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: PHOTOSYNTHETIC PERFORMANCE AND STEVIOSIDE CONCENTRATION ARE IMPROVED BY THE ARBUSCULAR MYCORRHIZAL SYMBIOSIS IN STEVIA REBAUDIANA UNDER DIFFERENT PHOSPHATE CONCENTRATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: New report on “Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations”

The authors have taken on board most of my suggestions, and I commend them for improving the manuscript.
A few minor issues to be attended to during editing:
There are still instances of content where concentration should be used, on line 109, 196, 386, 392 and 563.
Line 154 to read ..only one of them are shown.
Line 174 to read ..recorded. To minimize the..
Line 177 do you mean ..positioned on each side of all the nodes of the..
Line 302 mispelled described
Line 315 no comma after data
Line 316 to read .. were checked for normal distributions..
Line 376 to read Leaf number and foliar area were..
Line 377 to read fewer leaves than M+,
Line 378 significantly lower than what..??
I have checked response to the other reviewers’ comments and find them to be in order, but prefer the other reviewers to sign off on them.


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: PHOTOSYNTHETIC PERFORMANCE AND STEVIOSIDE CONCENTRATION ARE IMPROVED BY THE ARBUSCULAR MYCORRHIZAL SYMBIOSIS IN STEVIA REBAUDIANA UNDER DIFFERENT PHOSPHATE CONCENTRATIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript is much better than the original one.
Experimental design: More information is given which makes it better understandable.
Validity of the findings: OK, no problems.
Additional comments: No further 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: NURSING HOME RESIDENTS’ ADL STATUS, INSTITUTION-DWELLING AND ASSOCIATION WITH OUTDOOR ACTIVITY: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure of the article needs to be improved. Especially the method section is confusing to the reader, as details on measurement and data collection are given quite late in the manuscript, but which is needed earlier to understand everything that follows properly. Details on the statistics are given in the "Design" section that need to be placed in the "Analysis" section. These are just examples. I commented the structure in the pdf to make clear where improvement is needed.

In the results the authors come up with a result that was not part of the research questions and theoretical assumptions: the influence of the institution. If this result is presented it needs to be included in the research question, theoretically reasoned why the authors included this variable and discussed properly regarding the impact that this result has. Please also see my comments in the pdf.

The tables and ficures need to be revised (the number of tables shouls be reduced, captions added). Figure 1 need axis labels and a caption.
Experimental design: The meaningfulness of the original research question may be a bit questionable, as the investigated association is quite self-evident. This is also a reason why I recommend to include the influence the instituion has as a research question, as this is not so obvious and also not often investigated.
Validity of the findings: The discussion and the conclusion need to be broadened with respect to the finding that the institution has an effect on the dependent variable.
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: NURSING HOME RESIDENTS’ ADL STATUS, INSTITUTION-DWELLING AND ASSOCIATION WITH OUTDOOR ACTIVITY: A CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some sentence structure and American English issues in sentence structure remain. Literature sources are provided except where noted. The article is more cohesive with this review, however, major flaws remain.

The paper is unorganized. Adding a full literature review would be helpful.

A description of the variables should be consistent and located in the methods section

Content provided under the methods heading is unclear
Experimental design: The design of the study is stated as cross sectional with randomized selection of homes. No description of how the randomization was conducted is provided. As these homes are locations where nursing students perform clinical, the reader feels the randomization may have been somewhat biased. A full description of how the randomization was done should be included. For example, were all homes in a specifed area able to participate? You were required to obtain consent from all resients, however, a cross sectional study implies all residents in every home (except those who were exclused due to exclusion criteria) participate. Not knowing how many declined is a major flaw in this study design.

You have one variable ADL as measured by the Barthel Index is valid and reliable (although those validity and reliability statistics were not provided). No other variables are. Your description of the variables is unclear and presented in two locations which is confusing. Your description of the variable "visits" "going outside" and "activity" are believed to be the same variable, however, it is unclear.

The analysis is described as Poisson model or a multivariate model, then stated that you chose to use a negative binomial regression model instead. You should consistently describe what method of analysis you use. In addition, you have different homes with different ward types and different Norwegian municipalities. Each home is nested within each Norwegian municipalities. You need to review your analysis.
Validity of the findings: Parts of the discussion are overstated, stated incorrectly, or have English language wording issues that may help to confuse the meaning. See comments in paper.

Suggestions are provided to strengthen the strengths and limitations of this paper. Expand on strengths, there are more than one strrength. Weakness can be more clear and could include:

This weakness needs to be addressed as potential research bias. Measurement bias arises from a potential error the data collection and the process of measuring.You are trying to say that the students might not have assessed residents accurately. There are ways to address this in your data collection, but you did not so address as possible research bias.
How were the activities residents participated in counted. Just going outdoors is difficult to count unless you have someone outside to track 24/7 for the entire period, this is also a measurement bias

Did anyone decide not to participate or is it expected in this culture to participate in research activities. This may be a bias as well. Your homes were randomly selected but were the residents? Inclusive bias occurs when samples are selected for convenience.

Participants might be biased as well. Any self-reported data could be provided in a way the participant believed the students wanted to hear. response bias is a type of bias where the subject consciously, or subconsciously, gives response that they think that the interviewer wants to hear.
Additional comments: Thank you for the opportunity to review this paper. The authors have conducted a major revision of this paper and as a result, a new manuscript has been submitted. The authors would benefit from some guidance on research methodology and English language prior to the next submission.  Please see comments above and in the paper itself.


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: NURSING HOME RESIDENTS’ ADL STATUS, INSTITUTION-DWELLING AND ASSOCIATION WITH OUTDOOR ACTIVITY: A CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: x
Experimental design: The section „Limitations“ that is now placed in the Method section of the text should rather be placed in the “Strength and weaknesses” section.
Validity of the findings: x
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: A STUDY OF SOIL SEED BANKS ACROSS ONE COMPLETE CHRONOSEQUENCE OF SECONDARY SUCCESSION IN A KARST LANDSCAPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: From hypothesis to the discussion, authors want to say the "gathering effect" by rocks. However, this is not the point that your article should address since you did not have any design and results, the only evidence is "reference" sited from everewhere (temperate forest).
Experimental design: no comment
Validity of the findings: relathionship between soil seed band and vegetation.
Additional comments: Karst ecosystems are all over the world. Most of them had suffered great disturbances. Caring on a study to determine the relationship between soil seed bank (SB) and above ground vegetation have scientific meaning and practical implications. This article was written on SB and vegetation data from eight typical stages in secondary succession in Guiyang karst landscape of China to addressing the correlation of SB and vegetation. However, there are still some problem in.
Line 20: Guiyang.
Line 21 “tested” change to “determined”
Line 21-23: Resulted indicated that seed density of seed banks before field seed germination showed significantly different between each pair of succession stages” is not the same with your result.
Line 25:How do you define “neighboring regions”? Guiyang is in subtropical region, you even cited data from “temperate forests” and made conclusion to say SB in karst is higher than non-karst.
Line86-92: hypothesis 1 is not logical (richness in SB-aboveground diversity-habitat sustainability and stability-then richness in SB again?) and fully relevant to the study (do you want to approve this hypothesis thought your study). Do you have any design and result to approve hypothesis 2?
Line 99-100: Again ,do you have any design and result to approve “gathering effect”?
Line 340-341:“Seed density in seed banks of karst plant communities is significantly greater than of plant communities in which there are not bare rocks”. No data can support this conclusion.
Line 101-111. It should not be the last paragraph of the “Introduction”.
Line 345-347: “The low species composition similarity…needs to be dependent on species dispersal from outsides”. This low similarity is normal (larger seeds, distribution pattern, diverse seed rain pattern etc.). what do the author mean “natural recovery “? Does the SEBF and PEBF still need further recovery?
Line 347-348: Species richness in seed banks increases with increasing…indicating that conservation of diverse aboveground vegetation may maintain diversity of seed banks for restoration in practice”. What does the author want to say? What is the logic of “conserve vegetation” to “maintain SB” and then “restoration in practice”
Fig2. How can you made mathematic equations with your “succession stage” X axis? Bar chart may be more suitable.


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: A STUDY OF SOIL SEED BANKS ACROSS ONE COMPLETE CHRONOSEQUENCE OF SECONDARY SUCCESSION IN A KARST LANDSCAPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript studies the soil seed bank and above-ground vegetation in eight stages of secondary succession in a karstic landscape. Soil seed bank dynamic and its potential to restore degraded landscapes is an interesting topic. You recorded a great amount of very useful data. However, I found the manuscript difficult to follow and confusing regarding different aspects. For example, I would specify the main objective, hypotheses, and questions, and align them better to the analyses, results, and interpretation. Analyses description needs more details. Results should be limited to the questions. Discussion could be improved with a better interpretation of the possible reasons of the obtained results. I consider that correcting all these suggestions will increase the interest of your work. I also recommend checking the English and writing in all the sections to ensure the clarity of your work.

Introduction
Introduction gives a general background about soil seed bank dynamic. However, it repeats concepts in different paragraphs, making it difficult to follow. I consider that correcting the following suggestions could improve it.
-	I recommend stating the problem briefly in the first sentences of the introduction.
-	I suggest expressing the main objective more clearly. It could be rewording lines 93 to 95.
-	Hypotheses are confusing, consider rewording them. They should be affirmations stated in present tense.
-	In lines 51 it says “… in forest ecosystems (later succession stages)…”  and “… grasslands (early succession stages)…” what do you mean by that? Take into account that Hopfensperger (2007) makes a review in three ecosystems: grasslands, wetlands, and forests. The author evaluates the similarity between soil seed bank and vegetation, and in relation to succession at each ecosystem independently.
-	I recommend avoiding the use of words such as “obviously” (line 52), “evidently” (line 187).
-	In lines 54-56, what do you mean by “However, the relationships between plant diversity in above-ground vegetation and in its associated seed banks under the dominant paradigm are still unknown”. I found a contradiction with the well-known knowledge about the similarity between soil seed bank and vegetation reported previously.
-	In line 74 I would mention briefly which the main anthropogenic disturbs are in the karstic landscape.
-	I suggest finishing the introduction with the study questions. Thus, the paragraph in lines 101-111 should be previously.
-	From my point of view, in lines 61-71 you mention the main contribution of the study. I suggest focusing more in this aspect throughout the manuscript.

Materials and Methods
Sampling section is detailed, but I recommend improving writing and English in this section. Analyses need more details. I suggest aligning them with questions and results.
-	Line 124, I miss the meaning of SF.
-	Lines 128-129, I suggest changing “The name of each individual plant….” To “Each individual plant was identified…” or similar.
-	Lines 129-130, I recommend avoiding the use of “carefully” and “precisely”. Instead, describe how you measured canopy cover, diameter at breast height, etc. I also recommend including the references used for plan species identification.
-	I wonder how you measured important value of species, and why.
-	Lines 161-162, I recommend mentioning the anova analysis first, and then the LSD test, since LSD test should be only used if significant differences are detected by anova.
-	Analyses should be more detailed. You should include the software used and libraries if it corresponds. Detail the variables you used, especially for anova and regression analyses, and how you evaluated the assumptions, such as normality and homocedasticity, etc.
-	I suggest changing “arbor” to “tree” in line 169 and throughout the manuscript.

Results
Although results answer to some extend the questions, in some parts I found them confusing. I suggest you to check this section focusing in aligning the results with the questions and methods, and to improve the English and writing.
-	All differences have to be statistically significant to be mentioned as a difference. For example, in lines 176-177 you say “Seed density showed greatly different among recorded species”, are they significant different? I suggest to take this into account in all the result section.
-	I recommend avoiding the use of words such as “evidently” in line 187, “obviously” in line 210. As I have noted above, differences have to be statistically supported to be considered as differences. You could use the word “significant” in those cases.
-	Why did you compare the soil seed bank among different soil depths? I consider that the importance of soil depths should be mentioned in the introduction.
-	Lines 217-220, I miss p-values significance, is it 0.05? State it clearly.
-	I recommend to specify the area when you analyze species richness, since richness is dependent on area. For example, I understand that the area you are referring in table 1 is different from the area you are referring in figure 4. I consider that specifying it will improve the clarity of the manuscript, and could contribute to enhance the interpretation you do in lines 274-280.

Tables and Figures:
-	Table 1. I suggest changing “viable seeds” to “seedlings”. This table has a lot of information; check if all of it is relevant to answer the questions. Are you mentioning total number of species recorded at each successional stage? I recommend adding the complete name of each successional stage in the legend or somewhere in the table for understanding it independently from the text (the same for all figures and tables).
-	Table 4. I suggest using B and A to represent before and after field seed germination, instead of I and II for Jaccard index. Thereby, you will be consistent throughout the manuscript.
-	Table 5. What does * mean?
-	Figure 2, 4, and 5. Add p-value, or specify what * means.
-	Figure 3. I miss what dotted lines mean.

Discussion
Although discussion section compares the results with other studies, I suggest improving the interpretation of the obtained results by explaining which could be the reasons of what you found.
-	I suggest not mentioning again the figures and tables in discussion section.

I hope all of these suggestions help you to improve your manuscript.
Experimental design: Please, see comment above.
Validity of the findings: Please, see comment above.
Additional comments: Please, see comment above.


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: A STUDY OF SOIL SEED BANKS ACROSS ONE COMPLETE CHRONOSEQUENCE OF SECONDARY SUCCESSION IN A KARST LANDSCAPE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Fig.2 :We can make species richness comparison among different stages of secondary succession by ANOVA and then conduct post hoc test, we can also draw a trend line. However, I can not understand your mathematic equations. What does the X means in your equations?  Can succession stages be numbered?
Additional comments: It had improved, but fig 2. still need to be reconsidered.


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: A STUDY OF SOIL SEED BANKS ACROSS ONE COMPLETE CHRONOSEQUENCE OF SECONDARY SUCCESSION IN A KARST LANDSCAPE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: I found question 1 and 3 are well defined. I consider question 2 would be correct if the manuscript aim was to compare regions, and if such comparison was stated in methods and presented in results. In this case, I suggest to present the comparison among regions only in the discussion, as you did, but not as a question.
Data analyses are more detailed now, but I still find them a little confusing:
In line 166 you mention “important parameters”, I suggest to mention which parameters you are referring to. For the supplemental Information I, I found that you are referring to the Important Value of Species. As you said in the responses, it is basic information in plant community. So, if you mention it in the methods you should mention the contribution of this index to the aim of the manuscript too, and discuss the most relevant results you found from it.
In line 168 you say “the scale of 20 cm x 20 cm plots”. Are you referring to the germination trays? I suggest to refer to the field plot size, is it 10 cm x 10 cm for the three depths?
In line 169 you mention “seed viability” and “viable seeds”. I think it would be better to use some terminology as seedlings or germinable fraction of the seed bank, because the seedling emergence method only determines the germinable fraction of the seed bank and fails to detect dormant seeds and those seeds with specific germination environmental requirements, but which are still viable.
In lines 173-174 “Base on these data, ….” I suggest rewording the expression, because it is confusing. Also, I suggest to mention the scale at which you calculated the similarity, is it total species composition for each succession stage?
In line 180, I suggest change “ life forms” to “species”, maybe you could say “species were classified by their life forms into……”
In lines 180-182, I wouldn’t say that “…… is presented in figures”, but it should be clearer that you analyze the relationship between species richness and succession stages through regression.
Validity of the findings: The results are in line with the aim and questions and contribute to the hypotheses. I suggest you to check the supplemental information III. I found there are some significant values as .000, do they mean < 0.001?
I also found that levene test significant is .000, does it also mean  < 0.001? If it so, I would interpret that the null hypothesis is rejected, so there is no homogeneity of the variance. In that case you could try some transformation for example, until you check the homogeneity of the variance. Finally, the first column of each table, and the first and second in some of them, have numbers, are they the stages? I suggest clearly state to which stage each number corresponds, or change the numbers to the stages name.
Regarding the discussion I think it still needs a little bit of work.
In line 290 you say “species diversity”, are you referring to species richness?
In lines 290-292, you didn’t measure the seeds that fell to the ground, so it is a speculation and it should identified such as. I found the same kind of expressions in other parts, for example in lines 341-342 “The above-ground vegetation added many seeds to the seed banks”. Please, check them throughout the discussion section. Also, try focusing more in the interpretation of the results than in mentioning them again.
In lines 310-311, what did you mean by “….implies that seed dispersal outside of the stages of secondary succession is a natural regeneration strategy for restoring degraded karst landscapes to forests”?
Additional comments: From my point of view the manuscript “A study of soil seed banks across one complete chronosequence of secondary succession in a karst landscape” improved from the previous version. I consider it is more clear now, especially the background provided, which is in line with the aim, hypotheses, and results. Hypotheses are now well stated and results contribute to them.
I suggest to check it again, paying especial attention to the data analysis and the discussion. I also suggest to check some points in the abstract:
- I suggest including something about the aboveground sampling.
- I suggest not mentioning “Gathering effects of seeds occurring on naked stones in karst habitats explain the observed high seed density to some degree” because it is a speculation but it is not supported by the results.
- I couldn’t understand the last sentence “There may be negative feedback among areas of rocky desertification, high seed density and vegetation restoration.” What do you mean?

I hope all of these suggestions help you to improve your 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: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors of the manuscript “A robust semi-supervised NMF model for single cell RNA-seq data” present a novel approach to incorporate knowledge of tissue-specific marker genes by modifying an existing method called robust NMF to improve single cell RNA-seq data clustering accuracy. Authors demonstrate improved performance of the new method on ten single cell RNA-seq datasets that have true labels. However the application of this method to any other dataset of unknown cellular composition is not provided. Also there is no discussion in the manuscript what are the advantages and disadvantages of semi-supervised approach over commonly used unsupervised methods. The latter is important because use of the prior knowledge in clustering can lead to overfitting.

English language used throughout:
Text in 45, 47, 139 etc., should be rewritten to correct language errors.
Revise term “Hierarch clustering” and also provide parameters that were used with this clustering

Typographical and other errors in mathematical expressions:
Line 34: twelve datasets are mentioned, however in the main text analysis of ten datasets is given.
Line 118: variable sigma is not defined
Line 155, 241: “=” sign is missing

Figures:
Text on Fig. 2 is inconsistent with caption of this figure.
Supplementary Fig. 3 is similar in contents to Fig. 3 but for another dataset, and is not referenced or explained in the manuscript text.
Fig. 3 color coding is not explained.

All the questions and comments above need to be addressed in detailed.
Experimental design: Authors claim that the clustering accuracy is significantly improved when using rssNMF compared to NMF. This is justified by numbers in Tab. 2, where rssNMF is calculated with 1 group with 20 markers. However using only 1 group is not optimal, as suggested by Supplementary Fig. 1. Authors should comment why 1 group was chosen instead of 2 or 3, etc.

On line 220 authors state “Supplementary Fig. 1 shows the how the average performance of rssNMF varies with the parameters α and
Validity of the findings: In the abstract, Results subsection, it is stated that “rssNMF can identify key subclasses and latent biological processes”. However in the Results section of the main text there is no clear explanation how to identify biological processes with rssNMF method. Therefore the validation of the clustering results with pathway enrichment analysis should be clearly identified as such.

On line 266 authors reference Supp. Fig. 3, that is supposed to be Supp. Fig. 4, to exemplify genes participation in multiple biological processes. What particular gene is exemplified and what relevant processes should the reader focus on?

All the questions and comments above need to be addressed in detailed.
Additional comments: Material and Methods section needs to be rewritten to clearly separate the existing rNMF and proposed rssNMF methodology.

Derivation notes in section “Optimization algorithm for robust semi-supervised NMF” as well as Algorithm 1 are not essential for the definition of the rssNMF methods and could be moved to the Supplementary information.

Subsection “Dataset” of section “Results” contains background information on cell marker genes, and should be incorporated in the introduction section. The description of the used datasets should be extended to include information on how the clusters and true labels (annotation) in the analyzed datasets were obtained. The supplied datasets information can be improved by adding GEO accession numbers.

The mathematical definition of Frobenius norm in subsection “Original NMF algorithm” need to be moved to Supplementary Information.

The mathematical definition of ARI in subsection “Compared algorithms” need to be moved to Supplementary Information.

On lines 267-278 authors present “gene score” however the relation of this score to the rssNMF or the biological significance analysis is not clear. Also the Fig. 3 is intended to reflect the importance of this score needs more detailed explanation.
All the questions and comments above need to be addressed in detailed.


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: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The majority of the manuscript is clear, but the article can be improved with a better flow and transition sentences, and some typos need to be corrected.
Experimental design: Do not apply.
Validity of the findings: The authors need to provide the accession numbers of the single cell datasets.
Additional comments: Wu et al. presented the so-called “robust semi-supervised NMF” model to cluster single cells based on their gene expression data. The main contribution is proposing to add a regularization term representing local geometrical structure in the loss function of NMF. I think the idea of this model is interesting and could be useful, but the authors need to better justify the individual components of the model. As for the results, a more informative comparison with other scRNA-seq clustering tools are in need.

Line 24-26: Many clustering analyses will contain a feature selection step, which includes the identification and usage of marker genes. Therefore, it’s not proper to say cell marker genes haven’t been used in the clustering analysis of scRNA-seq data.

Line 100: Is it reasonable to assume the noises in scRNA-seq data are sparse? The authors need to provide evidence for this claim.

Line 103: The authors should introduce all the matrices (e.g., X, W, H) in the context of single cell expression data. Moreover, the matrix L should be briefly discussed here even though it is defined in line 128.
It looks like the authors choose to use the Frobenius form. Why don’t the authors use the L(2,1) norm as discussed in line 92, since it’s shown to be more robust?

Line 107: What does “this property” refer to?

Line 118: The authors need to justify the selection of this heat kernel and the parameters used in the kernel function. Are the results sensitive to the parameters? Will other options possibly lead to better results?
In addition, some cell types only have a few cell markers. The calculated cell distances would be unstable in this case. Did the authors consider identifying de novo cell markers?

Line 155 gives a definition, not a theorem.

Line 160. Why the threshold is alpha/2 in (3)? Should it be 2*alpha?

Line 161: Is this algorithm guaranteed to converge?

Results:

All the datasets tested in the results section have no more than one thousand cells, while large scale single-cell data with thousands of data are very common. The authors need to evaluate the method on larger dataset for a more comprehensive comparison.

There are more than 10 clustering methods that are specifically designed for single cell gene expression data, but the authors didn’t compare rssNMF with any of these methods. It is important to know if rssNMF provide improvement over existing scRNA-seq methods.

Minor:
180 “ a many” looks like a typo


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: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Wu et al presented a new method called rssNMF, which incorporate prior knowledge into the rNMF method. They compared their method to other NMF methods such as rNMF and original NMF, as well as two common clustering methods kMeans and hierarchical clustering, to show rssNMF is superior than all the other methods compared. The language is clear, and reader can follow the logic of the story. The figures and tables are well presented with scripts of the algorithm included.
Experimental design: I have major concerns about the design of the method comparison. Firstly, it is uncommon to define marker gene as the DE genes between different clusters because cluster identity is unknown when performing clustering analysis. Marker genes are usually obtained through previous study, such as bulk RNAseq experiment or fluorescence-activated cell sorting (FACS). For example, one could perform DE analysis of immune cell reference dataset (such as immgen.org) and then use the marker genes obtained on a PBMC dataset for better clustering performance. Secondly, while kMeans and hierarchical clustering are fundamental clustering algorithms, they rarely being used directly to scRNAseq data (Duo et al. F1000Res 2018. Freytag et al. F1000Res 2019), instead they might be a part of a clustering pipeline. As an example, SC3 (Kiselev et al. Nature Methods 2017) use kMeans in their consensus clustering pipeline. Therefore, the rational of choosing these two methods is unclear. The detail of these two methods used is not stated as well, such as the input and the parameters. Authors should either ignore the comparison to other methods thus limiting the scope of the comparison only to different NMF methods or compare against commonly used unsupervised clustering methods such as Seurat and SC3.
Validity of the findings: As mentioned above, the author should provide more detail on the method comparison, such as the parameter used in each method and how exactly the marker genes are selected, such as providing reproducible scripts for DE analysis.
Additional comments: typo:
"hierarch clustering" should be hierarchical clustering
there is formating error in line 201.


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: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Paper summary:
Authors build a method to incorporate marker genes via matrix Q to better cluster scRNA-seq data. To construct matrix Q authors first use true labels of cells to make groups of cells, then marker genes are selected for each cell group; differentially expressed genes are used as marker genes of each cell group. Then Q is used as input in rssNMF algorithm to obtain optimal H. Matrix H is clustered by KMeans into clusters of cells. These clusters are compared to the "true" labels via ARI. Clustering step is repeated 30 times, average ARI is reported as a performance metric.

Major comments:
(1) Author summary of analysis workflow should be added to the manuscript, focusing on the selection of marker genes.
(2) Tracked Word document is inconsistent with pdf-v1 document. Not all changes that authors claim to be done are incorporated in pdf-v1.
(3) Algorithm 1 missing input parameter L, the key parameter in rssNMF method.
(4) Gene score is determined from matrix W. Gene score is only necessary for selection of feature genes for biological significance validation. What is the cutoff for gene score value to use a gene in enrichment analysis?
(5) On line 300: "As shown in the Fig. 3, we assign all the genes to a specific cluster by different colors. The depth of the color indicates the value of gene score". The caption of Fig. 3 contains matrix W, H and consensus of C, which is not consistent with line 300.
(6) The last dataset ("Zeizel") in Table 2 has ARI of nearly zero, i.e. labels are assigned a random manner. Authors should verify and explain this result.
Experimental design: NA
Validity of the findings: NA
Additional comments: Minor comments:
(1) 57: Subsection title "Related work" is unnecessary.
(2) Any inconsistencies of names and terms need to be corrected; e.g. KMeans, Kmeans, RNMF, rNMF.
(3) Table 1 two bottom lines are not aligned with the rest of the entries.
(4) 215: term "Hierarch clustering" need correction.
(5) A reference for MSigDB is missing in the manuscript.


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

Input:
Title: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed most of my comments, but I still have following concerns after reading the authors’ response and revised manuscript.

Since the definition of weighting matrix is arbitrary, the authors should acknowledge this point in the discussion section and discuss possible improvement or validation approaches.

I didn’t understand this sentence in the authors’ response:
“Line 160: The reason is that the coefficient of Frobenius term in objective function is 1 but 1/2 in the theorem.”

The authors answered my question regarding the convergence but did not discuss this point in the manuscript.


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

Input:
Title: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 2
Reviewer: 3

Output:
Basic reporting: I have no further questions.
Experimental design: I have no further questions.
Validity of the findings: I have no further questions.
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: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Almost all of my comments have been addressed by the authors in the revised manuscript. One problem has not been addressed and one newly discovered error in the equation needs to be corrected.
(1) The issue of Figure 3 and its caption not being consistent with main text is not addressed. In the latest revised manuscript authors claim that “The depth of the color indicates the value of gene score and we can assign the cluster genes visually.” Matrices W, H, and C are displayed in Figure 3 as noted in the caption, however, the gene score is a function of W and is not shown in Figure 3. Therefore, in Figure 3, the depth of the color does not indicate the value of gene score.
(2) Additionally, the equation on line 64 contains two errors: first, the L denotes a functional and not a graph Laplacian, therefore should be changed to any other name, e.g. f; second, the right-hand side of the equation is missing the min operator, same as used in the left-hand side of the equation.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: A ROBUST SEMI-SUPERVISED NMF MODEL FOR SINGLE CELL RNA-SEQ DATA
Review round: 3
Reviewer: 2

Output:
Basic reporting: none
Experimental design: none
Validity of the findings: none
Additional comments: The authors have addressed all of my 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: BROAD SPECTRUM ANTIMICROBIAL ACTIVITIES FROM SPORE-FORMING BACTERIA ISOLATED FROM THE VIETNAM SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presents relevant information about the use of spore-forming bacteria to inhibit pathogenic bacteria growth. However, some sections of the presented data can be improved. For this reason, I considered that this manuscript needs minor changes for being considered for its publication in this journal.
Experimental design: Include more detail in enzymatic assays protocol performed in this manuscript.
Include more detail in the experimental design, highlight statistical factors and variables of response in the statistical analyses applied to the findings of this research.
Validity of the findings: Include a possible mode of action of spore-forming strains against pathogenic bacteria tested.
Try to compare the obtained findings with similar assays were spore-forming bacteria were applied to inhibit pathogenic bacteria growth.
Additional comments: Dear Author, I reviewed the manuscript (51002v1) entitled Broad spectrum antimicrobial activities from spore-forming bacteria isolated from the Vietnam Sea. This manuscript presents relevant information about the use of spore-forming bacteria to inhibit pathogenic bacteria growth. However, some sections of the presented data can be improved. For this reason, I considered that this manuscript needs minor changes for being considered for its publication in this journal.

Additional comments.
Highlight the advantages of using nowadays these spore-forming strains against pathogenic bacteria.
Check paragraphs extension in this manuscript.
Include more detail in enzymatic assays protocol performed in this manuscript.
Include more detail in the experimental design, highlight statistical factors and variables of response in the analyses applied to the findings of this research.
Include a possible mode of action of spore-forming strains against pathogenic bacteria tested.
Try to compare the obtained findings with similar assays were spore-forming bacteria were applied to inhibit pathogenic bacteria growth.
Include future trends to keep working with the obtained data.
Try to conclude with a general statement of the most relevant part of this study.


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: BROAD SPECTRUM ANTIMICROBIAL ACTIVITIES FROM SPORE-FORMING BACTERIA ISOLATED FROM THE VIETNAM SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article needs to improve, it presents an insufficient introduction and a background is missing to demonstrate how the work fits into the broader field of knowledge. Current references are missing.
Contaminated culture in figure 1; Low quality of figure 3; Figure 4 is a catastrophe, the phylogenetic tree must be rebuilt following all the required standards.
There are many tables, some unnecessary and incomplete essential information.
Experimental design: The results are very preliminaries. The information is not new in relation to marine bacteria, mainly for the genus Bacillus. Additional experiments are needed for the study, such as isolation and identification of bioactive compounds, tests for minimum inhibitory concentration and minimum bactericidal concentration, and cytotoxicity tests.
The reference list has many errors, this demonstrates a lack of care in preparing the document.
Validity of the findings: This study does not bring news in the area, it is a weak repetition of other publications.
In view of its lack of new information, I suggest that the manuscript be rejected.
Additional comments: The study would be interesting if more results were presented. The work must be continued in order to isolate the bioactive compound and show that it has some potential to be used in the treatment of any infection.


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: BROAD SPECTRUM ANTIMICROBIAL ACTIVITIES FROM SPORE-FORMING BACTERIA ISOLATED FROM THE VIETNAM SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This version of the manuscript followed all the suggested modifications and recommendations by the reviewers.
Experimental design: This section was improved in the revised version of the manuscript.
Validity of the findings: The obtained findings in this research are well described and compared with bibliographical references and justify the importance of this obtained data.
Additional comments: Dear Author, I reviewed the revised version of the manuscript (51002v2) entitled: Broad spectrum antimicrobial activities from spore-forming bacteria isolated from the Vietnam Sea. This version of the manuscript followed all the suggested modifications and recommendations by the reviewers. Besides, the findings obtained in this research are well described and compared with bibliographical references and justify the importance of this obtained data. For this reason, I considered that this manuscript can be accepted for its publication in this journal.


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

Input:
Title: HIGH-THROUGHPUT ANALYSIS OF ADAPTATION USING BARCODED STRAINS OF SACCHAROMYCES CEREVISIAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: GENERAL COMMENTS

The main challenge I found with understanding this manuscript was to follow the narrative. There appear to have been multiple apporaches taken, at various times, for various reasons. For example, in the Experimental evolution section, lines 459-472, there was a 25-day evolution experiment (two barcoded per well), and also mention of "532 longitudinal-evolutionary dynamics samples". I was confused. The dicsssino is much clearer, and does not need to change.

The Strains, media and culture methods section P9, line 109- do not explain well what the 92 diploid barcoded strains are.
For example, are they geetically different from noe another? Have they been sequenced? How heterozygous was the parent YPS163?
How different are they genetically from the ‘reference’ strain (barcode ID: d1H10) This is also the case in the results: P23, line 437.
STill at this point I don't know what the strains are. It was only in the discussion that I was sure that the barcoded strains started as clocal replicates.
hHis need to be explciit earlier, as it is critical to any readers understanding the experiment.

The 143 Experimental design section (P10, lines 143-) could be clearer.
Even with Figure 1 (which is good), I was not always clear about how many strains per well.
Perhaps show this to a colleage (that was not inolved) and check that they can understand samples, pools etc.

Data: there are no issues with the availabily of the raw data, or the code. All these are aspects are at or above the current standard.
Data analysis is also excellent. Sequence analysis and laboratory cuture also appear rigorous.

P24, line 453:
1% fitness differences does not seem very high, given that new alleles typically have effect sizes much smaller. How does this compare to other methods?

PS: Please provide figures in line with the text in future. Not at the end. Placing figures at the end is counter-productive as it makes the reviewers job harder. The journal should insist on this, as well (I know that most don't).

P24, line 453 "(n=22), we estimate high power (99.8%) to detect 1% fitness differences between treatments at a"
You estimate power for 22 strains, at this point in the ms. Why did you tehn conduct the 250 day experiments with two barcodes per well?
This should be explained in the ms.

SMALL CORRECTIONS
All page numbers ref to the page in the pdf.

P4, "fitness gains (adaptation) in a total of 76 experimentally evolving populations by conducting 1,216 fitness"
It would be useful to define what kind of populations here. Mitotic only or with meiosis? Starting from clonal, or a genetically heterogenous mix?

P7, line 69 "10,26,27]. However, the number of fitness assays, and thereby the resolution of those assays,"
reference style differs

P8, line 90: "Saccharomyces cerevisiae (hereafter, yeast) strains,"
At an early stage, please tell the reader what the strains are. A gene knockouts? A population of wild strains? etc.
Otherwise all the futher reading I am wonder what the experiments are doing to show.
(at the this point in the ms I am already convinced that bar codes are a good idea)
The Strains, media and culture methods section P9, line 109- still does not explain well what the strains in use are.
This is critical to understandin the experiment.

P9: line 134
"Saturated 24-hour culture was diluted (1:1000) into fresh medium at
134 the same time each day to initialize the next round of growth for all evolution and fitness assays."
What was the OD at this point? Did they reach stationary phase?

P13, line 201
"20, 22, 24 and 25) for a total of 532 evolutionary dynamics samples across 76 evolving two-"
Adding numbers such as "532 evolutionary dynamics samples across 76" does not help to clarify the experiment.

P11, line 170
Why 152 barcoded yeast strains used here?. At each point, please explain why certain numbers of strauis were chosen.

There are many sentences descrbing how many fitness assays can be conducted, for example:
"With four replicate fitness assays for each barcode, this amounted to 1,216 fitness assays (2 barcodes * 2 time-points 468 * 76 populations * 4 replicates)." P 24, line 267. And again P24, line 467, "fitness assays for each barcode, this amounted to 1,216 fitness assays (2 barcodes * 2 time-points".
I wonder how many of these statements help the reader to understand the experiment? We all apreciate, from an eary stage in them manuscipt, that the system allows for many repeats. That bar-seq is a cheaper option that full genome sequencing is well-known.


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: HIGH-THROUGHPUT ANALYSIS OF ADAPTATION USING BARCODED STRAINS OF SACCHAROMYCES CEREVISIAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: I felt that the article came a little short in terms of the background/context provided, specifically with reference to how this methods fits in with other similar methods. The authors should more clearly describe the similarities and differences. Please see more comments below.
Experimental design: Experimental design is sound.
Validity of the findings: The findings are supported by the data.
Additional comments: The manuscript “High-throughput analysis of adaptation using barcoded strains of Saccharomyces cerevisiae” presents a system to both measure fitness and conduct evolution experiments in budding yeast. The authors perform detailed analysis to assess the power of their system for detecting small differences in fitness. They also describe new experimental results confirming observations seen in previous studies, for example, that the rate of adaptation is faster in haploids than diploids. All in all, this is a nice study. My two major comments turned out to be very related, and so I really only have one main piece of advice that I hope improves the paper.

Major comments:
1.	Another similar barcode system (the Levy/Blundell system cited by this study) achieves many of the same goals and the authors should take the time to describe all of the similarities and differences with that system. Here are some examples:

a.	These systems have barcodes inserted into different regions of the genome. Is there any benefit to inserting into HO? Does the observation that barcode systems using a different barcode insertion locus are successful create possibilities for future multiplexing?

b.	The previous system measures fitness by finding the log-linear slope of barcode frequency change across multiple timepoints (see cited Venkataram et al reference from 2016). The system in this paper uses only the start and end point, which has been shown to yield reduced power in the following reference: https://www.cell.com/cell-systems/pdfExtended/S2405-4712(18)30390-9. Is there a future plan to gain power by considering more timepoints in the fitness measurements?

c.	The previous system does not pair the barcodes but combines hundreds of thousands. Is that something that you can do with your system? Is the max throughput of your system presumably similar to that seen in previous studies? Why did you choose to pair barcodes in the evolutions rather than combine hundreds of barcodes?

d.	Would having performed a two-step PCR, as in the previous system, have decreased noise due to PCR jackpotting? Is this going to become a requirement if the current system is used for evolutions tracking pools of thousands of barcodes?

2.	The authors argue that their system increases the throughput of fitness measurements and experimental evolutions. But a previous system (see cited Venkataram et al study 2016 and Levy & Blundell et al 2015) is actually much higher-throughput. The authors should be clear about this by making the following changes:

a.	In the beginning of the conclusion, instead of saying that this system improves throughput, perhaps add a qualifier, something like, “improves throughput relative to other evolution experiments using paired strains with fluorescent markers”.

b.	Are there specific questions that can be addressed with this system that are less amenable for study with the Levy/Blundell system?  Can you please describe these? Is your system basically another version of that system, or are there some nuances here that are different? Either way, this manuscript represents a valuable contribution. However, I wish this were clearer.

c.	I was very confused while reading the methods because I am familiar with the Levy/Blundell system and I made some assumptions about the system in this manuscript that were untrue. This was partly because the introduction reads as though the system proposed here improves throughput over Levy/Blundell. My confusion was about the hundreds of barcodes used for the experimental evolution studies. I assumed each of these barcoded strains was in fact a library, containing hundreds of thousands of unique barcodes. But this is not the case, and this system, as depicted, does not seem to improve throughput relative to Levy Blundell. I think the introduction needs to clarify what this systems does and does not do and how it is similar/dissimilar to Levy/Blundell’s.

d.	Pooling samples with different barcodes before DNA extraction is a clever idea! This is the kind of thing that might appear earlier in the paper, in some kind of comparison to previous systems. Alternately this could appear in the discussion. Performing the evolutions in 96-well plates is also different from Levy/Blundell and gives the opportunity to study many environments. This might help avoid batch effects, which were very high in Venkatarm et al.

Minor comments:
1.	The authors mention Blundell and colleagues in the introduction, but I believe Levy is the co-first author on at least one of the cited papers, maybe both. The text should probably read Blundell, Levy and colleagues.

2.	I was mildly confused when reading the methods section referring to the proof of concept experiments because it seemed to me that all of the strains should have the same fitness. Indeed, this was the null expectation. The authors should say so in this section of the methods, perhaps more than once.

3.	I love the power analysis! It is exactly the kind of thing I’ve been missing from previous studies.

4.	I’m confused about the orange lines in figure 3. It is a little strange that this figure appears before the experimental evolutions are discussed in the results section. Perhaps give a heads up about why the barcodes have different fitness (I assume because they receive beneficial mutations) and why power is weaker than in fitness measurements?

5.	Label the control group in figure 5.


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: HIGH-THROUGHPUT ANALYSIS OF ADAPTATION USING BARCODED STRAINS OF SACCHAROMYCES CEREVISIAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Lines 62 – In this paragraph, it might be helpful to address what is actually being measured during these fitness assays (what is fitness in a microbial context and in the traditional fitness assay context?)
Line 69 – correct references to proper format
Line 225-228 – Am I correct that for each evolved population, you pooled the time point 0 and competed it against an unevolved reference strain, and then pooled timepoint 250 and competed the pool against the unevolved reference strain? The wording here is a little confusing.
Figure 1 – could panels be included that illustrates the concept of the barcoding and pooled fitness assays? I think these aspects of the experimental design are more important to illustrate visually than a sampling regime.

There are many papers from the Lang and Desai labs that might be helpful to address sensitivity of fitness measurements, ploidy. For example:
Lang GI, Botstein D, and Desai MM. 2011. Genetic variation and the fate of beneficial mutations in asexual populations. Genetics. Jul;188(3):647-61.
Marad DM, Buskirk SW, Lang GI. 2018. Altered access to beneficial mutations slows adaptation and biases fixed mutations in diploids. Nature Ecology & Evolution. May;2(5):882-889.
Fisher KJ, Buskirk SW, Vignogna RC, Marad DM, Lang GI. 2018. Adaptive genome duplication affects patterns of molecular evolution in Saccharomyces cerevisiae. PLoS Genetics. May 25;14(5):e1007396.
McDonald, M., Rice, D. & Desai, M. Sex speeds adaptation by altering the dynamics of molecular evolution. Nature 531, 233–236 (2016).
A particular comment on the correspondence of barcoding and fluorescence based fitness measurements from Levy et al. 2015 would be helpful
Experimental design: What was the actual representation of the different barcoded strains in the timepoint 0 of the pooled fitness assays (e.g., how easy is it to ensure equal representation across all barcoded strains)? How does the initial abundance influence relative abundance over the course of the assay?

Can the authors address their rationale for including an unevolved reference strain in the competition vs. just measuring relative fitness of the pool by not including a reference strain and just looking at barcode abundance itself?

Line 315 – how do you know there were 20 generations?

Can the authors address their rationale for the paired barcoding in the experimental evolutions? What kind of considerations should be made for how many barcoded strains go in each population to track evolutionary dynamics?
Validity of the findings: There is some interesting data presented here about how bottlenecking and different environments influence evolutionary dynamics, but this section is very brief and all the data are buried in a bunch of tables and figures in the supplemental. I would suggest a Figure that includes some of this data in the main text. I would also suggest clarifying the biological relevance of the different measurements that were obtained.
Additional comments: Barcoding has been used to track fitness and evolutionary dynamics for many years now, and there are a variety of different systems in use. There is a tradeoff with the proposed system (or any barcoding system), the upfront labor to generate barcoded strains (e.g., transforming and verifying presence of barcodes, validation of neutral fitness) vs. the downstream payoff of pooled fitness assays. This compares to other commonly used systems, like competition against a fluorescently tagged ancestor strain, which requires no upfront labor, but fitness assays are more laborious. Ultimately, it comes down to what questions one is interested in asking. For example, if you were interested in different strain backgrounds, barcoding approaches are challenging. However, the ability to request the strains and use the scripts provided here to analyze data is a big benefit. I can particularly imagine the use of this system in undergraduate courses being very impactful.
Addressing the sensitivity of fitness assays for experimental evolution is an important problem in the field. The authors approach these issues very thoughtfully and methodically. It appears that the sensitivity of barcoded fitness assays presented here is similar to the sensitivity of fluorescent based approaches. In my opinion, the greatest utility of this approach comes from being able to pool strains for fitness assays. This could be particularly helpful for researchers interested in tracking fitness over the course of evolution experiments (e.g., questions about evolvability), which is very difficult to assay with current protocols.
 I think my struggle comes down to the framing of the manuscript. The framing is very technical and methods based, but I think it could benefit from highlighting the results and how they are innovative in a more biological context.


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: IMPACT OF COVID-19 OUTBREAK ON THE MENTAL HEALTH STATUS OF UNDERGRADUATE MEDICAL STUDENTS IN A COVID-19 TREATING MEDICAL COLLEGE: A PROSPECTIVE LONGITUDINAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the manuscript has been prepared in clear English with a good structure including appropriate use of figures and tables; literature review seemed to be adequate, and relevant results presented.
There are 3 comments for the authors to consider revising:
•	The numbering unit of ‘lakh’ is not an internationally well-understood concept, could the authors please consider revising this in Introduction.
•	It’s not very clear if there have been other longitudinal studies done before, on the effect of COVID on depression, anxiety, and stress in other populations/contexts. Please add something about this in the Introduction.
•	Figures 1 and 2 appear to be in low-resolution.
Experimental design: The study appears to be original research with well-defined research question. The Introduction highlighted the need for this study/more research on the impact of COVID-19 on high-risk populations such as medical students. The methodological design seemed to be adequate to address the research questions, and is described with sufficient details.
Validity of the findings: Although the study is based on a somewhat small sample size, the spontaneity of the study (or outbreak of the pandemic) and the longitudinal nature of the data would out-weigh this issue. Statistical analysis appears to be appropriate and thorough.
There are 2 comments for the authors to consider revising:
•	For Results, please consider adding effect sizes where-ever possible, especially to the changes in depression, anxiety and stress from  baseline to follow-up survey, regardless of whether the difference was p < .05 or not.
•	Line 355 – I believe ‘a relatively weak but significant positive correlation’ is more accurate than ‘a mild significant positive correlation’.
•	For Discussion, please talk about, whether there has been other longitudinal studies conducted on the change of depression, anxiety, and stress from before to during COVID, and if the magnitude of change found in the current study is similar/different to that found for other populations in the other studies.
Additional comments: Some typos noted:
•	Line 221 – literature search
•	Line 349 – the full stop should be placed after (table 2).
•	Line 450 – missing “and” in sentence: challenging and intense training


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: IMPACT OF COVID-19 OUTBREAK ON THE MENTAL HEALTH STATUS OF UNDERGRADUATE MEDICAL STUDENTS IN A COVID-19 TREATING MEDICAL COLLEGE: A PROSPECTIVE LONGITUDINAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study investigated the mental health of medical students in India. The paper was well organized and written. The research background was provided sufficiently. Raw data was shared. The major strengthening of the study was longitudinal study-design and validation of questionnaire for the COVID-19 related data. However, several concerns should be addressed.
Experimental design: Several details should be added, including the study site, sampling method at baseline, and the dependent and independent variables in the adjusted binary logistic regression. The limitations of small sample size should be addressed.
Validity of the findings: The results were provided sufficiently. However, the results should be separated into sub-sectionals for better understanding. The titles of all tables (including the supplementary tables) should be added. The statistic values should be added in both the results section and all tables. The P=0.000 should be written as P<0.001 instead.
Additional comments: No other 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: IMPACT OF COVID-19 OUTBREAK ON THE MENTAL HEALTH STATUS OF UNDERGRADUATE MEDICAL STUDENTS IN A COVID-19 TREATING MEDICAL COLLEGE: A PROSPECTIVE LONGITUDINAL STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: This is a prospective longitudinal study, which aimed to investigate the mental health of undergraduate medical students over a duration of 6 months by analyzing data collected before and during COVID-19 outbreak in India. However, I have some comments for the authors for their consideration.
Abstract
1. Authors should report 95%CI of prevalence.
2. DASS 21 should report the full name when authors first mentioned it.
3.  Authors should report specific statistic values about predictors (e.g., OR, 95%CI.)
Method
1. Authors should draw a flowchart to illustrate the sample selection from baseline to follow-up.
2. For a prospective longitudinal study, why authors do not examine the incidence of depression, anxiety and distress.
3. For a prospective longitudinal study, why authors do not examine the RRs?
4. What is the “Parallel analysis”? I did  not found any results from this analysis.
Results
1. Authors should add some subtitles in order to well understanding for readers.
2. Authors should re-design the table 1. A horizontal table is easy to understand.  Please turn the first column into the first row.
3. All supplementary tables did not present the titles. Please add them.
4. In all tables.  Please correct “p=0.000” to be “p<0.001”.
Discussion
1. As mentioned by authors in the limitation- “Majority of the study population (69.1%) were in the preclinical and paraclinical years which could have influenced the interpretation of results.” Why authors did not control this variable in the association analysis.


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: IMPACT OF COVID-19 OUTBREAK ON THE MENTAL HEALTH STATUS OF UNDERGRADUATE MEDICAL STUDENTS IN A COVID-19 TREATING MEDICAL COLLEGE: A PROSPECTIVE LONGITUDINAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for making the suggested revisions.
Experimental design: I was satisfied with this section in the earlier draft.
Validity of the findings: Thank you for making the suggested revisions.
Additional comments: I am satisfied with the author's revisions. Thank you.


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: IMPACT OF COVID-19 OUTBREAK ON THE MENTAL HEALTH STATUS OF UNDERGRADUATE MEDICAL STUDENTS IN A COVID-19 TREATING MEDICAL COLLEGE: A PROSPECTIVE LONGITUDINAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript was written clearly. Sufficient backgrounds were provided. Results and Tables have been modified as recommended.
Experimental design: Study design as been extended and detailed as recommended.
Validity of the findings: The longitudinal study design strengthened the interpretation of the results.
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: IMPACT OF COVID-19 OUTBREAK ON THE MENTAL HEALTH STATUS OF UNDERGRADUATE MEDICAL STUDENTS IN A COVID-19 TREATING MEDICAL COLLEGE: A PROSPECTIVE LONGITUDINAL STUDY
Review round: 2
Reviewer: 3

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: I am very satisfied with your 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: TESTING THE SHORT-AND LONG-TERM EFFECTS OF ELEVATED PRENATAL EXPOSURE TO DIFFERENT FORMS OF THYROID HORMONES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and has the correct structure. It is a well written manuscript with what I find a very interesting topic. They provide good context for the study.  All figures and tables are well done, although they should note what the box plots are showing (median and quartiles?).  I appreciate the inclusion of all data points along with the mean (median) values on the figures.
Experimental design: The experiments are within the scope of the journal.  The research question is well defined and is meaningful.  Our understanding of the influence of maternally derived thyroid hormones on development in birds is incomplete and this study attempts to address this by supplementing the yolk with additional thyroid hormones.  The methods are all well-defined.  I wonder how effective the dosing of the yolk is for actually delivering the hormone to the developing embryo - see comments below.
Validity of the findings: I hate to say this, as I really like the idea of the experiment, but given the low hatching success of the control group I tend to agree with the previous reviewers of an earlier version of this manuscript. It would be nice for them to repeat the hatching success portion of the experiment from initial exposure through hatching to confirm their findings on hatching success, especially given this is the only parameter where they found a significant response.  Your control group has a hatching success of only 25% and you are comparing it to 60% in other “control” studies. This means your control eggs had 2 to 3 times worse hatching success than other control studies.  How does taking into account those eggs with no development change the hatching success of the control?

I would like to see the data on mortality. When did the embryos die during development?
Additional comments: A major question I have is in regard to the dosing of the yolk with TH.  I know that these authors have used this method in the past with other species.  While I appreciate that the authors are attempting to elevate TH within a somewhat natural range, I wonder how well injecting in the yolk is as a delivery method.  Given this method and the lack of a response, we really don’t know when the excess TH are actually taken up by the embryo. Are the embryos actually experiencing “higher” levels of TH during development?  Do they become diluted in the yolk, so that it is a very minimal dosing or does it stay concentrated and they take it up all at once? Is there a study that has injected an inert dye into the yolk and watched how it diffuses and moves through development (not suggesting you do this, just wondering if you know what happens once it gets in the yolk)?  Can you provide the concentration in terms of ng T3 per ml (or mg) yolk?

Line 28: “These results suggest that yolk thyroid hormones are important in the embryonic stage of precocial birds…”

Line 60 – change “fasten” to accelerate

Line 179 - I am a little worried about the low hatching success in this study, as noted above.  While they show provide references of studies where others have a 50% hatching success, there are other studies with hatching successes between 80-95% for fertilized eggs (for one see Martin and Arnold, 1991, The Condor). Additionally, their control eggs had a 25% hatching success, which is almost half of the worst hatching success they reference. It would be nice to see the distribution of ages at which the embyros died. What was the distribution between the treatments of the 31 eggs with no developed embryo? If a larger majority of these eggs were in the control, this might skew your hatching success rates away from the controls.  Is the hatch rate you present in Figure 1 including the total number of eggs set or just those that showed developed embryos (discarded the infertile and those whose yolks may have been damaged resulting in no development) as the denominator? I would argue that it should be not include the infertile eggs that did not have a developed embryo. How far along were the additional 61 animals that did not hatch? I would recommend you repeat this aspect of the study to confirm the findings.

Line 291 – I applaud your use of a measure of effect sizes as a means to determine how large a response in the data is. I suggest you include the 95% CI for your Cohen’s d index.  There is a nice website: estimationstats.com which provides the R script to calculate Cohen’s d along with bootstrap 95% CI. Here is the citation they provide: Moving beyond P values: Everyday data analysis with estimation plots.  Joses Ho, Tayfun Tumkaya, Sameer Aryal, Hyungwon Choi, Adam Claridge-Chang. Nature Methods 2019, 1548-7105.

Line 326 – “… additionally tested early morphological traits apart from the growth curve…” Can you define early?  Is this during the embryonic period or neonatal period?

Line 358 – 362 – Did you remove the undeveloped (infertile) eggs from the denominator?

Line 363 – It might be useful for you to provide some data as to when the embryos died.  Was it early on in development or just prior to hatching?

Line 368 – add period at the end of the sentence.

Line 373 – I would change early to neonate or hatchling.  When I read early, I was thinking of during the embryonic stages.

Line 374-379 – might be nice to include the 95% CI for Cohen’s d. Will give us an idea of the range of d and how “strong” the moderate effect size is.

Line 380-381 – were there differences in the sex ratios for the three treatments?  Just curious.

Figure 4 figure legend – what do the boxes represent?  Please provide description in the legend.

Line 438 – Do they know at what point in the embryonic development that the embryo starts to produce T3 and deiodinase?  How far along into embryonic development? Can you add the timing to this sentence?

Line 446-448 – I am not sure what you mean by improving the age of embryo mortality.  If an embryo dies, does changing the timing of this improve it?  Because you do not provide the data for timing of embryo mortality, it is hard to judge this statement.

Figure S1 – what do the line and boxes represent? Provide in the figure legend.

Figures S5-S8 are not references in the text.


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: TESTING THE SHORT-AND LONG-TERM EFFECTS OF ELEVATED PRENATAL EXPOSURE TO DIFFERENT FORMS OF THYROID HORMONES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study sets out to investigate the effects of maternal thyroid hormones on hatching success and longer-term metrics in Japanese quails. While I disagree that this avenue of research is particularly novel, it would nonetheless be a welcome addition to the literature. Perhaps an enhanced focus on T3 vs. T4 would help set it apart — to this end, however, there is very little in terms of their endocrinological differences discussed at the outset. To this end, I think the introduction could use a restructure to showcase i) why we care about maternal thyroid hormones in Japanese quails, or why Japanese quails (or birds in general) represent a good model to investigate these questions. While the methods were selected for ecological relevance, the overall manuscript really lacked the endocrinological and ecological context to make it matter.

Introduction. We veer into avian territory petty quickly with not setup into why looking at maternal thyroid hormones in this system would be particularly interesting. In other words, there is very little context for how an avian system can help us understand this phenomenon. I assumed its largely because embryonic THs can be more easily manipulated? Other reasons? I would recommend strengthening this link, where you go from humans to birds.

43-44. Maternal effects of thyroid hormones is not a novel concept. Of the many examples in the literatures, Wilson and McNabb (1997) show that maternal thyroid hormones influence embryonic development in the same species of quail used here — perhaps this would be relevant to in the ms.

67-79. Now we’re back to all vertebrates. I would recommend restructuring the introduction so that there is a logical flow.

104-133. There are a lot of references to studies showing answers to these questions in other species. In this context, I think the introduction would benefit from justifying why these same questions are important in the Japanese quail — is the novel angle specifically looking at differences between T3 and T4? If this is the case, I recommend tailoring the introduction to this question and contextualising differences in T3/T4 action.
Experimental design: I also echo concerns over statistical power of previous reviewers. I do not necessarily have an issue with the smaller sample sizes themselves, but rather the unequal distribution in sample size across treatment. This means the chances of finding significant differences between treatment X and control for a given metric are substantially higher for some treatments than others — i.e. small samples more prone to type II error. Further, it is difficult to appreciate the findings on hatching success without the actual numbers of unfertilized eggs for each treatment. Could this be added to the results? Did I miss it? As mentioned by a previous reviewer, I also think further replication with a strict control treatment (in addition to the sham control used here) would be useful.

Methods + Results

146-147. Just use the range — 6.6 eggs is not particularly meaningful, but the range gives an idea of how sample sizes may be spread out.

I would expect methodology this invasive to include both a control and a sham control — in other words, injection into an egg alone does not in itself represent an ecologically relevant control. A strict control would allow you to test for the “normal” hatching success rate in these birds and investigate potential interactions between injection and thyroid hormone actions.

181-183. Good. This is an important point.

183-184. This is not a problem so much as the idea that manipulated hormone levels may allow some embryos to deal with injection trauma better than others. Again, this is where a strict control treatment would have been useful. Were all eggs injected by the same individual?

209. Was this observer blind?

220. Is this the sample size of males per treatment? Probably not, right? Unless I’m mistaken, this seems misleading. It would be more appropriate to denote the sample size of males per treatment (i.e. N=6-7)?

223-224. Use the sample size notation for the above.

281. Why only 1,000 simulations? 10,000 is commonly the “unofficial” minimum and would be much more believable, especially when p hovers around 0.05.

 292. I assume this takes unfertilized eggs into account?

296-299. Good.

337. I don’t understand this sentence — was the point to avoid over-parameterizing the model? Could you not have looked at each one separately?

360-362. I’m trying to understand how 21 out of 40 eggs for T3/T4 was significant, but 20 out of 40 for T4 was p>0.09? This is where more information in lines 179-181 would be more useful.
Validity of the findings: I would consider changing the title further to reflect that small sample sizes may have promoted type II errors — i.e. you did not find evidence that there are clear effects of thyroid hormones into adulthood, not that there “are no clear effects”.
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: TESTING THE SHORT-AND LONG-TERM EFFECTS OF ELEVATED PRENATAL EXPOSURE TO DIFFERENT FORMS OF THYROID HORMONES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all my major concerns.
Experimental design: The authors have addressed all my major concerns.
Validity of the findings: The authors have addressed all my major concerns.
Additional comments: When Figures 2, 3, S4, and S5 are printed on a black and white printer, one can not tell which treatment each line is for.

Figure legend for S1: it references itself (Fig. S1) for the description of treatments. I think, since you added a new S1, the material to be referenced needs to move from S2 up to S1.


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: TESTING THE SHORT-AND LONG-TERM EFFECTS OF ELEVATED PRENATAL EXPOSURE TO DIFFERENT FORMS OF THYROID HORMONES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The intro is much stronger and provides great justification for the study. It now runs a little long — now that you have the general bird justification, I recommend moving the Japanese quail justification (118-127) to the methods. I also recommend condensing the text in the intro thereafter so that it focuses on hypotheses but leaves much of the more specific details for the methods.
Experimental design: I have no major issues with the design itself though I think a true control would have been very informative.
Validity of the findings: In terms of the statistical problems, I maintain there is an issue with the unequal sample sizes. The imbalance is not just between the control and treatment groups as the authors assert but also between treatment groups themselves. In the cloacal gland recession analyses, for instance, there are 2-fold and 3-fold differences in sample size between T3 vs T4 and T3 vs T3/T4 treatments, respectively. This trend is similarly true for the other analyses. These differences, coupled with the especially low sample size of the control group, means that you have more power to find statistical differences between the T3/T4 treatment and control (for instance) than for the T3 treatment and control. The same is true for T3 vs T4 to a lesser but nonetheless worrisome extent. The added issue arises from the authors reporting largely negative results (as indicated by their previous title). In short, I simply have trouble accepting that the negative results may reflect anything but a lack of statistical power (or unequal power in the experimental design).

I also now read the statistical methods more skeptically. This is a lot of effort and work-around analyses for an experimental design that should be statistically straightforward to analyse via GLM ANOVA. It does not seem to be a particularly parsimonious way of analyzing the results. I also hesitate to even use descriptive statistics when N<5.
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: POSITIVE SELECTION WITHIN THE GENOMES OF SARS-COV-2 AND OTHER CORONAVIRUSES INDEPENDENT OF IMPACT ON PROTEIN FUNCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the manuscript is very well written, clear and easy to understand. The manuscript, when published, will definitely greatly aid in the ongoing research pertaining to the evolution and adaptability of SARS-CoV-2. However, I felt that the authors could have included more references in the manuscript, especially in the Introduction section. Also, some sentences are long and could have been broken into two to improve understanding.
Experimental design: The method section is very well written with sufficient details.

One query that I have is branch-specific dN/dS implemented in PAML package can also perform selection tests. It is unclear to me why the authors did not use this popular package. The authors should clearly mention the technical and biological superiority of their method over more commonly used Branch-specific dN/dS approach implemented in PAML.
Validity of the findings: The data are robust and statistically sound. All conclusions are well stated and linked to the original research questions.
Additional comments: 1. The first paragraph of the Introduction section should have a reference.
2. Lines 63-64: "A single nucleotide polymorphism (SNP)..." - Mention the nucleotide and amino acid substitution related to this SNP
3. Line 85: "Here we utilize a test for positive selection..." - Is there a name for this test? If so, can you mention it here and describe why it is superior to other similar tests?
4. Line 104: Please elaborate the gene by gene alignment procedure
5. Lines 147-152: Can you compare and contrast Zeta obtained from PhyloFit with Omega, generated in brach-specific dN/dS implemented in PAML?
6. Line 223: I could not find Protein 3a annotated in Fig. 2. Is it annotated in a different name?
7. Lines 230-233: Fig. 1A- For human SARS-CoV2, the signal of positive selection is not as prominent as in bats and pangolin. I would call it an intermediate signal between Pa-CoV-Gx and human SARS-CoV. Then how can you call it a 'prominent' signal?
8. From Fig. 1B it seems like the fourth signal near C-terminal domain is unique to SARS-CoV2. Is that true? If so, can you discuss the potential reason(s)?
9. Please annotate the ACE2 binding domain in Fig. 1B and IC
10. Please annotate and demarcate ORF1a and ORF1b clearly in Fig. 1A
11. Lines 275-277: "Of note, this is the only...non-synonymous substitutions" - It is unclear to me if there is no non-synonymous substitution, then how did you conclude it to be a signal of positive selection? Please explain.
12. Lines 279-281: "This suggests a complex...positive selection at the nucleotide level" - How is this possible? Please explain this.
13. Please avoid using adjectives such as 'Surprised'.
14. Line 352: "However, we did observe four SNPs..." - Please highlight these SNPs in Fig. 2C. By eyeballing, at least two of these SNPs does not seem to have AAF>0.6.
15. Also, two of these SNPs lie within areas with signals of strong purifying selection. It is unclear to me how hitchhiking is possible for these SNPs. Hitchhiking, by definition can operate on neutral sites or sites with slight negative selection. Please explain with references.


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: POSITIVE SELECTION WITHIN THE GENOMES OF SARS-COV-2 AND OTHER CORONAVIRUSES INDEPENDENT OF IMPACT ON PROTEIN FUNCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is excellently written - really well done.
Experimental design: One small question on the stability of predictions - more below in the General Comments.
Validity of the findings: One concern about the validity of the RNAfold predictions - more information in the General Comments
Additional comments: This manuscript reports the use of a new analysis method to identify selection pressures on the evolution of coronavirus genomes that recapitulates and enriches-upon/complements other results from protein-centered approaches. This is a well-crafted study (and extremely well-written manuscript) that provides interesting results that focus on evolutionary pressures that shape the RNA molecules comprising the viral genomic material. By focusing on features other than protein coding potential, the authors unlock insights into other aspects of coronavirus biology: e.g. functional RNA secondary structure. The results, particularly the identification and clustering of sites of positive and purifying selection, are of immense importance to the community and would make this a valuable contribution when published. The implications of RNA structure are absolutely worth discussing, however, the analyses of secondary structure presented here are (I believe) too speculative/preliminary for inclusion as a published Result.

Major Points:

1) As a non-specialist, it seems surprising that significant evolutionary pressures can be identified with an analysis of an alignment with so few genome sequences. Apologies if I missed this in the manuscript, but, it would be good to have an idea of how robust the identified sites are. For example, if you select different sequences from the same species (with some internal variation) to include in the analysis, would the results be the same: i.e. how volatile would the results be for resampled sequences?

2) There are two analyses of conserved/functional RNA structural motifs available for SARS-CoV-2: described in the Rangan et al paper, and in the preprint from Andrews et al, both cited in the manuscript. The sites of unusual selection identified in SARS-CoV-2 that were identified could be cross-referenced to structural motifs identified in these studies and that are available from the Das or Moss labs, respectively. For the Andrews, et al dataset, for example, selection sites can be loaded as a track on the RNAStructuromeDB SARS-CoV-2 genome (https://structurome.bb.iastate.edu/sars-cov-2) to aid in the analysis.

3) The results based off of the RNAfold predictions (e.g. lines 361-363 and 448-455) are not robust enough for making conclusions about potential structural differences between viruses. Minimum free energy predictions can be extremely volatile and may vary wildly based on the sequence length selected for inclusion. Importantly, they are also sensitive to even small sequence variations. For RNAs < ~700 nt RNAfold is expected to predict ~70% of base pairs correctly for an average RNA – but this value can vary greatly depending on the target. If the authors want to perform a rigorous analysis of RNA structure they can attempt to use the available biochemical and in silico structural results for the S region to build a model for this domain of SARS-CoV-2 and attempt to predict the effect of variations in sequence with respect to this model. While interesting, however, I would recommend potentially removing these analyses from the Results section but to discuss the potential roles of RNA structure in these processes in the Discussion, which is already done. I’d not delay the publication of this paper for these analyses, but perhaps include them in a follow-up publication.

Minor Points:

Line 107: This sentence would flow better if "Despite" was replaced with "Although”.


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: POSITIVE SELECTION WITHIN THE GENOMES OF SARS-COV-2 AND OTHER CORONAVIRUSES INDEPENDENT OF IMPACT ON PROTEIN FUNCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been substantially modified according to the reviewers' suggestions. I think this manuscript can be accepted in the current form.
Experimental design: Looks great now. I have no additional comment.
Validity of the findings: Again looks fine
Additional comments: Nicely written manuscript. Detailed and great to read. I wish you all the best for your future endeavours.


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: POSITIVE SELECTION WITHIN THE GENOMES OF SARS-COV-2 AND OTHER CORONAVIRUSES INDEPENDENT OF IMPACT ON PROTEIN FUNCTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have done a great job here and I think the revised manuscript is good 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: THE FUNGAL COMMUNITY IN NON-RHIZOSPHERE SOIL OF PANAX GINSENG ARE DRIVEN BY DIFFERENT CULTIVATION MODES AND INCREASED CULTIVATION PERIODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: - Some parts (for example, L83-86, 213-215, 253-255, 258-259) are not grammatically correct or sounds awkward. Please revise.
Experimental design: - Please provide more information on how the ginseng fields were selected.
Validity of the findings: - L253-255 in the Conclusion is not clear.
Additional comments: 1. Please make it clearer why fungal communities in non-rhizosphere soil were studied. What did the studies conducted on rhizosphere soils find? What is the importance of non-rhizosphere soils? Providing answers to these questions would help the readers better understand the rationale of this study.

2. The Discussion is mainly reiteration of results, and thus needs significant improvement. Literature on studies of soils from ginseng fields or other crops need to be incorporated. Comparison of previous studies with current study will enrich the Discussion.

3. Please use alternative terms for 'artificial cultivation' and 'soil micrrobiome community imbalance'. They are confusing terms.

4. The figure legends need to be more specific. Please indicate what C2, C5, W15, W35 are, even if they were explained in the Materials and Methods section. This will help the readers better understand the contents in the figures.