIN DOMAINS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Hockenberry and Wilke describes the tool BACPHLIP. Overall, the manuscript is very well written and the results are also convincingly and clearly presented in a logical way, with the exception of one of the figures where the data does not seem to match the text (see "general comments"). I have also downloaded and used BACPHLIP. Its usage is fairly intuitive and straightforward, and I had no issues obtaining a lifestyle classification for a dataset of phage genomes.

The literature is well referenced, but one of the points in the introduction would benefit from further detail. This referes to lines 32-33 ("Phages are extraordinarily diverse and this dichotomy is an over-simplification"). I concur, but perhaps this could be expanded a bit more. Are the authors referring to virulent phages that can remain in pseudo-lysogeny within a cell? To phage-plasmids that blur the lines between mobile elements?

There is also an issue (possibly a typo) in Figure 1. The number of phages in the independent dataset is shown as 157, but shouldn't it be 172 phages (line 129 and line 153)? Or did I miss something? If it is a typo, check also the number of related phages (266), since 172+266 does not match the 423 phages used to test the model(s).

Finally, the raw data (protein domains used for classification) is included, but it is not referenced in the main text (at least nowhere I could find).
Experimental design: BACPHLIP performs the very difficult, but necessary task of assigning a lifestyle to phages based on their genomic features. The authors illustrate well the need for this type of classification, specifically based on the fact that the only other available tool was based on a limited set of genomes. The methodology developed to achieve this goal is clear and, as far as I can discern, looks robust.

The authors provide the protein domains that underlie the Random Forest model, and also provide the code to test and build the classifier in BACPHLIP (although I have not tested this specific code), which is great for users that want to re-train the model.
Validity of the findings: The authors find, and convincingly show, that BACPHLIP outperforms other approaches to classify phage lifestyle. They also control for a potential statistical inflation of their results by taking into account phage relatedness in an additional analysis.
Additional comments: Overall, I enjoyed the manuscript and I find it very close to be suitable for publication. My main point is not even really a criticism, but it concerns the link between BACPHLIP and the dataset of Mavrich and Hatfull. The authors mention that (lines 176-177) "[the] similarity [between BACPHLIP and Mavrich] is perhaps not surprising given that both of these methods rely on targeted identification of protein domains associated with lysogeny". But in fact the two methods are linked in a more intrinsically fashion, because the model beyond BACPHLIP was trained by using the lifestyle assignment of Mavrich, and not just because they use the same type of genomic features. That is, if I understood correctly, any misclassification of (e.g.) a temperate phage in Mavrich will have an impact in how the RF model is scored during training, which will itself influence future lifestyle assignments in the testing dataset.

To be clear, I don't see this as a major issue, or at least not one that can be resolved. It is already explicit in the methods that the Mavrich dataset underlies the training of the model, and there really is a lack of other sources of data for phage lifestyle (it is impossible to experimentally or manually classify them all). However, I do feel it is important to reinforce this (perhaps in the discussion?) as an idiosyncrasy of the method, beyond the biased phylogenetic composition of the Mavrich dataset (lines 244-246).

There are two other suggestions that I think would improve the manuscript, but neither is critical:

	1) It could be interesting to show how PHACTS fares when considering only phages whose lifestyle was classified as "confidently" assigned. Is the inaccuracy vastly different? Note that I am aware that PHACTS very often results in non-confident assignments, which by itself is an issue. But this could be important to understand a) whether previous works that used this classification but limited their results to the "confidently assigned" phages are robust, and b) to further reinforce that BACPHLIP outperforms even the most stringent PHACTS classification (if that is the case). I think a supplementary figure/table, or even a mention of this additional analysis in a sentence in the text could be informative.

	2) Regarding the prediction confidence, the authors state that 333 genomes had >=95% probability for a given lifestyle, with only one error in this set, which is quite good. However, I wonder how this distribution looks like for the whole testing dataset. Is it binomial, i.e., do the probabilities cluster around 95% for those 333 genomes and then at low values (~50%) for the remainder genomes? Or is it more evenly distributed? And where do the lifestyle mis-assignments tend to occur in this distribution? The authors suggest that the users of BACPHLIP should treat predictions between 50% and 95% as uncertain, but being aware of this distribution could help them make a more informed decision regarding the cutoffs for significance.

Additionally, I have a couple of two other minor points:

1) Line 62: "parA|ParA|parB|ParB". I was initially a bit confused as to why these term were used as genomic feature. I had to search in the literature to find out that they refer to the partitioning systems of actinobacteriophage, which was unknown to me. A sentence on what are these genes, and why are they potentially relevant to discriminate temperate from virulent phages would be useful for other readers.

2) Lines 221-223: "However, we note that varying degrees of confidence are (and should be placed by users) on the predicted lifestyle of individual phages that have 51% vs 99% probability, for instance." The phrasing of the sentence seems confusing to me. Rephrase?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF ULTRAFILTRATION AND IRON CHLORIDE FLOCCULATION IN THE PREPARATION OF AQUATIC VIROMES FROM CONTRASTING SAMPLE TYPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clear and well written, it has lots of side experiments, which enrich the experimental structure. Data included in figures and tables, as well as in supplementary material is really well presented. But, the introduction needs more detail on different ultrafiltration applications enhancing the references using rexeed ultrafilters and the background they got. Rexeed ultrafilters are dead-end ultrafiltration devices, that can be also modified to perform axial ultrafiltration (see Gallardo et al 2009). To my knowledge, Rexeed ultrafilters are not tangential flow ultrafilters, and the reference given by authors (22) does not use rexeed filters, use Fresenius filters which are different from rexeed.
Although relevant results are presented, authors should verify and correct this information in order not to confuse readers.
Experimental design: Research question is well defined and experiments rigorously performed. Although it is relevant to the audience that authors have included 6 different non-enveloped phages, they should point that the inclusion of an enveloped virus is lack on the study.
specific comments:
all p in p-values should be in italics
line 269: above instead of below?
line 387: delete "and" before river
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF ULTRAFILTRATION AND IRON CHLORIDE FLOCCULATION IN THE PREPARATION OF AQUATIC VIROMES FROM CONTRASTING SAMPLE TYPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: A few citations are needed.
Line 56: According to the citation provided by the authors, viruses outnumber other microbes by up to two orders of magnitude, but in the following paragraph (lines 63 to 64) “viral DNA comprises a small fraction of total community DNA.” Include a literature citation for lines 63 to 64. Also, it is important to clarify how both ideas are true. Provide a description with an example that relates genome sizes of viruses to other microbes.

Line 541: Provide literature citation for “T3 and HS2 have negative surface charges in this pH range.”
Experimental design: The agarose gel electrophoresis experiment was challenging to interpret. Figure S5 did not contain information to determine the sizes of the marker DNA. The figure needs to be amended or better described in the text. Pulse field gel electrophoresis with appropriate ladders is the recommended method for environmental DNA virus genome quality analysis.
Validity of the findings: no comment
Additional comments: For authors
The authors compared two methods for purifying DNA viruses (ultrafiltration purification and iron chloride flocculation purification) present in wastewater (influent and secondary effluent), river water and seawater. They concluded ultrafiltration purification performs better than the iron chloride flocculation. This study provides much needed new information for preparing DNA viruses for metagenomic sequencing.

General comments/questions per section

Introduction
Line 56: According to the citation provided by the authors, viruses outnumber other microbes by up to two orders of magnitude, but in the following paragraph (lines 63 to 64) “viral DNA comprises a small fraction of total community DNA.” Include a literature citation for lines 63 to 64. Also, it is important to clarify how both ideas are true. Provide a description with an example that relates genome sizes of viruses to other microbes.

Materials and Methods
Lines 130 to 142: Check that the statements concerning virus spikes are consistent with Table 2. Add Table 2 for: “In freshwater matrices, Enterobacteria phage T3 (GenBank accession no. NC_003298, ATCC® BAA-1025-B1TM) was spiked into samples (Table 2).” The virus spikes for the various matrices are described except for wastewater influent. Add this missing description to this paragraph.

Line 289: Provide line equations and LOQs for the qPCR assays. How many non-template and negative extraction controls were included in the qPCR assays? How were false positives dealt with if they were observed?

Line 305: Did storing the droplets at 4C up to 60 hours cause any artifacts prior to running the plates? Such as those described in lines 308 to 310; i.e., rain or % compartmentalization?

Line 306: Provide more details for setting thresholds for classification of positive and negative droplets. Provide LOQ for 16S rRNA gene ddPCR. How many non-template and negative extraction controls were included in the ddPCR assays? How were false positives dealt with if they were observed?

Lines 318 to 323: The DNA virus spikes ranged in size from 5 to 168 Kb (see Table 1). Pulse field gel electrophoresis is a better approach for assessing the quality of spikes as well as endogenous DNA viruses. Explain why 0.3% AGE was used instead of PFGE for assessing quality of DNA.

Lines 470 479: Figure S5 did not contain information to determine the sizes of the marker DNA. This makes it difficult for the reader to interpret the data. The standard practice for gel electrophoresis is to indicate on the gel image the kilobase sizes for molecular standards. The figure needs to be amended or better described in the text. Some of the agarose wells appear to contain stainable DNA, while others didn’t. What is the size of this DNA? A related question, what is the resolution of 0.3% agarose? As stated above, PFGE with appropriate ladders is the recommended electrophoresis method for genome quality analysis. GeneRuler High Range DNA ladder has fragment sizes ranging between 10 and 50 Kb. Why was this ladder used for environmental DNA viruses known to be larger than 50Kb? Two of the virus spikes (Table 1) have genomes approximately two (HM1) or three (T4) times larger than 50Kb.

Line 541: Provide literature citation for “T3 and HS2 have negative surface charges in this pH range.”

Lines 584 to 604: 16S rRNA gene ddPCR assesses the removal of bacteria. Explain why eukaryotes (via 18S rRNA gene ddPCR) were not assessed too.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF ULTRAFILTRATION AND IRON CHLORIDE FLOCCULATION IN THE PREPARATION OF AQUATIC VIROMES FROM CONTRASTING SAMPLE TYPES
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, Langenfeld et al compare two different virus concentration methods (tangential flow filtration and iron chloride flocculation) used prior to DNA extraction for viral metagenomic studies. They compare the efficiency of recovery in different samples, from wastewater treatment to seawater. To measure recovery, they spiked the samples with known concentrations of viral isolates and used droplet digital PCR to measure the recovery of these viruses at each step of the process.

The manuscript is well written and is easy to follow. The references cited are appropriate. The figures are clear.
Experimental design: No comment.
Validity of the findings: As the field of viral metagenomics keeps on expanding, this study is important to make sure that the highest quality of the viral assemblages is maintain during the concentration process.
Additional comments: While the spike and recovery approach is good to be able to monitor each step, it would have also been interesting to have total viral counts of before and after viral concentration, as well as after the DNAse step, which would better represent the impact of the methods on the viral assemblages. Omitting the remaining of the viruses lowers the impact of the publication.

It is unclear why they used T3 on freshwater samples and HS2 on seawater samples. While I understand that they did this because T3 was isolated from a freshwater host (E. coli) and HS2 a marine host (Pseudoalteromonas), it makes the comparisons difficult between the two environments as each phage/virus behaves differently.

Line 214: It is unclear why they decided to filter out the microbial host using a 0.45 µm filter since it is well known that many prokaryotes will go through these filters. While they did look at 16S rRNA gene contamination, when proposing benchmarked protocols, even though this is not what was not the step the study focused on, each step counts in the process and it seems that this one (filtration for removal of microbial hosts) was slightly overlooked.

Here, only one phage was added at a time. It would have been interesting to have all the phages added at the same time and measure the recovery of each. What works for a phage may not work for another one. Without redoing all the experiments, it could be worth expanding in the discussion.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF NAC GENES IN BRASSICA JUNCEA VAR. TUMIDA
Review round: 1
Reviewer: 1

Output:
Basic reporting: NAC proteins are a large family of transcription factors, participating in diverse physiological and developmental roles as well as a defence response against biotic and abiotic stresses. Authors report the genome-based survey of NAC gene family in Brassica juncea var. tumida.

In addition to their in silico study, they made expression analyses of selected NAC genes in different temperature stresses. This study may be the first step for further analysis of NAC’s function and application in the tumorous stem mustard
Experimental design: The authors followed a classic methodology for this kind of study. The experimental part (wet bench) could be more complete.
Validity of the findings: To enhance the impact and novelty of their study, the current version of the manuscript needs revision.
Additional comments: The work of Jiang et al. presents an in silico genome-wide analysis of the NAC proteins in the crop plant in the tumorous stem mustard. The authors have used published genome sequence information from in Brassica juncea var. tumida to mine for NAC genes where 300 have been found. They performed classification and phylogenetic analysis with other species. Overall the manuscript describes a very important and interesting development in this particular research field. Moreover, it gives some interesting scientific perspective on this topic, which is a major concern for the next years (ie, global warming). NAC gene family is assumed to have different roles in diverse plant function and process. However, authors do not discuss the precise and specific role of BjuNAC gene family. Authors should emphasize and clearly discuss the role and function of such genes, it is not clear how BjuNAC genes are regulated under different stress condition. Yet authors may think of following suggestions:

-	Your introduction needs more detail. I suggest that you improve the description at lines 58- 67 to provide more information regarding NAC proteins and temperature stress.
-	Line 69: move(AABB, 2n = 36) after allotetraploid
-	Line 72-75: if possible, add some references.
-	Line 76-90: this paragraph can be more concise or moved to the Discussion part
-	Line 93: typo (a space is missing before the reference)
-	Methods: a description of your script (supplemental?) can be useful (line 140)
-	Line 161: why did you choose Actin as housekeeping gene?
-	Results: it may be interesting to add HSE cis-element because they are key element in the heat-shock response (line 221)
-	Discussion should be carefully revised. The manuscript needs to further discuss and enrich the functional role of BjuNAC genes, compare results with the existing literature (what is similar, what is different), explain why did you choose treatment of 24h, 48h. It is a very strong treatment affecting many physiological processes. Authors need to be careful with their interpretation. Performing qRT-PCR on the hallmark of HS/cold stress in theses conditions may add strength to the manuscript (in supplemental?)
-	Size-wise, BjuNAC proteins varied a lot. Can you talk a bit about that? What would be the adaptive benefits of such diversity?
-	Line 282: add “in rice, “ before “The OsNAC7 subgroup”
-	289-296: Can you elaborate more what are the function of these NAC during the heat and cold stress?
-	Line 303-305: Can you detail in the Methods part of you obtained this result?
-	Line 309. I will be more careful with the statement. Only a few BjuNAC genes were experimentally tested.

Figures:
- Fig1. Because you compare NAC proteins in different species, it can be useful to add other crop species such as rice (and wheat?).
- Fig4. Adding labels 39°C, 5°C can improve the figure.
- Adding all clustalW alignment in supplemental figures may be interesting as we can see if the sequences are similar or not.

English and typo error: a careful proofreading may correct the few mistakes in the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF NAC GENES IN BRASSICA JUNCEA VAR. TUMIDA
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is a nicely written manuscript and literature review is upto date.
Experimental design: Adequate, but a comparison with basic diploid progentors would have been very useful.
Validity of the findings: Conclusions are adequate and based on data generated. I missed comparison with diploid progenitors.
Additional comments: It is a very informative and well compiled. However, one would have liked to include phylogenetic comparison of BjuNAC proteins with those from  Bni NAC and Br NAC . Same should be true for chromosome distribution, copy number variation and gene expression analysis across ploidy.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VARIANCE OF VEGETATION COVERAGE AND ITS SENSITIVITY TO CLIMATIC FACTORS IN THE IRTYSH RIVER BASIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Overall, the work is good.Please check the attached comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VARIANCE OF VEGETATION COVERAGE AND ITS SENSITIVITY TO CLIMATIC FACTORS IN THE IRTYSH RIVER BASIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript adopted a vegetation sensitivity index (VSI) based on high-resolution remote sensing
datasets to study the sensitivity of vegetation to climatic factors in Irtysh River basin. It is a beneficial trial for the vegetation cover monitoring. But the manuscript need major revision before publication.
Experimental design: Figure 1, What is the white regions of the land cover data?

Figure 1, It is better to add the stations' name.

Line 126, How about the interpolation accuracy?

Line 132, Why do you use Savitzky-Golay filter? Add the reference for this method.

Line 140, How to resample?

Line 143, How about the data filling accuracy?

Line 206, What is the
Validity of the findings: Figure 2, line 214, The NDVI value need to be divided into more levels especially in the low values.

Figure 6, The correlation analysis is meaningless for the whole pixels.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VARIANCE OF VEGETATION COVERAGE AND ITS SENSITIVITY TO CLIMATIC FACTORS IN THE IRTYSH RIVER BASIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	The introduction is rather vague and needs more detail. I propose that you enhance the description at lines 45-48 to provide more justification for your research gap.
2.	In line 52 consider removing “that”.
3.	Line 58; “, that is,”  to  ‘and that is’?
4.	Line 64; consider to clarify “in that its state” or alternatively rewrite the sentence.
5.	Line 65 to 67; what is the “memory effect”? First define it and then refer to it later on.
6.	Line 67 to 69; I suggest sticking to one of the definitions.
7.	Line 80; consider to rewrite it.
8.	Line 84; rewrite, please.
9.	Line 85 to 90; please clarify the point you want to make.
10.	Line 95; “At the same time” is not necessary.
11.	Line 98 to 101; rewrite it and make your point clear.
12.	Figure 1; please explain the reason that one of the meteorological stations are outside of the study area? Maybe you can overlay a hill shade to the zoomed area? The north sign is not necessary as your map has already N-E labels, this also applies to other graphs to avoid redundancy.
13.	Line 126; reference is needed here.
14.	Line 131; please add a reference.
15.	Line 131; please substitute the word “pix” to “pixel”  throughout the study.
16.	Line 144; make a complete sentence, rewrite.
17.	Line 174; provide a reference, please.
18.	Line 200; make a complete sentence.
Experimental design: 19.	Line 132; please explain your choice of smoothing filter and add a reference and also please elaborate the parameters ( you can use THIS LINK as a source; https://www.mdpi.com/2072-4292/9/12/1271/pdf or https://opengeospatialdata.springeropen.com/articles/10.1186/s40965-017-0038-z)
20.	Line 143; You can add one or two more sentences here as the gap-filling approach is vital to this study. Here are some links that might be helpful. https://www.mdpi.com/2072-4292/12/3/361/htm and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008475/.
Validity of the findings: 21.	Line 372; Please consider changing “comprehensive” as this study didn’t provide evidence about the comprehension of the method. Alternatively, you can provide evidence that the method is comprehensive e.g., by comparing to similar topics like https://www.mdpi.com/2072-4292/11/21/2475, https://www.mdpi.com/2072-4292/12/19/3150 and or https://www.mdpi.com/2072-4292/12/11/1758, https://www.mdpi.com/2072-4292/12/7/1113.
22.	Line 374 to 376; Is this a conclusion of your study, if yes, please explain it?
23.	Line 384 to 385; please either bring a sentence about the mentioned analysis or consider removing the sentence.
Additional comments: In general, I like the research the technical part was interesting and all the codes and function were well written and well documented. I encourage the writer(s) to continue writing code as small functions that can be used later easily. I also encourage the writer(s) to be more careful about scientific writing and paying more careful attention to grammar.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VARIANCE OF VEGETATION COVERAGE AND ITS SENSITIVITY TO CLIMATIC FACTORS IN THE IRTYSH RIVER BASIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have looked through the response and found the authors have successfully addressed my comments. Therefore, I recommend to publish it in the present form.

The manuscript has been greatly improved and all reviewers' suggestions have been applied. I believe that the paper is now ready for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: VARIANCE OF VEGETATION COVERAGE AND ITS SENSITIVITY TO CLIMATIC FACTORS IN THE IRTYSH RIVER BASIN
Review round: 2
Reviewer: 2

Output:
Basic reporting: - Please consider removing the abbreviations in the abstract as they are not needed there but in the body text.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HISTOPATHOLOGICAL ANALYSIS OF ZEBRAFISH AFTER INTRODUCTION OF NON-BIODEGRADABLE POLYELECTROLYTE MICROCAPSULES INTO THE CIRCULATORY SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “Histopathological analysis of zebrafish after introduction of non-biodegradable polyelectrolyte microcapsules into the circulatory system”

The paper contains information of potential interest considering the relevance of the histolopathology of zebrafish species after introduction of non-biodegradable polyelectrolyte microcapsules. However, some critical points still need to be addressed
Experimental design: Abstract:

-	The histopathological data in the abstract is a bit confusing! I suggest that histopathological data in several organs of zebrafish as brief sentences should be included.
-	Could author(s) please say something about the benefit of this study?

Introduction:
-	It is well-written and showed the good introduction; however, Lines 44-48, 51-52, 91-95; some relevant references to quote these sentences should be provided.

Materials and methods:

-	Line 115: I am confusing about why the age of zeabrafish, as 4-12 month!
-	Line 152: why did author(s) collect data the day 1, 14 and 22 post-injection?
-	Regarding the fluorescent protocol, I recommend separating this method into new paragraph! provide information about the antibodies, provide details on the antigens (native, recombinant or peptides) and specificity of the antibodies. Refer to previous publications that validated and used this antibody.
Validity of the findings: Results and Discussion:
-	Please include the figure (s) in Lines 271-273.
-
-	Please add the figure in Line 275 (after.., while they were abundant in the sinuses in the organs). Additionally, they … refer to?
-
-	Line 294-296: I would recommend adding some discussion to support!!! like literature reviews or scientific reports.

-	Many figures displayed as Fig. 3C-3D (Line 299), Fig. 4A,4B (Line 332) or Fig. 9A, 9D (Line 573). All words “Fig.” should be replaced with “Figs.” instead.

-	What does it mean about … with dissociation and atrophy of hepatocyte ? (Lines 358-359)
-	Is this statement correct? I did not see the figure in the abdominal cavity (Line 369).

-	Please add some relevant references in Lines 399-401.
-
-	Figure 3: What does “f” in Figure 3D stand for? Why do you think that the release of microcapsules is exhibited? Is it artifact?

-	Figure 5: Better replace ‘oocyte’ in Figure 5A with ‘ovary’. What do you mean that it is the focus of inflammation in Figure 5C? Could you please explain in more detail?

-	Figure 6: It is not easy to identify the macrophage phagocytizing renal cell and mast cell !!. please add the higher magnification of the 6F. Importantly, I recommend that the mast cell should be replaced by the eosinophil granulocyte instead.

-	Figure 7: Could you please indicate the dilated capillaries in this figure?

-	Figure 8: What is the mucus in Figure 8B?. I guess that the mucus cell is shown; however, I think that this is the fiber of connective. Moreover, Figs. 8C-8D, they are the lamellar fusion together with epithelial hyperplasia.

-	Figures 9: Accordingly, two figures displayed the mast cell; however, I recommend that they should be considered as eosinophil granulocytes as mentioned above. These cells had a diameter of 9-10 µm and large eosinophilic granules in the cytoplasm. Could you please show in higher magnification of this cell?
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HISTOPATHOLOGICAL ANALYSIS OF ZEBRAFISH AFTER INTRODUCTION OF NON-BIODEGRADABLE POLYELECTROLYTE MICROCAPSULES INTO THE CIRCULATORY SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript needs recheck due to linguistic errors.
The gap of knowledge should be explained carefully.
Experimental design: Ethical code should be provided.
Validity of the findings: Study limitations should be provided.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HISTOPATHOLOGICAL ANALYSIS OF ZEBRAFISH AFTER INTRODUCTION OF NON-BIODEGRADABLE POLYELECTROLYTE MICROCAPSULES INTO THE CIRCULATORY SYSTEM
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is quite clear statement of the rationale for your approach to the problem studied.
Experimental design: Fluorescent PMs-PEG detection is quite specific technique. The author(s) may set this method as a subsection and all details concerning antigens and antibodies used in this examination should be given.
Validity of the findings: -“Fig” or “Figs” Could the author(s) recheck throughout the paper?
-Lines 294-296 and 399-401, could author(s) should quote these sentences?
-What dose “f” in figure 3D stand for?
-Few words are ambiguous or incorrect. Could author(s) recheck them?
Like “oocyte” in figure 5A (ovary?) or “mast cell” in figure 6F and figure 9 (eosinophil granulocyte?)
-Some descriptions are missing or not related to the figures, Like…abdominal cavity in figure 5 and …dilated capillaries in figure 7. Could the author(s) point them out?
Additional comments: This paper is quite clear and understandable. The researcher conveys research in proper and concise manner. However, some valuable data should be provided and amended before publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HISTOPATHOLOGICAL ANALYSIS OF ZEBRAFISH AFTER INTRODUCTION OF NON-BIODEGRADABLE POLYELECTROLYTE MICROCAPSULES INTO THE CIRCULATORY SYSTEM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear observations are found throughout the manuscript.
Experimental design: This part in the paper is clear.
Validity of the findings: All data are satisfied for publishing.
Additional comments: All of the comments have been addressed and revised. The data provided in the paper is satisfied for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HISTOPATHOLOGICAL ANALYSIS OF ZEBRAFISH AFTER INTRODUCTION OF NON-BIODEGRADABLE POLYELECTROLYTE MICROCAPSULES INTO THE CIRCULATORY SYSTEM
Review round: 2
Reviewer: 2

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: -


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TORQUEMADA: TOOL FOR RETRIEVING QUERIED EUBACTERIA, METADATA AND DEREPLICATING ASSEMBLIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presents a tool (named TQMD) for removing redundant bacterial genomes from a set of assemblies. Specifically, the tool dereplicates genome assemblies by removing genomes for which there are sufficiently close relatives (defined by a distance threshold), resulting in a smaller set where the assemblies are more unique and maintain taxonomic diversity of input genomes. The topic is highly relevant to the genomics and metagenomics fields as the number of microbial genomes sequenced each year is expanding rapidly.

Overall, I think this is a nice piece of work, which can be potentially an important contribution in its field. I especially appreciated the care with which the representative genomes are selected by TQMD and the way the tool uses an internal database to store information about already analyzed genomes (only new genomes are added to the existing database). Nevertheless, I do have some concerns that I would like to see addressed by the authors.

1. Introduction needs more focus on the topic of genome dereplication and current tools designed for this purpose (e.g., dREP, Assembly-Dereplicator), rather than a general description of alignment-free methods for pairwise sequence comparison. I suggest the tuthors shorten the description of alignment-free methods and just provide the reader to the review (reference #1). For example, the paragraph about information theory-based methods (lines: 57-77) seems irrelevant to the study as all the tools for genome dereplication (including TQMD) are word-based (use k-mers) and do not use information theory concepts such as sequence complexity or compression. I suggest that the authors provide more justification for the study in the Introduction (specifically, by describing the existing tools for dereplication and presenting their advantages and disadvantages if possible).

2. I commend the Authors for their extensive 5-year work on the TQMD tool. However, throughout the manuscript, the authors describe TQMD as it is still unfinished. For example: "For now, we do not use all the metrics available in the TQMD database" (line: 232). In a similar manner, the authors state that 13 metrics of genome quality are retrieved by TQMD from the QUAST tool (line: 183), however, in the next sentence the authors stay that only three metrics were used (line: 192, "Among these metrics, we eventually decided to take into account...")
Experimental design: 3. My main concern, which is also confirmed by the Authors, is that TQMD is only optimized for dereplicating genomes at high taxonomic levels (i.e., class and phylum) but not at lower taxonomic levels such as family, genus, and species. In NCBI RefSeq or GenBank, more than half of all available bacterial genomes belong to only several species (e.g., Escherichia coli, Staphyloccocus aureus, Bacillus subtilis), each present in thousands of almost identical copies. According to GtDB, which is an excellent curated resource for bacterial taxonomy built upon the latest RefSeq database (v. 95), there are 191,527 bacterial genomes encompassing about 16,000 representative species belonging to 111 phyla. Given the number of genomes belonging to one species is many times larger than the number of phyla there is a pressing need for efficient dereplication tools at the lowest taxonomic levels. As the authors admit in the manuscript, at low taxonomic levels TQMD does not work as accurately and efficient as the other two tools, dREP and Assembly-Dereplicator. It is clear to me that this low performance of TQMD is a result of using relatively short k-mers (~12 nucleotide), which are the upper bound to calculate the exact distance metrics (JI and IGF) in feasible time. However, I think TQMD might be improved to be at least as accurate as dREP at low taxonomic ranks (I elaborate my suggestions in the next two comments).

4. Calculating the exact JI and IGF distances in TQMD limits the size of k-mers that can be used. The authors are aware of this issue and concluded that "replacing Jellyfish by Mash would be interesting to compare the efficiency with the current version but this would imply dropping the IGF distance (since Mash only implements JI) and would require Mash to accept using different k-mers sizes". I think that the Authors can easily replace Jellyfish by Mash, preserving both JI and IGF distances. In addition to JI, Mash also outputs the number of shared k-mers in a sample (by default, the sample size is 1000, which is under the "-S (sketch size)" parameter in Mash). The authors could use this number to estimate IGF by slightly modifying the equation from line 256 to the following one: IGF(A,B) = |A ∩ B| / min(length of genome A, length of genome B). In this way, both the metrics would be approximated, allowing the selection of longer k-mers, thus making the TQMD tool also appropriate for low taxonomic levels. Of course, I am not forcing the Authors to do this, but I would appreciate the Authors to comment on this idea.

5. TQMD analyzes all 12-mers from one strand of genome sequence assuming the input genomes have the same strand orientation. I suggest the authors to use the "--canonical" parameter in Jellyfish to count k-mers on both strands and perform JI and IGF calculations on canonical k-mers only. This would also ease the calculations since the number of all possible 12-mers is 4^12, the number of canonical ones will be somewhere about (4^12)/2.

6. In line: 161-163 the authors state that Jellyfish crashes when using a size below 11 nucleotides. It is a known bug in Jellyfish v. 1.X, which was also confirmed by developers of Jellyfish. It is recommended to use Jellyfish in version >= 2.

7. When dereplicating genomes people usually want to cluster sequences that are at least XX% identical. For example, when clustering bacterial genomes belonging to the same species many studies use an average nucleotide identity (ANI) of 95% as a threshold. It would be useful to explain to readers how to set proper IJ or IGF thresholds in TQMD to obtain a desired sequence identity threshold for the clustering.
Validity of the findings: no comment
Additional comments: I very like the idea that representative genomes are expected to be fully sequenced, correctly assembled, richly annotated, and devoid of contaminating sequences.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TORQUEMADA: TOOL FOR RETRIEVING QUERIED EUBACTERIA, METADATA AND DEREPLICATING ASSEMBLIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The analysis of Assembly Dereplicator is problematic.
Validity of the findings: no comment
Additional comments: Léonard et al. propose a method for dereplicating genomic datasets. There is a need to dereplicate genomes given the large and rapidly growing number of available genome assemblies. However, the proposed method is computationally intensive and it is unclear that dereplication at higher taxonomic ranks is a problem. A common approach to building phylum or class level data sets is to manually select a single genome from each of these taxonomic groups. This ensure representation across known diversity and a representative from each named lineage. A tool to select a suitable representative for each higher-level taxon (i.e. phylum or class) would be useful, but this decision can be based purely on information pertaining to the quality of the assembly and available annotations. Generally speaking, assignment into different phyla or classes is sufficient to indicate genomes are diverse and that their incorporation into a dereplicated data set is warranted.

Major:
•	As of this review (Dec. 1, 2020), TQMD did not work for me. I downloaded the source code, extracted the tarball, and ran “per Makefile.PL, “make”, and then “make test”. This failed with the message below. The README file on BitBucket would benefit from indicating the minimum Perl version required and all dependencies. Ideally, the installation script would install all required Perl modules. I am not familiar with Perl and was not able to install the software. A Conda package would be welcomed.
#   Failed test 'use TQMD;'
#   at t/00-load.t line 10.
#     Tried to use 'TQMD'.
#     Error:  Can't locate Modern/Perl.pm in @INC (you may need to install the Modern::Perl module)
•	The Introduction relies heavily on the prior work on Zielezinski et al. which was published in 2017. Can the authors comment on additional methods and software tools which may have been developed since 2017? Along these lines, the Introduction would benefit from introducing dRep and Assembly-Dereplicator in order to clarify why a comparison against these two tools is sufficient to establish the benefits and need for TQMD.
•	 Does TQMD only work on systems with SGE/OGE? This is restrictive as many labs do not run HPCs with this scheduler. I appreciate supporting multiple schedulers is not practical, but I would expect TQMD to be able to be run on a machine without requiring a scheduler. For example, we make common use of 64 CPU machines with >500 GB of RAM that are run without a scheduler.
•	 TQMD would prove useful to a much wider audience if there was also an option to work with genomes in NCBI’s GenBank assemblies instead of only RefSeq assemblies. My understanding is that RefSeq does not contain any MAGs or SAGs. Currently GenBank contains an additional 100,000+ genomes relative to RefSeq. Admittedly, these genomes are often of lower quality than those in RefSeq with many being metagenome-assembled genomes (MAGs) or single-cell genomes (SAGs). However, these genomes also span a substantial amount of diversity missed by the higher quality isolate genomes in RefSeq which is critical for many downstream analyses.
•	Line 224: It is indicated that genome contamination will be estimated with the program Forty-Two. CheckM and BUSCO are established programs for calculating contamination which should give more accurate estimates than those based on just 42 ribosomal proteins. However, my major concern here is that it is indicated that Forty-Two “is not yet part of the automatic preparation pipeline”. How does TMQD obtain this information? This seems like an essential statistic, especially if MAGs are to be considered, and believe TMQD so incorporate this before it is made available to the wider research community.
•	The global ranking of genomes used by TMQD is based determined using an “equal-weight sum-of-ranks approach”. It would be helpful to readers and myself if this statistical approach could be explained given that the ranking of genomes in a critical step. I am also unclear on what metric 5 is “number.enriched.alis / 90”.
•	I have no trouble following the explanation of how the dereplication strategy works, but do not find the workflow given in Figure 1B clear. The flowchart makes no mention of genomes being processed in a specific order or clustering being performed in a greedy fashion based on specific threshold.
•	TMQD dereplicates by comparing the current query genome to all genomes currently in clusters. It would substantially reduce the amount of computation if the query genome was only compared to the representative of each species cluster. This has the benefit that the resulting clusters are genomes that will guaranteed to be similar to the selected representative. Is there a disadvantage to this more computationally efficient dereplication strategy given the purpose of TMQD? For many purposes, it seems undesirable to have cluster where genomes may only be connected via a single linkage (i.e. genome A is similar to genome B is similar to … is similar to genome Z, but genome Z is highly divergent from genome A).
•	I am unclear on the exact methodology used in the “Phylogenomic analyses” section. Earlier in the manuscript it is indicated there are 155,519 genomes as of RefSeq 202. However, the dereplication of the full bacterial sets appears to have started with only 63,863 genomes (datasets A and B in Table 1).
•	The dereplication of the bacterial sets results in 49 and 151 representative genomes for a threshold of 0.1 and 0.12, respectively (Table 1). According to the latest version of the GTDB (https://gtdb.ecogenomic.org/) there are >300 bacterial classes. Can the authors comment on the benefits of TMQD as opposed to a taxonomy driven dereplication strategy where the best representative of each class is selected? This would seem to be far less computationally intensive and ensure a tree contains representatives across the current known diversity of the bacterial tree.
•	The comparison to Assembly-Dereplicator starting on Line 535 is problematic. It appears that either Assembly-Dereplicator has a fundamental flaw in which case the comparison is not informative, or the program is not being run correctly. At a Mash distance threshold of 0.5 (~50% ANI), a substantial amount of dereplication should occur. Either way it is unclear what benefit providing that analysis has unless this issue can be addressed.

Minor:
•	Is the software called TQMD or ToRQuEMaDA? I strongly prefer the former. It is unclear why the abbreviation ToRQuEMaDA requires its own abbreviation, and it is confusing that the title of the manuscript uses a different abbreviation than the Abstract.
•	The Abstract has the phrase “…high taxonomic level…”. I think this should be “higher taxonomic levels”.
•	The README file on the BitBucket page would benefit from removing the standard template sentences regarding the purpose of a README file and information about CPAN. It would be beneficial to make it clear that TQMD is a Perl program and thus requires Perl (certain version?).
•	I do not believe that the Proteobacteria and Firmicutes are necessarily “hyper-sampled”. Perhaps these phyla just contain substantial diversity compared to other phyla.
•	Line 222: I believe the clause “which could rule out some metagenomes” should be “which could rule out some metagenome-assembled genomes”?
•	dRep and Assembly Dereplicator should be referenced when first mentioned.
•	Line 220: it is indicated that “the presence of at least one SSU rRNA 16S predicted by RNAmmer is a requirement for the genome to be selected”. Is this true? Where is the presence of 16S rRNA genes used as a criterion by TQMD?
•	Line 341: I believe the phrase “inter-distance genome” should be “inter-genome distance”.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TORQUEMADA: TOOL FOR RETRIEVING QUERIED EUBACTERIA, METADATA AND DEREPLICATING ASSEMBLIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: I congratulate the authors for this very nice and thorough revision. I am delighted to recommend the manuscript for publication in PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TORQUEMADA: TOOL FOR RETRIEVING QUERIED EUBACTERIA, METADATA AND DEREPLICATING ASSEMBLIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for their revisions and well articulated responses to my previous concerns.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TCF7L2 RS7903146 POLYMORPHISM ASSOCIATION WITH DIABETES AND OBESITY IN AN ELDERLY COHORT FROM BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Bride et al examined the TCF7L2 rs7903146 (C>T) association with T2D and obesity in a ten year cohort of 1,023 elderly Brazilians. They found the T allele increased T2D risk in the normal-weight group. The C allele increased the obesity risk, while the T allele showed protective effect against obesity.
The manuscript is clear and well-written, and the statistical analyses were performed appropriately. Appropriate references are cited.
Experimental design: Research questions are well-defined, methods and experimental approach are clear with sufficient detail and information to replicate.
Validity of the findings: Conclusions are based on the findings.
Additional comments: 1.	The T2D group was diagnosed based on respons to questions “has a doctor or nurse ever told you that you have diabetes or high146 blood sugar levels?” Have the authors confirmed the T2D grouping based on the result of fasting plasma glucose and glycated hemoglobin measurement?
2.	The authors found that based on logistic regression analysis, the risk for T2D varied according to BMI status (Results line 217). The pathophysiology of obesity is different in male and female. Did the authors observed any difference in T2D risk in male vs. female? Could the authors please do separate analysis for male and female, and compare the results?
3.	The median age is significantly different among the 3 groups (normal-weight vs. pre-obesity vs. obesity). Could the authors kindly elaborate whether their finding that the T allele increased T2D risk in the normal-weight group was not due to the older age of this group? Has the author performed interaction analysis of the T allele and age in increasing the T2D risk?
4.	The authors described an increase in the TT genotype in the ΔBMI second tertile when compared to the first tertile in Supplemental Table 3 “Genotypic distributions by tertile intervals from ∆BMI values of volunteers without type 2 diabetes mellitus”. Does this increment also observed among subjects with T2D?
5.	Dietary factors such as fiber (Hindy et al. Genes & Nutrition. 2016. 11:6 doi: 10.1186/s12263-016-0524-4) and dessert (Ouhaibi-Djellouli et al. BMC Genet. 2014. 15:134. doi: 10.1186/s12863-014-0134-3) intakes could also play an important role in T2DM etiology, and gene-diet interaction could be present in T2DM pathogenesis. Therefore, it would be appropriate if the authors could include the lack of assessment of dietary intakes as one of the limitation of their study.

Minor comments:

1.	The sentence in Discussion line 242-244 is not clear, could the authors please rephrase their sentence: “We confirmed that the genetic susceptibility conferred by rs7903146 to T2DM is influenced by BMI status and the TT genotype, at risk for T2DM, is associated with a protective effect against obesity.”
2.	The sentence in Discussion line 295 is not clear, could the authors please clarify what they mean: “...and it difficult the recognition of eutrophic versus obesity status.”
3.	Sentence structure in Discussion line 299-300 need to be revised: “In elderlies without T2DM, was observed an increased number of TT genotype carriers...”


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TCF7L2 RS7903146 POLYMORPHISM ASSOCIATION WITH DIABETES AND OBESITY IN AN ELDERLY COHORT FROM BRAZIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: According to the WHO definitions, use "overweight" instead of "pre-obesity" to define the group with BMI of 25.0–29.9 kg/m2.
Experimental design: Sample size should be added (based on the expected prevalence of this SNP in Brazilians/closest population).
Validity of the findings: No Comment.
Additional comments: Thank you for submitting your interesting study. Please comment on the other TCL7 gene variants that may be in linkage disequilibrium with the one in your study, or maybe significant in obesity, metabolic syndrome, and/or Diabetes mellitus.  Only a few editing points and language final check are required.