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: ANTI-COVID-19 MULTI-EPITOPE VACCINE DESIGNS EMPLOYING GLOBAL VIRAL GENOME SEQUENCES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Some topics of the article are not clear:
line 65 to review the comma position - Scientists are working tirelessly to find a cure, but as of now the impasse
line 77 to change diseases state to disease state
line 78 to put a comma in: "The genome contains six open reading frames (ORFs), including ..."
line 80 to change "envelop" to "envelops"
line 82 to remove "in" and to put a comma in: "cases, even death ..."
line 86 to change "to treat" to "in treating" and put a comma in: "Currently, ..."
line 125 to change "on predicted protein" to "of predicted protein"
line 147 to put "a" before strong
line 229 to change "about the unique" to "into the unique" and change "aspects about protein" to "aspects of protein"
line 371 to change "water refined models" to "refined water models"
line 391 to put "t" in capslock "Supplementary table 2" to "Supplementary Table 2"
line 395 to change "Eigen value" to "Eigenvalue"
line 403 close the parentheses
line 420 to remove one "of", in this line you have double of
line 439 to change "Our subunit vaccines are therefore projected" to "Therefore, our subunit vaccines are projected"
line 466 to change "silico analysis indicate" to "silico analysis indicated"
line 477 to change "was" to "were"
line 493 to change "is" to "are"
line 503 to change "nonstructural" to "non-structural"
line 503 504 to put a comma in: These structural, as well as nonstructural proteins, play an important
Experimental design: Your methods need a better description in the servers that you used. Please, check them:
line 130 In CTLpred, what is the predict approach? (p.e. Quantitative matrices based; Only ANN Based; Only SVM Based; Consensus Approach; Combined Approach) What are the parameters? (p. e. QM Cutoff &nb sp; ANN Cutoff Score; SVM Cutoff Score). Please, answer this question for all tools used in this section.
line 166 In IFNepitope, what is the approach used? (p.e. Motif based; SVM based; Motif and SVM hybrid)
line 201 In AlgPred, what is the method chosen in the server? (p.e. Mapping of IgE epitopes and PID; MEME/MAST motif; SVM module based on amino acid composition; SVM module based on dipeptide composition; Blast search on allergen representative peptides (ARPs); Hybrid Approach (SVMc+IgE epitope+ARPs BLAST+MAST)). Please, answer this question for all tools used in this section.
line 212 In AntigenPro, what are the parameters used? (p.e. ACCpro: Solvent Accessibility (25%); SSpro: Secondary Structure (3 Class); ABTMpro: Alpha Beta Transmembrane; DISpro: Disorder; CONpro: Contact Number; CMAPpro: Contact Map; 3Dpro: Tertiary Structure; SOLpro: Solubility upon Overexpression; ACCpro20: Solvent Accessibility (20 Class); SSpro8: Secondary Structure (8 Class); DOMpro: Domains; DIpro: Disulfide Bonds; SVMcon: New SVM Contact Map; COBEpro: Continuous B-cell Epitopes; ANTIGENpro: Protein Antigenicity; VIRALpro: Capsid & Tail Proteins)
line 223 What are the parameters used in 3Dpro?  Please, answer this question for all tools used in this section. Check this website (http://scratch.proteomics.ics.uci.edu/) in line 224, is it the site of 3DPro?
line 237 What are the configurations in HADDOCK 2.2? (p.e. in the first: molecule Active residues, Passive residues, Segment ID to use during the docking and What kind of molecule are you docking?); and in the second molecule: Active residues, Passive residues, Segment ID to use during the docking and What kind of molecule are you docking? Please, describe all parameters used in the server ...)
line 251 Do you use Additional Options in server JCAT?
Validity of the findings: no comment
Additional comments: Your article has an interesting topic in our pandemic time and these methods are very important to find a solution to this problem, but I need to know more details about your methods. I have some doubts.


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: ANTI-COVID-19 MULTI-EPITOPE VACCINE DESIGNS EMPLOYING GLOBAL VIRAL GENOME SEQUENCES
Review round: 2
Reviewer: 3

Output:
Basic reporting: This manuscript is about immunoinformatics approach of COVID-19. It describes the in silico approaches for the development of multi epitopes based vaccine against COVID-19. The work presented is of standard quality comprising bioinformatics research with all the important data and subsequent analyses included.
Experimental design: The approach is straightforward, however, there are few comments, observations need to be addressed and/or elaborated.
Validity of the findings: The result has novelty and may aid in the development of an effective vaccine against the target virus in future
Additional comments: Comments for the author
This research has significance in the current scenario of the field, but I would recommend some major revision as I found a few major and minor issues throughout the manuscript.
Comments for the author
This research has significance in the current scenario of the field, but I would recommend some major revision as I found few major and minor issues throughout the manuscript.

Major

•	In order to global vaccine, a population coverage analysis would generate more acceptable epitope candidates with the ability to confer immunity in different endemic/pandemic regions of the world.
•	Authors should mention the references for the linkers and why those linkers are appropriate and were used for vaccine construct.
•	There is no mention of EAAAK linker in the manuscript, just in Figure 2.

•	In the discussion section mention few similar approaches that have been utilized for in silico vaccine design against other pathogens. Some recent publications are worth citing:
•	As described in link provided "Purpose: The aim of this server is to predict MHC Class-II binding regions in an antigen sequence, using quantitative matrices derived from published literature by Sturniolo et. al., 1999.  The server will assist in locating promiscuous binding regions that are useful in selecting vaccine candidates". CTL epitopes bind with MHCI molecules, not MHCII.
•	Line 140-141: “strong binding affinity” Please specifies the cut-off applied here.

•	Line 148-149: “MHC binding …” Please specify the difference on prediction methods among Net MHC 4.0 server and IEDB server as both predict binding affinity with HLA super family alleles.
•	Authors should give a in details about the comaprative immunogenic properties the two vaccine construct.

In Table 1 (CTL, mapped epitopes into 6 ORFs - 1AB, S, M, 6A, 7A, and 7B), and in Table 2 ( HTL, they mapped epitopes into 4 ORFs( envelope, 8, 9 and 3a) - covering 10 ORFs, not 11. In addition, it should be clear if they aimed this distribution (because the ORFs are not repeating in each table). If yes, would be good to explain if author selected some epitopes instead of others for this reason.

•	In the result section, (Table 1) there are 23 epitopes described as the short-listed CTL epitopes. However, in figure 2 there are only 12 epitopes. Confusing please clarify.

•	I would suggest author to give a proper workflow of his analysis with number of epitopes used and discarded.

•	Same for (Table 2) HTL epitopes. It is written 11 epitopes in text, there are 9 in table 2 and 10 in figure 2.

Minor

Abstract:
Line 29-30: The aim of the article was find the diversity, maybe "cover the diversity of SARS-CoV-2  strains".
Line 35-37 : “All Open Reading Frames  ORFs) were subjected to Cytotoxic T-lymphocyte (CTL) epitope and Helper T cell lymphocyte  (HTL) epitopes using CTLpred and HLApred, respectively”
Kindly re-phrase the sentences
Line 64-64: SARS-Cov/(MERS-CoV) is the abbreviation for the virus not the illness.

Line 100: Line 107, Line 108: Author should check trough out the manuscript to maintain the nCOV-19 or SARS-CoV2
Similar problem is multiepitope, multi-epitope etc. please check.
Spacing and end of the sentences are clear eg.
Line 244:  Plasmid UC19 was used for in silico cloning of both multi epitopes.  More
Line 356: BspQI-sapI (Fig 9. Should be BspQI-sapI (Fig 9).
Line 373: The overall structure of of 475 genomes of COVID-19 …

Please check thoroughly throughout the manuscript.

Line 158: IFN … Which type of IFN?Please specify.
Line 227 : Molecular Docking with Immunological Receptors it should be “Molecular Docking with TLR8 ”as authors is not using other TLRs .
Line 250: “The Pakistan ..” clad should be clade.
Line 258: Please standardize nomenclature of Pakistani strain. “CDS of MT240479” Here its different from Methodology section "genome from Pakistan Accession number GWHACDD01000001"
Line 265-267: “For. Potentially immunogenic  “Sentence too long and confusing. re-phrase the sentences


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: EFFECTS OF COST SURFACE UNCERTAINTY ON CURRENT DENSITY ESTIMATES FROM CIRCUIT THEORY
Review round: 1
Reviewer: 1

Output:
Basic reporting: a.	Introduction
i.	The introduction was the strongest piece of the manuscript, in my opinion. I felt like the flow from one paragraph to the next was coherent and made significant progress in preparing me for the rest of the paper. Topic sentences were generally on target and logical. The last paragraph in the introduction (115-134), however, needs to be expanded and split into at least two paragraphs (115-126, and 126-134).
ii.	115-126: In 115, you begin by suggesting that you will explain the various sources of uncertainty in developing a cost surface. Then 116 to 123 explain how variability in cost weights are one source of variability. 123-126 brings in thematic resolution and other differences in layers as sources of variability. However, as I see the paper, you are fundamentally most interested in understanding how current density is affected by four variables or factors: (1) varying cost weights, (2) thematic resolution, (3) spatial arrangement/ fragmentation, and (4) range of cost weights. You build up cost weights strongly, but I think it is important to build a stronger basis for why you are studying thematic resolution, spatial arrangement, and range of cost weights.
iii.	126-134: I appreciate that you clearly laid out which variables you are studying in sentence 126-129. I also appreciated the clear expectations for what they expected to find. With a stronger paragraph preceding this, we’ll have a better idea of why we should expect these things. The only addition I would like to see to this paragraph is a statement of expectation/hypothesis of how changing cost values will affect current density. Basically, strengthen the sentence in 128-129.
b.	Structure
i.	Overall, the structure of the Introduction was very good, aside from the issues I mentioned above. The structure of the Methods section worked well for me as well. However, structural issues and omissions in the Results and Discussion made it more difficult to follow. Inconsistency in the order in which you address elements of the study makes it more difficult to follow. In the Introduction, you introduce the three factors you are studying, in an order that makes sense, but the lack of parallel structure in results and discussion is a major hindrance. Please revisit each of your hypotheses in the same order in both Results and Discussion and ensure that there is sufficient detail about each.
ii.	Results:
1.	The fragmentation and thematic resolution paragraphs are good, but should switch places. This will maintain consistent order with the introduction.
2.	I would also strongly recommend to begin the Results section with an introductory summary paragraph about how varying absolute values of cost weights affected current density in general. Certainly you discuss this in other paragraphs in the context of other variables, but I don’t see why you couldn’t discuss this in general terms. For example, across all types of scenarios, how often did changing cost weights have a significant bearing on the current density?
3.	The third paragraph focuses on cost value range, but is inadequate at describing your results. Give more specifics, e.g. numbers rather than simply “In general …”
iii.	Discussion:
1.	If you divide up parag. 1 based on which hypothesis it focuses on, it’s nearly all fragmentation, and only one sentence is on thematic resolution. Overall, there is little on thematic resolution in the discussion. Please remedy this.
2.	You would be well served to stick with the same order for your hypotheses throughout the paper: 1) cost weights, 2) thematic resolution, 3) spatial arrangement, 4) range of cost weights. Without this structure, it is hard to follow and to know what the key messages are for each one.
c.	Figures
i.	Figures 1-5 worked fine for me in the sense that they gave a depiction of what the landscape models were like. The only change I would like to see is to show the nodes. You describe that in the text but do not show the nodes in the figures.
ii.	Figure 6 was good. Additional figures that summarize the results in more ways than simply listing mean RS values would improve the paper.
d.	English
i.	The English is very good overall.
ii.	“large scale” (line 259) is ok, but many conservation practitioners do not come from a strong geography background, and therefore I favor fine/coarse scale instead of large/small scale.
iii.	The only typo I noticed was on line 50: “costs weights.”
Experimental design: a.	Original primary research
i.	The research question is original.
b.	Research question defined, meaningful, fills knowledge gap
i.	This is valuable for conservation – circuit theory has as stronger theoretical basis than least cost path analysis, so expanding our understanding of the behavior of circuit theory in various contexts is important.
c.	Rigorous investigation
i.	I understand that rank order of cost weights is probably more important than the relative differences between those cost weights. The numbers that are above 50% or even close to 90% seem great, but how do they compare to other models that do not maintain rank order? I am not certain that you have used baselines/controls/null models, against which we can compare these results. How do all these heterogeneous surfaces compare to a homogeneous surface? How much worse is the RS of a homogeneous surface than a bad model that maintains rank order? What if the rank order of cost weights is NOT preserved – how different would the results be? How do we determine at what point it is relatively unimportant to develop precise cost weights? The phrase “provided that the rank order of the costs is accurate” could become more meaningful in that case.
d.	Methods detailed
i.	Please explain the concept in lines 195-198 in more depth.
Validity of the findings: a.	Data controlled, statistics sound
i.	It was light on statistics, and only included Spearman correlations. These are appropriate and are rightly the main results. It is possible that there are other statistics that could draw additional inferences from the results, but I can’t think of any others.
b.	Speculation identified
i.	I did not notice any speculation.
c.	Conclusions well stated, linked to research question and results.
i.	Conclusion was appropriate.
Additional comments: This is a valuable study and look forward to seeing it in print. Please see my comments for details on what changes I think you should make to strengthen this study.

Karl Jarvis
Southern Utah University


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: EFFECTS OF COST SURFACE UNCERTAINTY ON CURRENT DENSITY ESTIMATES FROM CIRCUIT THEORY
Review round: 1
Reviewer: 2

Output:
Basic reporting: See "General comments".
Experimental design: See "General comments".
Validity of the findings: See "General comments".
Additional comments: When I read the abstract, I thought that this study would be assessing the sensitivity of current density or resistance distance to "mis-assignment" of cost values across classes (in terms of rank order). For example, if expert opinion is used to parameterize the resistance surface, and the expert decides that class A has higher cost than class B when the converse is true. Or equivalently, how sensitive outputs (passage probabilities) are to a non-monotonic transformation of inputs (conductance values/class). This sort of error is probably quite common in practice, and so seems useful to qualify the conditions under which inference is dramatically impacted.

Instead, what is investigated is the sensitivity of outputs to the relative difference in cost between classes, maintaining rank order. Equivalently, the sensitivity of outputs to monotonic transformations of inputs. Sensitivity is measured as average rank correlation (Spearman) of outputs, across transformations of inputs. So, the question being asked can be roughly boiled down to: does a monotonic transformation of the inputs produce a more-or-less monotonic transformation of the outputs? The answer seems to be yes (average Spearman correlations ~0.9), but I don't find this nearly as interesting or useful a the hypothetical scenario in the first paragraph. In fact, I'm surprised the average Spearman correlation is not closer to 1. I'm curious if there are parts of the graph that on average show a greater discrepancy that could be artefacts; e.g. regions nearer to the border of the raster (I realize the authors included a spatial buffer, but the 20% rule of thumb is not a hard guideline and depends on the size of the raster).

The second part of the study assesses sensitivity of output to "fragmentation", which corresponds to spatial autocorrelation/degree of clumping in cost values. There are 10 simulated surfaces in each "fragmentation treatment". For the first part of the study (assessing changes in magnitude of cost, discussed above), the outputs were compared across different transformations ("cost scenarios") of the same simulated surface: this is valid. For this second part, outputs are compared (via rank-correlation) across different simulated surfaces. I don't see how this is meaningful-- of course outputs from completely different spatial configurations will have a correlation of 0 on average, provided you generate enough simulations. Am I missing something?

The third part consists of comparing simulations where the cost classes are subdivided to create a more "continuous" resistance surface. This seems useful, and relates to the distinction between discrete land use classification and continuous spatial covariates.

The method used to simulate landscapes doesn't seem ideal, because:
  A. The high-fragmentation landscapes are essentially just white noise (every cell is assigned a different class independently). That's going to result in connectivity that's quite similar to the case where all cells have the same cost, I believe. So doesn't seem like a very useful test-case.
  B. The low- and mid- fragmentation landscapes are generated by assigning contiguous blocks of cells to a class. That's OK, but how does it give insight into real landscapes?
Given that only a single real landscape is included in the study, a lot of the oomph is riding on the generalizability of these simulations.

In summary, the general theme -- sensitivity of predictions about connectivity to assigned costs -- is useful. I'm less convinced that the particular questions asked here are useful. The manuscript is well-written and the figures are fine, although six figures is a bit much given the short length of the manuscript (especially as several of them are purely illustrative).

Ln 103: I suggest you use the interpretation of resistance distance as commute time (rather than voltage): it's proportional to the expected time needed to get from point A to point B and back again.

Ln 123: "unaware of similar analysis evaluating current density" -- I find that surprising given that commute time and passage probabilities are used heavily in other fields. Did you look into the computer science/graph theory literature? Commute-time distance (or variants thereof) is ubiquitous there and heavily employed in practical applications (e.g. Google's PageRank).

Ln 125: "landscape models" -- by this do you mean generative models of landscape, e.g. simulations from random fields?

Ln 132: Define what you mean by "themes"

Ln 133-134: "changing the spatial arrangement of relative cost weights" -- not clear what you mean here. Any sort of rearrangement of the raster would alter the spatial configuration of relative cost weights, no?

Ln 139: Given that the cost value scheme is central to the analysis, and that Rayfield et al 2009 is behind a paywall, please provide a bit more description of why you chose these values as a starting point.

Ln 144-151: this seems like a really simplistic way to simulate landscapes: why didn't you simulate via a random field model, were you could control the degree of spatial autocorrelation (for example, simulate a continuous-valued Matern process and "discretize" it)? For the "high-fragmentation" treatment, assigning each pixel independently will generate a surface that looks like white noise. This will, I think, result in resistance distances that are effectively very similar to log Euclidean distance.

Ln 167: "16" -> "6"

Ln 174: nitpicking ... "simulating" will mean stochastic simulation to most people. What you are doing is solving a large, sparse linear system: it's totally deterministic.

Ln 192: "Spearman rank correlation" ... so, you're asking whether a monotonic transform of the inputs produce monotonic transforms of the outputs? I'm frankly surprised that the Spearman correlation coefficients aren't closer to 1. I wonder if they would be, if you compared passage probabilities averaged across sliding windows (e.g. larger geographic areas than just individual pixels).

Ln 201-206: this seems like a pretty cursory analysis of the simulations. Are there particular parts in the graph where the change in cost value has a disproportionately high impact on the outputted passage probabilities?

Ln 206-209: But ... for comparing across fragmentation treatments, you're calculating correlations between outputs from completely different spatial configurations, right? Why would you expect the average correlation to be non-zero?

Ln 215-217: Again, I think this could benefit from a more thorough examination: *why* is the rank correlation in passage probability sensitive to range in cost values?

Ln 231-234: in line with previous comments-- this seems self-evident. Why test it?

Ln 243-244: interesting, and I see why Pearson correlation between outputs would be sensitive to concentration of current in particular cells ... but why would this change rank correlation?


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: EFFECTS OF COST SURFACE UNCERTAINTY ON CURRENT DENSITY ESTIMATES FROM CIRCUIT THEORY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have done an excellent job of communicating their findings
Experimental design: Methods are sound, and based on the approach of an important parallel study
Validity of the findings: I agree that the interpretation fits with the results
Additional comments: I have no additional comments. The authors addressed my previous comments thoroughly.


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: EFFECTS OF COST SURFACE UNCERTAINTY ON CURRENT DENSITY ESTIMATES FROM CIRCUIT THEORY
Review round: 2
Reviewer: 2

Output:
Basic reporting: See 'general comments'
Experimental design: See 'general comments'
Validity of the findings: See 'general comments'
Additional comments: You've adequately addressed my concerns, except for one point I am still confused about. When you say 'the spatial arrangement of costs is varied, while other aspects of the cost surface are maintained' (line 130) and 'changing spatial arrangement of costs is one form of rearranging cost ranks' (line 167-168), do you mean that cost values were swapped across classes while maintaining the spatial arrangement of classes? (e.g. the same landscape classification map, the same cost values, but different choice of which class was low/mid/high).

When I read the first version of this manuscript, I understood this analysis as something different: comparing current density across entirely different maps (pairs from the 10 simulations within a fragmentation level) for a fixed cost-weighting scheme ... for which case I fail to see why the expected rank correlation would be different from zero, or how comparisons across entirely different maps equate to 'rearranging cost ranks'.

In the methods, please clarify which of these is the case. Also, if possible, could you please include the code used to generate the simulated landscapes as a supplement (would help allay confusion such as the above)? Otherwise the manuscript is OK for publication, in my opinion.


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: THERE IS NO MAGIC BULLET: THE IMPORTANCE OF TESTING REFERENCE GENE STABILITY IN RT-QPCR EXPERIMENTS ACROSS MULTIPLE CLOSELY RELATED SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: A few minor comments on the manuscript:

1) Line 45 is vague and needs more description.
2) Line 47 - are they referring to gene expression?
3) Line 167 - RNA extraction from insect tissue typically requires glass-bead disruption.  It is not clear from methods whether this technique was used.
4) Line 232 - the authors state that they used three primer pairs for each gene. How did they determine which of the primer pairs to use in their analysis? In line 403, they indicate they used single primer pairs per each gene.  This needs to be clarified in the methods.
5) Line 236 - For real-time reaction mixes, the authors indicate they used 5 ml of cDNA, 10 ml of SYBR green, and 5 ml of primers. This seems to be an error as it should be microliter quantities.
6) Table 2 - it would be helpful to indicate the size of PCR amplicons for each gene/species
7) Figure 2 - the figure legend is minimal and needs some more description.
Experimental design: No comment
Validity of the findings: Lines 327-344: The authors attempt to provide the rationale for why they selected 2 or 3 references genes based on stability.  This is a confusing section and I suggest a re-write for better clarity and rationale.
Additional comments: In the manuscript, by Foquet and Song, the authors use a combination of RNA-sequencing data and quantitative real-time PCR to assess and validate the stability of reference genes used to quantify gene-expression changes in grasshoppers during the process of density-dependent phenotypic changes. This short report represents an important reference document for those studying gene expression changes in grasshoppers and can be used as a model for the determination of reference genes in other experimental systems. The manuscript is well-written and the analysis is comprehensive with appropriate literature references to support their findings. The experiments are well-designed and the data generated has been expertly analyzed. The presentation of the data in both figures and tables is sufficient. The conclusions are sufficient; however, a more clear description of the rationale for selecting reference genes would improve 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: THERE IS NO MAGIC BULLET: THE IMPORTANCE OF TESTING REFERENCE GENE STABILITY IN RT-QPCR EXPERIMENTS ACROSS MULTIPLE CLOSELY RELATED SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is clear and concise, well referenced, and the figures are well-constructed and easy to understand.  Tables 4 & 5 are a little confusing at first review, but I am at a loss as how to better organise them, as they are presenting (incorporating) multiple concepts and comparisons, which all need to be included. The RAW data appears to all be correct and support the figures and tables in the main text & figures.  The hypotheses are quite clear and understandable.
Experimental design: Good paper in general (please see General Comments for suggested revisions & clarifications). Well constructed description of a neglected issue and topic, likely to have more than a trivial effect on many studies. Methods have been describe in proper detail, mostly.
Validity of the findings: This reviewer has encountered many of the same issues and concerns that the authors have presented, working in a non-model animal system(s).  While there are some well-reviewed reference genes in model systems, in wild animals, or in non-traditional model systems, there are not well-documented reference genes.  It is often difficult to include these unique reference genes in publications, however this paper strongly argues for the proper assessment of chosen reference genes and more importantly details why and how this might be done.  Another important aspect of this paper is to point out the discrepancy in some of the software used to choose the "correct" reference gene(s).  Well explained as to why they may not give the best result in specific situations.
Additional comments: Overall, a good, solid paper.

Suggestions:

(1) Why did the authors choose not to include the MIQE guidlines worksheet as a supplemental?

(2) Consider using ng*mL^(-1) as opposed to ng/ml

(3) Consider including specific catalogue numbers for some of the PCR reagents, especially the Taq enzyme.  Was the Taq enzyme a hot start (HS) variant?  If not, why not?

(4) Please consider a brief discussion on the nature of the discrepancies between RNA-Seq & RT-qPCR data results.  Because these are two totally different techniques as described in your paper it is unlikely that they would give similar results.  RNA-Seq without UMIs is prone to significant PCR artefacts that RT-qPCR is not, and RNA-Seq has positional effects (3' bias) that RT-qPCR may or may not have depending on the positioning (choice) of the qPCR primers within the gene/exon.

Minor corrections:

(a) Was a value of 3.9 RQN considered "good"? Is that scale from 0 to 10?  If so, for locusts, are the scores skewed because of the nature of the totalRNA? Perhaps an explanation would be warranted?

(b) Why were the reads "converted" to Sanger format? The CASAVA 1.8 pipepline (by Illumina) onwards uses a standard Phred score system that is compatible with Sanger - unless I'm mistaken.

(c) What were the contaminating sequences? (line 191)

(d) What was the nature of the in silico normalisation method used (line 193)

(e) Lines 236-237, I believe that the mL should be microL (the "m" symbol got incorrectly encoded).

(f) Were there any primer-dimers as seen by agarose gel analysis (line 287-288).

(g) Line 362-363: "qPCR results agreed well ..." Not really, see point #4 above.

(h) Figure 2: The colouration of the bars is difficult to discern with the black being very close to the dark grey on a printout - can these be maybe switched to patterns (hashes/dots/etc).

(i) Table 4 & 5: You took the geometric mean, did you consider "voting" to choose the best gene amongst the three different software evaluations.


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: THERE IS NO MAGIC BULLET: THE IMPORTANCE OF TESTING REFERENCE GENE STABILITY IN RT-QPCR EXPERIMENTS ACROSS MULTIPLE CLOSELY RELATED SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Thank you for addressing the concerns and suggestions of the reviewer.


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: THERE IS NO MAGIC BULLET: THE IMPORTANCE OF TESTING REFERENCE GENE STABILITY IN RT-QPCR EXPERIMENTS ACROSS MULTIPLE CLOSELY RELATED SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
Additional comments: Thank you for the corrections.  Please include the version of R that the script was written for in the remarks (REM Statement) of the script, so that in case subsequent iterations render the script non-functional, one can benchmark the code.

Other than that, manuscript is ready-to-go!


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: GUT MICROBIOTA OF OBESE AND DIABETIC THAI SUBJECTS AND INTERPLAY WITH DIETARY HABITS AND BLOOD PROFILES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is presented in an intelligible fashion and written in standard English. The article include sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. However, there are several aspects of format that should be improved before publication:

1)	The scientific name of the taxa at any taxonomic level must appear on italic, for example, line 250-251 and line 295.
2)	In Table 2, in which the sPLS analysis is shown, the authors should please specify in some way, which associations correspond to sub-index a and which to sub-index b, since the relevance is not the same.
3)	The authors reveal that they have observed significant differences were noted regarding consumption of chicken (0.005), rice vermicelli (0.028) and fermented fruits or vegetables (0.038) between groups, but could the authors be clearer and to specify between which groups in the text (Line 201)?
4)	Line 263: Faecalibacterium showed significant difference between OV-OB and OB-T2DM comparisons (Fig. 3C), while [Prevotella] differed in L and OV comparison (Fig. 3D). What is the P-value and the P-value adjusted? Please, there are more cases like this in the text, please include this information.
5)	The clarity and resolution of figure 4 is insufficient and tends to confuse, I recommend an improvement in it for a more intuitive observation of the results.  In this same figure, DiabeM refers to the T2DM group? Why was it named differently in the first graph of figure 4?
6)	Please check the nomenclature of the groups of study, for example in the Line 271, groups V and B are mentioned, when you should put OV and OB, It is not like this?
7)	There is an error with respect to figure 6, in the text (Line 301) it is indicated "Dim 1 and 2 with large eigenvalues (2.28 and 1.96, respectively)" while the figure 6 shows other values Dim 1 = 12.8% and Dim 2 = 14.3%.

On the other hand, the authors provide in the supplementary material, the patients data (age, gender, weight, HDL-c, fasting glucose level, etc.), sequencing data processing of each sample, OTU richness and diversity indices of each sample, the food frequency questionnaire and the results of FFQ of each patient, by contrast, the authors not mentioned data deposition statement or at least the deposition information not is noted in the manuscript. According to Peer J policy the raw data, in this case fastq files must be publicly deposited.

Finally, the article complies with the ethical requirements and with the data protection of the subjects.
Experimental design: The topic of the manuscript is interesting but there are some problem.

The aimed of this paper to explore whether microbial composition and diversity has an association with host BMI and T2DM in a population of Thai subjects. However, the sample size is very small to each group of study with 8 subject in the lean and overweight groups and 7 subjects in the obese and T2DM groups.

Furthermore, the authors have not taken into account the homogeneity of the groups in terms of gender, especially it should be noted that the obese group is made up of 4 men and 3 women and the T2DM group is made up of 6 women and a single man. On the other hand, the ages are highly variable, finding a 71-year-old individual in the obese group, the youngest of this group being a subject with 24 years, so the average age of the group is 42 years. However, the T2DM group has an average age of 60 years, which added to the fact that it is made up of women almost exclusively, we could say that they are postmenopausal women.
These gender and age aspects are especially important when dealing with the analysis of the intestinal microbiota.

To this we must add that in the T2DM group we found 4 thin, 2 overweight and one obese. Therefore, when attributing the changes in the intestinal microbiota in this group with respect to the obese group, we cannot assure that it is due solely to the fact of having diabetes. Furthermore, if we see in Table 1, the characteristics of the study subjects, both the obese group and the T2DM present 3 criteria for metabolic syndrome, hypertension, high triglyceride levels and elevated fasting glucose.

Taken together all these aspects, the authors must better define the population under study, in order to reach the objective they are addressing in this article.
Validity of the findings: The topic of the manuscript is interesting and It is also novel because the intestinal microbial community is investigated in a population of Thai subjects, not previously described.

The methodology is described in sufficient detail and the analysis of the data. The statistical analysis appears to be technically sound. However, I would appreciate a more detailed explanation about the MFA analysis, as well as its results and its importance in the manuscript.

One aspect that authors should address is the use of QIIME 2 instead of QIIME 1. QIIME has been shown not to work optimally with baseline settings, furthermore this paper from 2010 does not show default settings for MiSeq. Why is the QIIME2 version not used? Please elaborate.

The same goes for the used version of the Greengene database (is not mentioned in the manuscriopt), even if it was the latest version of it, this is years old and in the meantime a lot has changed in taxonomy especially in phylotypes associated with the human GI tract. I would suggest the latest SILVA database.

The authors also recognize the weaknesses of the study population and the need to complement with other techniques for a deeper understanding of the subject.

Perhaps the real goal of this study should be to describe the gut microbiota in relation to BMI and T2DM in Thai subjects and to evaluate possible microbial patterns associated with their eating habits and blood profiles. Please consider this on Line 69.
Additional comments: The work in this study describes a cross-sectional 16S rRNA analysis of the bacterial community structure of the gut microbiota in 30 subjects stratified into three categories based on BMI and a group with diabetes as well as the relationship with their eating habits and blood profiles in a population of Thai subjects.

The first issue stems from the fact that the sample size is very small for a microbiome study that was designed to assess the role of BMI and T2DM on the composition of the gut microbiome. Indeed, the authors themselves point out that there have been many conflicting results published on changes related with obesity and T2DM  in the gut microbiota that likely reflect differences in the populations under study, small cohort sizes, complexity of the obesity phenotype, confounding factors such as age, gender, diet, etc.  Other published reports have described gender-related differences in the microbiome from the gut (Mueller et al., AEM, 2006; Markle et al., Science, 2013; Santos-Marcos et al., Mol Nutr Food Res., 2019; Kim et al., World J Mens Health, 2020). In addition, In this manuscript both the obese and T2DM groups have metabolic syndrome, according to the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATPIII) the subjects have elevated fasting glucose, hypertension and elevated triglycerides levels. In this sense, the recent literature supports the gut microbiome’s potential influence on the various risk factors of metabolic syndrome (Gildner., Evol Med Public Health, 2020;  Dabke et al., J Clin Invest, 2019). Finally, the group of diabetics is much older, this could be influencing the composition of the microbiota (Nagpal et al., Nutr Healthy Aging, 2018; Maynard et al., Subcell Biochem, 2018) also the majority of subjects in T2DM group are women and have been observed changes in the intestinal microbiota  in post-menopausal women (Vieira et al.,  Front. Microbiol, 2017). All these works suggest the potential involvement of differences in gut microbiota in the unequal incidence of metabolic diseases. How might these findings influence your results? The differences between and significance of the findings from this study in light of these previously published results should be discussed. At a minimum the authors should discuss the power calculations that were used to determine the size of their cohort. If this cannot be done, then the conclusions need to be tempered to say that their conclusions are valid for this set of subjects only and may or may not be generalizable to a larger population study.

Last, antibotic usage in the analysis of the gut microbiota is also important.  Have subjects taken antibiotics or prebiotics / probiotics or some type of drug prior to evaluation of the gut microbiota?


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: GUT MICROBIOTA OF OBESE AND DIABETIC THAI SUBJECTS AND INTERPLAY WITH DIETARY HABITS AND BLOOD PROFILES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Grammar and punctuation corrections needed
Experimental design: 1. Are there any sampling bios? If so how to rectify that.
2. selection criteria for the subject are required.
Validity of the findings: Faecalibacterium prausnitzii, a butyrate-producing bacterium, significantly higher in OB than that in other groups-discuss about this
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: GUT MICROBIOTA OF OBESE AND DIABETIC THAI SUBJECTS AND INTERPLAY WITH DIETARY HABITS AND BLOOD PROFILES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript is an observational study of Thai adults. Subjects were classified according to their obesity and diabetes, to compare the differences between their eating habits and gut microbiota.

1) The language is not bad.
2) To clarify the purpose of the study, it is necessary to modify the paragraph structure of the introduction.

1st: An overview that obesity and diabetes are continuously increasing in recent years and are becoming a socioeconomic burden.
2nd: Overview of the growing interest in research on intestinal microbiota and correlation with various diseases
3rd: Explain that obesity is universally described as BMI, along with existing studies showing that westernized eating habits are related to some diseases
4th: Explaining that there is a need to check the relationship between eating habits and gut microbiota depending on the obesity and diabetes of Thai citizens.

3) Nevertheless, this study seems to have one major flaw in the hypothesis. This study compared an average of 60's obese/overweight diabetics with the general population, and it is difficult to say that these subjects represent diabetics.
Experimental design: In the extension of 3) mentioned above, I'd like to add more details.

Subjects were divided into 3 groups according to the obesity level.
How about comparing the difference between diabetes and non-diabetes in each group after diabetics are included in the group according to obesity level?

If the baseline characteristics start with the existence of differences between groups, the meaning of comparison seems to be diminished.
Validity of the findings: Gut micriobiome(OR microbiota) studies in populations that do not focus on westernized diets have been conducted in several countries. In particular, I'd like to recommend that the authors search for additional studies targeting populations in East Asia.
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: GUT MICROBIOTA OF OBESE AND DIABETIC THAI SUBJECTS AND INTERPLAY WITH DIETARY HABITS AND BLOOD PROFILES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Manuscripts were written in an easy-to-understand manner and in standard English.

The composition of the paragraphs pointed out in the INTRODUCTION section has been revised as appropriate to reflect the reviewer's points.
Experimental design: The sample size is still very small, which will remain the biggest weakness of this paper.
No matter how gender and age are considered, it is difficult to say that these analysis results represent typical Thai adults.
Validity of the findings: Despite the methodological limitation, the results described in this paper are interesting. This is because the authors have observed changes in the gut microbiota of Thai adults that have not been previously identified.
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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF LACTIPLANTIBACILLUS PLANTARUM RS66CD BIOFILM IN HIGH-SALT CONDITIONS AND PLANKTONIC CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The first part of the introduction contains multiple examples of cited articles which are not appropriate for the statement that they are cited for. This undermines the scientific credibility of the statements. Please correct these. Here are three examples:

1) Line 29-31: George et al 2017 is cited to support the claim that L. plantarum is used in many different fermentation processes. The cited article however is a study on sub-inhibitory concentrations of gentamicin for L. plantarum, which is irrelevant for this statement. Please cite either a good review article or a studies that actually proved that L. plantarum is found/used for such a fermentation.

2) Lines 31-34: Two papers are cited after the claim that “L. plantarum plays a vital role in human health” (see also below). However, none of these papers actually discuss the role of L. plantarum in health, they instead focus on other lactobacilli. Lactobacillus is a very broad genus and therefore I am not sure that all these health effects also apply to L. plantarum. Your text does not say this exactly, but by placing these examples behind the claim that L. plantarum plays a vital role in human health, you do seem to insinuate that these health effects apply to L. plantarum as well, which I believe is false. Furthermore the second cited manuscript describes the role of a Lactobacillus EPS in colon cancer and is totally unrelated to the claim that you make regarding “reduce fasting blood glucose levels in diabetic patients”.

3) Shi et al, 2018 is cited to support the claim. The cited article does not mention number of live L. plantarum during production and storage of fermented foods. It instead discusses L. plantarum numbers in a simulated gastrointestinal tract, which is a completely different environment than fermented foods. In addition, I do not believe that this claim is true as many L. plantarum strains start to thrive during the production of fermented foods such as fermented vegetables.

In addition I think that the following claim definitely needs revision:

Line 31: “L. plantarum plays a vital role in human health”. I think this statement is too strong. I believe that L. plantarum could be linked with some health effects, but stating that it plays a vital role seems to be a bridge too far. If you disagree, please provide a good research paper or review that supports this claim.

Finally, the English of the manuscript is of sufficient level to understand the manuscript, but should be proofread a couple of times before resubmission because it does contain several errors that could easily be caught by proofreading:

Lines 13-4: “Lactobacillus plantarum (L. plantarum) that is commonly used producing fermented foods plays a vital role in human health.” should be “Lactobacillus plantarum (L. plantarum), which is commonly used for producing fermented foods, plays a vital role in human health.”
Line 18: “[..] and analysis of differential genes with compared to [...]” should be “[..] and analysis of differential genes compared to [...]”
Line 29: “hetero fermentative” should be “heterofermentative”
Line 31: L. plantarum should be in italic
Line 48: “Mg2+ can promoted the formation of initial biofilm” is grammatically incorrect.
Line 90 -91: Please rewrite this sentence to make it more easy readable.

Apart from this, the goal of the manuscript is clear and seems interesting to pursue.
Experimental design: In my opinion, the methods section fails to meet the standards set by PeerJ. It is missing crucial information which would be necessary for reproduction. Starting with the fact that the sequencing data is not publicly accessible. In addition, the bioinformatic analysis is not thoroughly described, scripts that were used to process the data are missing, tools used are not properly cited and manufacturers names of reagents are often missing. This section needs to be updated and especially the bio-informatic protocols need to be described in more detail.

Here are some specific comments which could help to improve this section:
- The sequencing data is not published in an acceptable discipline-specific repository. In fact, it is completely missing. Please upload your data to ENA and provide an accession number in the manuscript.
- To improve reproducibility please add manufacturers names after each product/platform. E.g. lines 80 -85 miss a lot of information.
- Line 68: GenBank MN160322 points to the 16S rRNA gene sequence. I believe that the whole genome sequence would be better here.
- Line 68: Why was this L. plantarum strain chosen? Because of its probiotic effects or because of its biofilm formation capabilities? Please elaborate
- Line 85: What was the read length used for sequencing?
- Lines 88 -89: What do you mean with “Based on read quality, alignment, saturation, and distribution of reference reads.”? It is unclear what action has been performed on these parameters and I also do not understand the meaning of the word "saturation" in this context.
- Line 92: “The result of the test is in accordance with the difference heterogeneousness criterion (p＜0.05 was used to 93 filter statistically significant results)”. I do not understand what you mean with this. Please elaborate.
- Lines 94-95: Please cite the manuscripts coupled with the Goatools and KOBAS tool instead of linking to the software’s homepage.
- Lines 95 - 96: “Biofilm formation of differential expressed genes were used for hierarchical cluster analysis using multiple experiment viewers, and the heat map.” This sentence is unclear to me. I believe that you mean that biofilm formation in the conditions that these genes were differentially express were used for… Please correct.
- Lines 99 - 100: Why was a different method used for RNA extraction and cDNA synthesis for the RT-PCR section? It would make more sense to perform the RT-PCR on the exact same samples used for sequencing. If this was not possible, please explain why.
- Line 107 - 108: “Data was tested using duncan's multiple range test in SPSS 22.0 for Windows”. This is a broad statement that is unclear. What data was tested? And what for?
- Line 109: edgeR software was used to “analyse bioinformatics data”. First, please properly cite this package with the relevant manuscripts. Second, the fact that edgeR was used for differential expression analysis should be mentioned in that section and not here. Third, scripts that explain this analysis should be provided or otherwise please provide a short summary of what steps were performed within the edgeR analysis.
- In general: A complete section is missing which describes how the RNA reads were further processed. To what genome were the reads mapped? How was this genome annotated? How were the mapped reads counted?
Validity of the findings: Due to the shortcomings of the above two sections, I do not feel that I can properly evalute the findings of this manuscript in its current form and have no feedback in the current stage.
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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF LACTIPLANTIBACILLUS PLANTARUM RS66CD BIOFILM IN HIGH-SALT CONDITIONS AND PLANKTONIC CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the current manuscript, the authors have investigated the effect of different concentrations of NaCl on biofilm formation by a probiotic L. plantarum strain. The 48 g/L concentration has been then used for further experiments including SEM and the differential gene expression analysis. Finally, qRT-PCR has been performed for validation of transcriptome data which showed a good correlation.

Introduction:
The aim of the study is not reasonable and the problem statement could not vindicate the necessity of the study. Particularly when we see that they have ignored the available data on the topic such as:
De Angelis, M., Siragusa, S., Campanella, D, Di Cagno, R. and Gobbetti, M., 2015. Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200. Proteomics, 15(13), pp.2244-2257.
Ramírez, M.D.F., Smid, E.J., Abee, T. and Groot, M.N.N., 2015. Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates. International journal of food microbiology, 207, pp.23-29.
Muscariello, L., Marino, C., Capri, U., Vastano, V., Marasco, R. and Sacco, M., 2013. CcpA and three newly identified proteins are involved in biofilm development in Lactobacillus plantarum. Journal of basic microbiology, 53(1), pp.62-71.

Results:
Line 113 and Fig. 1: What has been the control treatment? The effects of various concentrations of NaCl have been compared to each other. The lack of a suitable control has led authors to select high NaCl concentrations which are not applicable in food industry. Moreover, the letters above the columns should be reordered in Fig. 1.
Line 114 and Fig. 2: The SEM images are not informative and have not made any sense!
Fig. 3. What is log2(FC)? How should we interpret this figure? The figure caption should be written in a way that everybody can understand it without referring to the paper text.
Fig. 4. Please provide an explanation on how to interpret the figure.
Fig. 5. Please add information on the genes investigated to the figure caption. An explanation on how to interpret the figure should be also provided.

Discussion:
The discussion is a literature review rather than making links and comparisons between results of the study and previous works. The reports on the biofilm formation mechanisms in L. plantarum have been completely ignored which is the main weak point of the discussion part.

References:
The references are listed in order of their first mention in the text and the reference list is not alphabetical.
Line 156. Neuhaus and Baddiley et al.: et al should be omitted.
Line 162. Typas et al., 2011: in the reference list the year of publication is 2012.
Line 190. The reference "Bourret and Silversmith, 2010" was not found in the reference list.
Line 354. A.M Stock, et al. was not found in the text.

English syntax:
English throughout the whole manuscript should be revised. Here are some examples:
Line 48. Mg2+ can promoted the…
Line 54. … these result provide a…
Line 60. …under biofilm forming conditions and  nisin resistance operon…
Line 62. Treatment…
Line 88. . Based on read quality, alignment, saturation, and distribution of reference reads. (is it a sentence?)
Line 189. HPK on the plasma membrane of the cell to sense external signal stimulation.
Experimental design: The manuscript fits within the journal scope. however, considering the available reports on the mechanisms involved in biofilm formation by L. plantarum  especially that of Angelis et al. (2015) entitled "Comparative proteomic analysis of bioﬁlm and planktonic cells of Lactobacillus plantarum DB200", the only novelty of this ms seems to be the in detail investigation of NaCl effect on formation and development of biofilm by this species. However, some questions arise here:

Biofilm formation is important in relation to food spoilage and pathogenic organisms. Although some L. plantarum strains contributing to food deterioration, the strain examined in this study is a probiotic one and the authors intended to induce biofilm formation in this bacterium as a tool to overcome the food-related stresses. But are we allowed to enrich high-salt foods with probiotics? According to CODEX, it is not recommended for foods containing more than 450 mg sodium per serving or 100 g. The recommended daily intake of salt is 6 g/d (=2.4 g/d sodium). The in vitro concentration of 40 or 48 g/L is too high to be used in healthy foods and the alternative methods such as encapsulation seem to be more efficient and reasonable!

Materials and Methods:
Line 73. The MRS medium has been used as a control of Mg and Mn free MRS with added NaCl. But we know that even trace amounts of Mg and Mn pose effects on biofilm formation and the growth ability of bacteria. To exclude their effects, why have not the authors used the MRS medium with added NaCl as a treatment medium?
Line 86. More details should be provided on the differential gene expression analysis.
Table 1. Please provide the target genes and references for each primer set.
Validity of the findings: The main aim of this study is questionable, as increasing biofilm formation by a probiotic strain under high osmotic pressure and such high amounts of NaCl can not be considered as a good tool to improve probiotic stability in food products! how can the food with 4-5 percent NaCl be considered healthy?
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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF LACTIPLANTIBACILLUS PLANTARUM RS66CD BIOFILM IN HIGH-SALT CONDITIONS AND PLANKTONIC CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Minor comments:
1.	The English language could be somewhat improved to ensure that an international audience can clearly understand the text.
Some examples where the grammar could be improved include lines in the Abstract:
-	“analysis of differential genes with compared to planktonic cells by RNA-seq technology (Illumina sequencing)”
-	“Lactobacillus plantarum (L. plantarum) that is commonly used producing fermented foods plays a vital role in human health.”
-	It is also not clear what you mean with “activity” in the sentence “The biofilm formed by the bacteria can effectively increase the activity of the cells”? Do you mean transcriptional activity?
Line 180: “acids, which play a central role in increasing the ability of to form biofilms (Sílvia et al., 2008).”
Line 257: “This study has no supported by funding sources.”
2.	EPS abbreviation should be explained.
3.	Please provide more explanation in the figure legends. For example:
Figure 1: Please indicate if the data columns represent means or medians of the three repetitions. For the letters above the bars: these are statistically significant differences, but compared to what (which column is compared to which other column)?
Figure 5: Please explain in the legend what P1, P2, P3 and B1, B2, B3 mean.
4.	Figure 2: Please indicate where the extracellular matrix is located in the photo.
Experimental design: Minor comment:
1.	In the section “2.2 RNA extraction, library preparation, and sequencing”, manufacturers of the reagents and kits should be mentioned.
Validity of the findings: Major comments:
1.	Biofilm formation was promoted by the addition of 48 g.L-1 NaCl. It should be discussed how relevant this is in real life, for example in the industrial setting. Is 48 g.L-1 NaCl a relevant concentration?
2.	Biofilm was grown in 48 g.L-1 NaCl in modified, MgSO4 and MnSO4-free MRS medium, while the control condition was grown in MRS. This might be a limitation of the study. Is the transcriptome change due to the high levels of NaCl and absence of MgSO4 and MnSO4, or due to biofilm formation?
3.	In the manuscript there is a statement: “KEGG pathway analysis showed that genes coding for D-Alanine metabolism, peptidoglycan biosynthesis, two-component system, carbon metabolism, bacterial secretion system, lysine biosynthesis and fatty acid metabolism were crucial for biofilm formation.” However, changes in gene expression during biofilm formation do not necessarily indicate that these genes are crucial for the biofilm formation itself. These changes might be signs of metabolic changes happening in parallel with the biofilm formation, due to high NaCl concentration, etc.
A more critical view of the findings and additional discussion is encouraged. For example, it should be mentioned what additional in vitro experiments should be performed to validate the findings (e.g. making L. plantarum knock-out mutants in the described genes).
Additional comments: The article poses a relevant research question, as less is known about biofilm formation by beneficial lactobacilli compared to pathogens. An advanced transcriptomic approach is used to reveal the gene expression changes linked to L. plantarum biofilm formation. A wide array of potential hits is identified, and a detailed overview is provided of the genes and their function in the discussion section. This article provides novel data of scientific interest and can pave the way for future studies.


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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF LACTIPLANTIBACILLUS PLANTARUM RS66CD BIOFILM IN HIGH-SALT CONDITIONS AND PLANKTONIC CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: - During the revisions of this manuscript, a new nomenclature for Lactobacillus has been published (see https://doi.org/10.1099/ijsem.0.004107 or http://lactobacillus.uantwerpen.be). I strongly encourage the authors to update their manuscript according to this new nomenclature. In this case, Lactobacillus plantarum, would need to be changed to Lactiplantibacillus plantarum.

- Line 46: Lactobacillus rhamnosus is mentioned for the first time so should not be abbreviated. Also according to the newest nomenclature this species is now called Lacticaseibacillus rhamnosus

- Lines 50-60: Several biofilm-promoting agents are presented here, but it is unclear whether these are all related to L. plantarum. Please add the organism names next to each claim for clarity. E.g. Mn2+ promoted biofilm formation in L. plantarum….

-Line 123: “DGEs” should be corrected to “DEGs”.

-Line 241: These species should first be written in full format instead of abbreviated. Also please update to new taxonomy.
Experimental design: - Lines 78-79: Are these experiments published? If so, please add a reference, if not maybe add a statement between brackets (unpublished results).

- Line 108: The data was submitted to NCBI but lacks good medata, which makes it hard for others to reproduce. I believe more effort should be put in providing decent metadata. For example the abstract reads “The RNA-seq for L. plantarum biofilm and planktonic cells”. Providing the abstract from this manuscript would already give better context. In addition, each sample was submitted with another strain isolation name (e.g ”a1”, “a2”, …) which is not correct as only strain RS66CD was used for the experiments, please correct. Furthermore the “sample type” is incorrect. You state “single-cell” while it should be “cell culture” (see https://submit.ncbi.nlm.nih.gov/biosample/template/?package=Microbe.1.0&action=definition). The same goes for the sample description, wherein it would be useful to provide the Na+ concentrations.

- Line 110 - 118: Citing bio-informatic tools is of importance for the developers to be able to keep attracting funding. Please cite the resources you use. There are missing citations for Trimmomatic, Rockhopper, EdgeR, GOAtools and KOBAS.

- Line 159 - 160: Please cite the KEGG and GO databases.

- Line 164: What twelve pathways are putatively involved in the formation of biofilm?
Validity of the findings: - Line 167: figure 5 should be updated. It is hard to interpret the short gene names only and matching them to Table3. Also some genes shown on the figure are not in table 3 (FabZ). The readability of this figure would be improved if the full gene annotation is shown next to the short gene names.

- Lines 170-177: It is unclear to me what the motivation for selecting these specific gene is. Please provide a reasoning on why these genes were specifically targeted with qPCR.

-Lines 178-...: The whole discussion is hard to follow. It reads more like a literature study than a discussion of the research results and is hard to interpret. Please update this section by guiding the reader through your results. For example it is hard to distinguish which genes were upregulated in this study and which were found in literature. You could start each by more specifically pointing out which genes were upregulated and then start discussing them.

-Line 207: Why is dacA relevant here? I do not see it in Figure 5. Is it also a DEG?
Additional comments: I would like to thank the authors for revising their manuscript. I have reevaluated the work and now also reviewed the results and discussion sections, which I did not assess previously. The main weakness of the manuscript in its current state is the discussion. It is really hard to follow and sometimes seems unrelated to the results of the manuscript. Although all genes discussed there are DEGs, it is sometimes unclear how they are related to biofilm formation. Maybe a reduction of this section to only keep the relevant information would be useful.


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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF LACTIPLANTIBACILLUS PLANTARUM RS66CD BIOFILM IN HIGH-SALT CONDITIONS AND PLANKTONIC CELLS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscripts has been well structured. In the revised version, the aim of the study has been well explained. Now, we know that the test strain is not a probiotic one, but still it can be used as a fermentative strain in high-salt fermented foods like the traditional one mentioned in this manuscript. Relevant references have been added to the revised version.
The English syntax has been improved; but there are minor errors in the revised version:
-formation biofilm or biofim formation?
- lines 80-81: Previous experimental results show that L. plantarum RS66CD was able to form biofilm
Experimental design: The revised methods have been expalined in details.
Validity of the findings: The revised version has provided explanations on how to interpret the figures and the questions have been well explained.
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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF LACTIPLANTIBACILLUS PLANTARUM RS66CD BIOFILM IN HIGH-SALT CONDITIONS AND PLANKTONIC CELLS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors made significant efforts to improve the manuscript regarding methods and discussion, as well as language and appropriate references. A few aspects remain:

1. The parts added in the discussion as tracked changes are relevant, however they would benefit from another proofreading, as it is not always clear what the authors mean (e.g. “The biofilms of L. plantarum were reduce the density by destroy extracellular matrix (Fernández Ramírez, et al., 2015), which further indicates that the formation of related proteins can promote biofilm formation”).

2.	Figure 1 legend is now better explained, however the meaning of a, b, c... is still not clear. If there is a letter “a” above the column, the result is significantly different compared to what? Please define what each letter means.
Experimental design: No comment
Validity of the findings: 3. I am still not entirely convinced by the authors’ reply in the rebuttal to my comment “These changes might be signs of metabolic changes happening in parallel with the biofilm formation, due to high NaCl concentration, etc.”.
The authors should emphasize in their manuscript that this work is focused on gene expression during the formation of biofilm specifically as a result of high salt concentrations, and biofilm formation without such high salt concentrations might involve different mechanisms. It might be appropriate to change the title from “Comparative transcriptomic analysis of Lactobacillus plantarum RS66CD biofilm and planktonic cells” to “Comparative transcriptomic analysis of Lactobacillus plantarum RS66CD biofilm in high-salt conditions and planktonic cells”.
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: INCREASING METHANE (CH4) EMISSIONS AND ALTERING RHIZOSPHERE MICROBIAL DIVERSITY IN PADDY SOIL BY COMBINING CHINESE MILK VETCH AND RICE STRAW
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are a few sections that need to be rewritten for clarity and consistency.

    • What is green manure? Define in introduction (first use line 38). try to be consistent with usage of terminology throughout manuscript.
    • Line 40 should read ‘… is also unclear’
    • five-point sampling method … citation needed (line 122)
    • missing quality control steps in methods, including parameters used (line 179)
    • What R packages are you using to perform CCA, Bray-Curtis, etc. calculations? (line 179)
    • Provide citations for the R packages when included
    • Were there any post processing of the data? Counts turned to relative abundance? This information should also be present in the methods.
    • ‘If’ should be ‘if’ (line 206)
    • sentence on line 206, “These results indicated ...” should be rewritten for clarity.
    • Dilution curve should be rarefaction curve (line 225)
    • Line 258 is better written: ‘...because soil fertility and crop yield are improved...”

Figure 2. It may be better to show all data points on your plot to more intuitively visualize the variation in the data. I would recommend boxplots here with your individual data points overlayed.
Experimental design: In general, describing your methods in more detail, including those used in data analysis, would help the reader understand in more detail what was done with the data without assumptions.
Validity of the findings: There is some inconsistency or a lack of clarity in the presentation of the results in the discussion. Please see below. In general, I think elaborating further in the discussion would be beneficial as there are many results that are not discussed.

• It is possible to perform an an analysis of similarity (anosim R package) on the microbial community composition data (Fig 3C/D) to test if the groups had significantly different compositions. This would strengthen this area. Additionally, perMANOVA (adonis2 package) would also work here.

• line 220 .. Please comment  on the lack of differences between experimental treatment. It would seem the statement is correct relative to the control, however additional nitrogen in the form of Chinese milk vetch seems to reduce the methane emissions in the presence of straw.
• The section ‘Effect of green manure and straw on rice yield and CH4’ seems to contradict the results of the present study. The results of this study indicate that additions of straw and Chinese milk vetch increase methane emission (figure 2), which contradict the statements throughout this section. Elaborating further on why these results contradicts expectations from the literature should be included.
• It is a recurring statement that the short term application of the Chinese milk vetch may have contributed to the observed results of the present study. A comment on what the mechanism is that contributes to a difference over longer application periods would be beneficial to the reader.
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: INCREASING METHANE (CH4) EMISSIONS AND ALTERING RHIZOSPHERE MICROBIAL DIVERSITY IN PADDY SOIL BY COMBINING CHINESE MILK VETCH AND RICE STRAW
Review round: 1
Reviewer: 2

Output:
Basic reporting: Rice is one of the most important staple foods worldwide, yet it is known that most of rice cultivation, carried out under flooded conditions results in high amounts of methane emitted during waterlogged conditions. Only a fraction of applied chemical fertilizer is utilized by plant and the microbes, whereas the rest leaches into the groundwater. Finding a green alternative, which does not result in large yield loss of rice is scientifically as well as socio-economically important matter.
Current manuscript investigates an important environmental problem from the microbiological diversity perspective. Most of the methane is produced by methanogenic archaea, anaerobes known to thrive under waterlogged conditions. Furthermore some aerobic methanotrophs are dependent on nitrate-such as ANME-2d archaea and nitrite, such as M. oxyfera bacteria, as electron acceptor. The healthiness of soil is dependent on the microbial community of the soil. Novelty of this manuscript lays in investigating the fungal community alongside that of the bacterial. Fungi as the ultimate degraders of complex organic matter, supply the soil with bioavailable carbon, yet have not been addressed as often as bacteria even though they play a key role in the carbon cycle.

The written english language would benefit from a second check as at times verbs are missing in the sentences. It is clearly structured, although in the introduction the link between methane and microbes is not discussed, although majority of methane under waterlogged conditions is produced by methanogenic archaea. Nor are important microbial groups considered.
The sequencing data referred to at lines 185-186 does not have an accession number under which it was deposited.
The manuscript would be easier to read if treatments wre referred to with always same terms, currently f.e. green manure and effcts f chinese milk vetch are used interchangably.
The figure legends should be rechecked. On figure 1, the error bars lean to different sides.
Experimental design: Research question is defined, and surely authors have done a lot of experimental work as well as sequenced the data and checked chemical properties of the soil.
The section of materials and methods needs some more clarifying as authors refer to local practices which are unknown for the reader or us terms as reasonable amount of data. The way statistical analysis was conducted in R needs more clarification and details.
Validity of the findings: Despite of a lot of work being carried out, the findings are not very well presented, analysed or in depth described. For methane emissions, it is known that once fields become flooded and rice is cultivated, methane emissions do increase. Rice straw addition does increase methane emissions as well. What is the role of the vedge addition remains unclear. The manuscript needs a bit more structuring as it is not clear how the pot and field experiment on different years and soils can be compared. One of the large analysis methods relies on 16S rRNA gene to address the microbial community composition. For 16S one can go down to atleast to order and family level. Authors have a lot o sequencing data, yet results contain a very small section on most abundant phyla with the second one remaining unassigned. This should be rechecked. As these results are presented in context of methane, the methanotrophic bacteria found in this dataset deserves a closer look.
In the discussion section, a lot of comparable research has been carried out, as paddy fields are well studied ecosystem, though it is no referenced and should be revised carefully.
Additional comments: The current manuscript has potential as it adresses an important topic of rice as staple food, grenhouse gas emissoins and unerlaying microbial community in the rhizosphere.
In the introduction covering the aspect of methane and microbes should be included.
In the section of materials and methods, to use only one term to refer to a treatment would make the manuscript more easily readable. Possibly adding a table instead of description.
In the result section, major part of the manuscript is sequencing data, yet if the second largest phylum remains unassigned, the data should be rechecked. As it is possible to look into the abundance of methanotrophic bacteria, one should go to that level instead of phylum.
Discussion should berevised as about paddy fields and sraw addition many studies have been conducted. To describe the effect of chinese vedge  and how we can mitigate methane or use less nirtogen fertilized could be the niche of this manuscript.

I have found several specific points whic improved, could benefit the manuscript.

Introduction:
L39-40 sentence lacks a verb. Suggestion: Instead of also use is
L51: changes over the cultivation period
L59-60: after the return alter to addition of Chinese milk vetch to paddy fields
L60: paddy soil or rhizosphere soil?
L60-61: Our goal in this study->The goal of this study or our goal; in this study is redundant. It is a long sentence with not clear message, e.g under the condition of straw. I suggest to restructure
L63-64: Which inputs? Microbial and fungal communities instead of components. As the title is about methane emissions, it would be good to add that to the importance.
General suggestion for the introduction:
As this manuscript addresses methane emission and microbial community changes, the authors could add a paragraph to explain how green fertilizer addition affects the methane emissions via microbial community changes. Explain that most of emitted methane originates from the methanogenic microorganisms present in the paddy field soil.

Materials and methods:

L71-73Authors could condense the lines by in average the annual sunshine was X, rainfall Y, etc. Instead of repeating average.
L74: How is yellow mud soil developed by river impact material. What is the river impact material?
L94: Check the formula for calcium magnesium phosphate
In the field experiment, nitrogen fertilizer was added to the experimental plots as well. How do the amounts of nitrogen in the form of urea compare to the amounts of fertilizer supplied to the fields where no vetch is cultivated over the winter?
L107: Could the authors explain, what is meant by irrigation management being the same as local routine? The reader very likely has no idea which is the local routine there.
L112-113: Could the authors explain the pesticide and herbicide local practices. How often, which ones and in which amounts were these added?
L115-117: Rice root microbiota is a different microbiome than the one in the rhizosphere and soil. Please use same term throughout the manuscript, especially for the samples you investigated.
L120: for microbial analyses->until further processing. Either DNA extraction or soil analysis.
L129: Add a space before reference
L150: Could the authors add the packages which were used in R for analysis?
Results:
L192: what is early rice ear stage?
L193-196: It is unclear what is meant under rice winter fallow, green manure rotation. Please rephrase.
In comparison to the control, the higher methane emissions when rice is planted, with or without straw could be expected. Could the authors elaborate more on the effect of the straw on methane emission or different nitrogen treatments?
L205-206: For clarification: in case the fertilizer was applied, treatment a, with or without straw addition, the rice yield was the highest?
L206:capital letter I the middle of the sentence
L213: space needed before reference
L225: Do authors mean rarefraction curves? What is meant by data was reasonable?
L235: How was the data analysis carried out, which resulted on the phylum level unassigned group to be second most abundant? Were these the unclassified reads pooled? On phylum level it is unlikely to have a large fraction of unassigned reads I these passed the quality control.


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: INCREASING METHANE (CH4) EMISSIONS AND ALTERING RHIZOSPHERE MICROBIAL DIVERSITY IN PADDY SOIL BY COMBINING CHINESE MILK VETCH AND RICE STRAW
Review round: 1
Reviewer: 3

Output:
Basic reporting: English
The English language could be improved in the text. More in detail:
-	Line 91-92: substitute the original sentence with: “the two factors were combined to yield six treatments (i.e Va, Vb, Vc, SVa, SVb, and SVc). A control treatment (NSV) was established with winter fallow before rice cultivation”
-	Line 95: substitute the original sentence “normal phosphate and potassium fertilizers” with “usual phosphate and potassium fertilization management”
-	Line 132: substitute the original sentence with “as reported by Avnimelech et al., 2001.”
-	Line 157: substitute with “air-dried and sieved at 6 mm size.”
-	Lines 305-309: the structure of this sentence should be improved. You could divide in two separates sentences.

Literature references, field background/context.
Your introduction provides the right context and background for many of the points. However, it needs to be improved for the following general aspects and specific points:
-	Lines 33-40: the reasons and the strategies for the reduction in the use of chemical fertilizers, for the increase in crop NUE and for the use of green manure and straw return to the soil should be presented in a more comprehensive way, more related to the experiment carried out and bringing more references from the literature. More in detail, the relationship between the above-mentioned themes and the expected effects on soil fertility properties, soil and rhizosphere microbial and fungal population, and greenhouse gases and methane emissions, especially for paddy systems, should be described. You may include the following references: Dawson, et al.. "Characterizing nitrogen use efficiency in natural and agricultural ecosystems to improve the performance of cereal crops in low-input and organic agricultural systems." Field Crops Research 107.2 (2008): 89-101; Bhattacharyya, P., et al. "Effects of rice straw and nitrogen fertilization on greenhouse gas emissions and carbon storage in tropical flooded soil planted with rice." Soil and Tillage research 124 (2012): 119-130; Bertora, Chiara, et al. "Dissolved organic carbon cycling, methane emissions and related microbial populations in temperate rice paddies with contrasting straw and water management." Agriculture, ecosystems & environment 265 (2018): 292-306.
-	Lines 115-117: move this sentence to the introduction section.
-	Lines 44-48: you should provide references that could explain why the experiment focused on microbial and fungal population in the rhizosphere and not also soil, the microorganisms investigated and which are the expected effects for plant nutrition and methane emissions due to microorganisms structure change. For reference, also see Conrad, R. (2007). Microbial ecology of methanogens and methanotrophs. Advances in agronomy, 96, 1-63; BERG, Gabriele, et al. Unraveling the plant microbiome: looking back and future perspectives. Frontiers in microbiology, 2014, 5: 148.
-	The scientific context (and reference) for the methane pot experiment is not provided, as well as the illustration of available methodologies used in this field.

Article structure, figures, tables. Raw data.
Specific comments on Tables, Figures and Raw data are:
-	Table 2: the comparison of CH4 emissions in the different treatments are well provided in Figure 2, as emissions rates. For this reason, it is better to remove this parameter from the table and to include the results in terms of kg/ha in the text, comparing this data with data from literature.
-	Table 3 should be structured as Table 2, with parameters in column and treatments in rows. Also consider to improve the visualization of the table by  separating bacterial and fungi data.
-	Figure 1 shows different p(F) values. However, the ANOVA should be performed as for Table 2 data;
-	Figure 1 shows the pot rice production data with different findings compared with field data, which causes confusion and are not discussed in the paper. You should explain the findings from the pots experiment in the text and maybe removed them from the figure, while providing them as supplemental materials;
-	Figure 1: It is not clear if the yield data refer to one rice variety or if an average of the two varieties. This aspect is very important, if rice varieties show a similar or different effect.
-	Figure 3 should be simplified, for helping readers focusing on the main aspects. Graph A to D, that shows results not discussed in the text, could be removed from this figure and included in supplemental materials.
-	Raw data for Table 2 should provide the replication numbers for each treatment

Hypotheses and results.
-	The hypothesis formulation should be improved in the introduction section to be coherent with the results presentation in the tables and figures (e.g. mid-term effects on: rice production, soil properties, microbial and fungi structure and diversity, methane emissions).
Experimental design: The introduction section should be improved for providing a broader context for a wider audience in biological and environmental sciences (see also specific comments).

Research question and knowledge gap.
The objectives of the experiments are stated in Lines 60-65 of the paper. You should improve the clarity of the paragraph and include the following aspects highly relevant for the experiment meaning: 1- the effects are studied after four years of treatments (rotation) establishment (Line 83: field experiment started in winter 2015, Line 117: soil collection in June 2019); 2 – if also rice varieties effects are investigated or not (line 106-107); 3 - treatments also refer to different fertilizer strategies for reducing total N supply; 4 - the research question also includes the effect of treatments on rice yield and methane production.
Moreover:
-	Line 61-63: according with Table 2, you should also state that “the changes on soil properties” due to treatments are evaluated, not only the relationship with microbial and fungal changes.
-	Line 62: it is not clearly stated which parameters are “environmental factors”, which seem to only refer to soil parameters.
-	Lines 64-65: it is not clear if “mechanisms” refer to microbial or chemical driven effects.

Rigorous investigation performed to a high technical & ethical standard.
Following main methodological aspects should be better explained:
-	it is not clear how the two rice varieties were treated in the experiment in the soil collection and results analysis. The effect of the different rice varieties is very important and should be presented if available.
-	the pot experiment should present in a clearer way for the following aspects: Lines 152-155: It is a parallel field incubation experiment? Four pots for each treatment were incubated in a field closed to the main field experiment, and the results of the four pots were merged for having one replication? Lines 158-161: it is not clear if you placed the plastic pots in the soil or on the surface and how you added water to the dry soil to reach the water conditions for the experiment and how these conditions were maintained during the experiment. Possibly add a reference for this experiment.
-	The soil properties before the experiment (Lines 74-77) should be included in the experiment and discussed or, alternatively, only a brief description could be provided in the “site description” section, in order not to generate possible confusion on the experiment’s objective.

Methods described with sufficient detail & information to replicate.
Materials and methods section could be improved:
-	Lines 73-74: A better description of the soil profile should be provided, using an international classification and/or literature reference. Moreover, soil texture data are lacking.
-	Lines 78-80: the data on vetch should be provided in the “ field experiment” section, with more information on vetch cultivation and soil incorporation technique, methodology utilized for estimating C and N supplied, the biomass yield if available, year-repetitions if measured .
-	Lines 100-101: the plant stage of the second top-dress fertilization is not clear (i.e booting or heading stage?). Please use international standard for definition and description, as BBCH identification keys.
-	Lines 105-106: you should describe sample collection (the area of harvest) and samples management (e.g dry matter determination, etc.).
-	Lines 106-107: the presence of two different rice varieties and growing cycle durations should be presented at the beginning of the paragraph.
-	Line 172: a briefly description of the methodology for calculating methane emissions should be provided
-	Line 174: add a reference to this sentence from a similar agro-environment
-	Lines 192-193: Was the rotation maintained from  the establishment of the experiment (winter 2015) to soil and rhizosphere sampling (June 2019) at rice heading stage? If this is the case, you should emphasize this aspect (i.e mid-term effect of rotation/treatments)
Validity of the findings: The presence and data collection and analysis of two different varieties must be clarify.

The results section and related disccusions needs to be improved:
-	Lines 193-202: the presentation in the text of table 2 results, which are high relevant for the experiment, should be expanded. For instance, which treatments are statistically different? Moreover, methane results should be separated from soil properties results.
-	Lines 205-206: Figure 1 results should be presented in a clearer way (see comments on Figure 1);
-	Lines 206-208, lines 212-216 and lines 219-220: these sentences should be moved to the discussion section.
Additional comments: The paper needs a deep improvement, especially in the following points:
-	how you include the presence of the two rice varieties in the experimental design;
-	the pot experiment should be presented as a further parallel investigation;
-	The results section should be expanded (see specific comments).
-	The discussion section should be improved. It is well structured as paragraph division, but you should illustrate the connection between your experiment results and what you are discussing in the text. More numeric data, examples and comparisons with the results from the literature should be presented in this section, before conclusive statements.
-	Tables and Figures should be simplified as illustrated in specific comments of this revision


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: INCREASING METHANE (CH4) EMISSIONS AND ALTERING RHIZOSPHERE MICROBIAL DIVERSITY IN PADDY SOIL BY COMBINING CHINESE MILK VETCH AND RICE STRAW
Review round: 2
Reviewer: 1

Output:
Basic reporting: Basic reporting
There are still some problems with English and many typos throughout the manuscript that make it hard to follow sometimes, however I suspect PeerJ's language editing service to be of great help here. My suggestions have been addressed in part and thus have some followup issues.

Discussion
(Line 288). Sentence seems incomplete.
(Line 290). Unclear what is being said in this sentence.
(Line 291). This conclusion, in whole, doesn’t seem to be supported by the data in Figure 2. Yes, the CH4 emission were highest in SVa which had high levels of CH4 emissions compared to the other treatments, but the others are all similar. Thus, how can you say that it is related to N input? If it were, you should see a similar effect in the Va-c treatments, where Va would be the highest CH4 emissions relative to Vb-c. I would change this statement so that it better reflects the data.
(Line 303) Space between Methanomicrbia and after. Sentence also unclear.
(Line 305) Capitalization of long-term.
(Line 299). This whole paragraph should be given a better look for English corrections. It is quite hard to follow. Also, there are many statements about microbial abundances higher in a certain experimental group without statistical support. This needs to be included.
(Line 302). It is quite difficult to see the % differences of Methanomicrobia in this plot with the current colors. If emphasizing a particular group of microbe, perhaps it is better to show that group’s abundance in a separate plot across the treatments being compared.
(Line 305). Sentence incomplete.

Conclusions (line 325): The first sentence is more clear when it is apparent to the reader that the statement is in relation to the control condition.
(Line 328) When abundance comes into play, indicating that these are relative numbers is important. Thus, the relative number of methanogens may have increased (or specific methanogen clades) but it is unclear from the current data whether the total number was affected. In addition, there is insufficient evidence from the current study to suggest any of these organisms is 'beneficial.' I suggest more cautionary language such as 'punitively beneficial' or remove 'beneficial microorganisms' entirely.
Experimental design: No comment.
Validity of the findings: I have commented above on some statements that are not justified given the data and would need to be adjusted to better fit the data.
Additional comments: Thank you for addressing my previous concerns. There are a few followups that I feel are necessary before the manuscript is suitable for publication. I feel there are certain areas that can be clarified in order to match the statements and conclusions made and what the data supports. This is particularly true in the discussion and conclusion sections.