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: KELPIE: GENERATING FULL-LENGTH ‘AMPLICONS’ FROM WHOLE-METAGENOME DATASETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes a piece of software which solves a worthwhile problem, gene-targeted assembly from short reads in the metagenomic context.  Provided it works as well as the authors think it does, and that it is supported/maintained, it may facilitate lower-cost targeted-gene analysis in large datasets by limiting the scope to a handful of molecular targets.

This paper should cite and offer a one-sentence comparison to EMirge (Miller https://doi.org/10.1186/gb-2011-12-5-r44) , which does something similar.

Excessive conservation not the only cause of assembly failure; the inverse problem--insufficient coverage--is also severe but not fixable. You handle this by simple thresholding (like everyone does) but the crude effects of low-abundance signals are not described clearly.  I can't tell reading the text how completely the kelipe assemblies appear to represent the short reads.  I didn't catch how many different kelpie amplicons were produced for each of the datasets, and how many short reads mapped to them without digging into excel tables.
Can you mention the amplicon/shotgun quantity-quality tradeoff?

"that rarer organisms in the community will not be represented in the set of extended reads if their coverage is incomplete"
This is an important point, both to clearly define "incomplete coverage" -- here you seem to mean coverage gaps that are impossible to asseble across--and to articulate this as a limitation of the technique.

"generally good agreement"  -- how good?  You should find a statstic suitable for evaluating the concordance of multinomial samples for comparing kelpie to amplicons and gold standard.  Euclidean distance on the p's or the multinomial generalization of Jaccard index, (called Ružička index) would both serve.  Either would give a number between 0 and 1 for how similar the distributions are that you should report for all the relevant comparisons (and in the text).

"...is more flexible than amplicon sequencing"  ??? This isn't really what you mean.  It's more expensive than amplicon sequencing.. it targets almost all of the genes, at a sequencing depth cost, but this isn't flexibility, it's a tradeoff.

"down to very low WGS coverage depths"  --  Statements like this require reference ranges.  How low is very low?  How do depths constructed using kelpie from WGS compare to amplicon?

"only a single viable next kmer"  implies that the approach is sensitive to [sequencing] error rate.  Is there a model for this, or an SOP that calls for kmer-based error correction?
Experimental design: I don't think the validation convincingly shows equivalence to amplicon analysis, and the visualizations are not inspiring, but as the authors point out, convincing validation in the wild is exceedingly difficult.

The comparisons that focus on the difference between 0 and nonzero overlook the fact that every taxon has a number from both datasets.  The .03% amplicon abundance, for instance, is less relevant than the corresponding shortread mapping abundance on the SSU, which is difficult to estimate from the text.

There is a lengthy discussion in lines 410-435 about data cleaning that seems out of place.  The CAMI data has some low-abundance things, and you know their abundance from the gold standard, so identifying the low-abundance taxa by mapping seems unecessary.   It would be more helpful to describe the abundance levels that keplie retrieved SSUs at and the abundance levels where it failed to assemble--identifying the sensitivity boundary for keplie using the variation in CAMI abundances--rather than describing the CAMI dataset by mapping.   I hope this isn't to justify suppressing the low-abundant taxa from the comparison.

Table 6 seems to evaluate presence-absence of taxa, which overlooks the numbers of reads mapped.  The numbers matter, and the taxa not detected shouldn't matter much for the reason they weren't detected.

"only a single viable next kmer"  implies that the approach is sensitive to [sequencing] error rate.  Is there a model for this, or an SOP that calls for kmer-based error correction?
Validity of the findings: How do the results of keplie amplicon assembly retain abundance information?  Are there any reads that end up unmapped to kelpie assemblies?  You probably wrote it but I missed it.

Source:
The repository linked does not contain how-to style installation instructions.
The repository does not contain sample data or a walkthrough invokation on sample data.  This is extraordinarily helpful for scientific programs, and will increase the audience of people who will be willing to use the software.

License:
The repository does not contain a copy of the relevant MIT license.

"down to very low WGS coverage depths"  --  Statements like this require reference ranges.  How low is very low?  How do depths constructed using kelpie from WGS compare to amplicon?
Additional comments: Most generally, I would like to see more numbers in the text (as opposed to the figures) when the numbers are critical to the results.

The text inspires the question "Why does keplie work better for repeated regions than other assemblers?"  You might speculate about what sets kelpie-assembly apart.

inherent presence --> presence    (too many uses of inherent this paragraph)

Stylistic complaints: Does kmer really need camel case styling if it's not in a product name?   Do amplicons really require "scare quotes" unless you are referring to virtual amplicons, in which case, isn't adding an epithet more clear than punctuation ?  Similarly 'gold' taxonomic profiles can be gold-standard taxonomic profiles.

The abbreviation "CSM" for Coal Seam Microbiome used in the captions to the figures should be spelled out in the text.

The names of underlying in-house tools (FilterReads) need not be buried in footnotes.


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: KELPIE: GENERATING FULL-LENGTH ‘AMPLICONS’ FROM WHOLE-METAGENOME DATASETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Literature references, sufficient field background/context provided

In general I found the introduction clear but a bit poor of reference. As example:

Sentence at line 53-54 need more support, please provide some reference to support it or improve the description of the phenomena.

Sentence at line 63-64 need more support, please provide some reference to support it.

The sentence at line 67 is a bit redundant with the previous one (Line 53-54) and need to be supported by references.

The paragraph from line 75 to line 84 is a good summary of amplicon method but need to be improved with some references.

2) Professional article structure, figs, tables. Raw data shared.

In general the structuring of some paragraphs should be revised to make the reading more fluid and  the results more comprehensive.

Some issues should be stressed more in the introduction to highlight the results. For example, the problem of PCR artifacts have a key role in environmental molecular studies but this problem is introduced in the results section, passing into the background (Line 334-336)

The section Results contains several paragraphs that I think should be moved in "Materials and Methods" section.
The section "Materials" is not present  and the description of the database are in the result section. This type of structure makes the reading more cumbersome.

More in details:
Line   276-295, Line 304-312 both paragraphs sounds more as “materials”. For the sake of clarity I suggest to move in this section the descriptions of the datasets used for the benchmarks.

Line 319-323 This description should be moved in Methods
Line 369-373 This description should be moved in Methods
Experimental design: I advise the authors to carefully review the materials and methods section and structure it more clearly as suggested in the previous box, maybe using a drawn workflow.
Validity of the findings: no comment
Additional comments: Kelpie seems to be a very interesting tool, with several possibilities of applications not only for the 16S (as suggested by the authors but unfortunately not tested or not showed). A more linear text structure could make reading more usable even by readers outside the bioinformatics field.


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: KELPIE: GENERATING FULL-LENGTH ‘AMPLICONS’ FROM WHOLE-METAGENOME DATASETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for the remarkable improvement in the linearity of the text. The parts added or repositioned made the reading smoother. This clarified the potential of the software and highlighted both the positive sides and the possible limits, making the reader more aware of the instrument he is using.
I still say that even if it does not bring upsetting news in the field of metagenomics (and without having pre-existing ones) it is a simple and well-made tool that fulfills the one for which it was written.
Honestly, I've never seen it as an alternative to the amplicon base approach but more like a parallel approach to fill some gaps. In the ecological field (i.e. metabarcoding) it can be an interesting tool to test, especially in cases where the length of the marker is a limit in resolving the taxonomy.
In my opinion the work is certainly worthy of publication.


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

Input:
Title: SPATIAL VARIABILITY IN SOIL PH AND LAND USE AS THE MAIN INFLUENTIAL FACTOR IN THE RED BEDS OF THE NANXIONG BASIN, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Your points are clear enough but there are significant language usage problems throughout this manuscript. The following are some examples and my reasons for pointing them out: "Element imbalances" (line 34) is too vague to convey that soil pH is important to plants. "Inhomogeneity" (line 35) should be avoided in this case because it is inappropriate to use an antonym negated with a prefix of "un-" or "in-" to define a term, and also some complex systems can be homogeneous. This and other paragraphs of the introduction clearly are tackling the problem of establishing causation between soil pH and vegetation, but the contrived language tends to mask the valid points you have raised.

You could avoid colloquialisms like "hot topics" (line 42) or "so far" (line 75) in scientific writing.

Many of the paragraphs appear truncated, leaving me wondering why the claims you make are true and relevant. A good example: Why did "scholars worldwide" (line 44) being applying geostatistics? This is a great place to make a concise lists of the pros (and minor cons) of using this suite of statistical tools.

I've often seen geostatistics incorporating or correlating two or more properties, which contradicts your claim (line 52) that geospatial properties "are rarely analysed in combination".

I'm confused by the phrase "soil parameters are highly variable in space and time" (line 55) because soil properties tend to change very slowly. Short-range variability of soil pH is very high but not over time (on the human scale).

I understand your points, which are appropriate, but adverbs customarily come before the word they qualify. The sentence from lines 58 to 60 maybe should read, "Because the spatial distribution of soil pH is stochastic, accurately measuring the spatial distribution of soil pH has implications for crop production." This avoids convoluted and/or redundant wording to convey your point.

It is atypical for any scientist to "prove" a hypothesis (line 60) but rather suggest a hypothesis that has not been disproved.

"Structure" (lines 59, line 65, etc.) has a number of very different meanings in statistics and soil science. It is good that you have suggested this as a factor, but you could define this term so readers of different backgrounds can better understand you. Likewise, the term "rational" can be used in many different ways, and you could include a definition or exclude the term as it can be assumed your readers are rational.

"Redbed" (line 66, etc.) requires definition, additionally "purple soil". These are not terms that are internationally used.

The term "eco-security" is somewhat unusual and vague. It is unclear whether you mean the security of the ecosystem or security of the soil, which you mention erodes. This is, of course, another very valid point to raise, but your wording makes it unclear what exactly you wish to convey regarding the security of an ecosystem.

"Past studies have demonstrated that the extent of soil erosion by water varies with pH" (line 72) is a very important point, but a description of the mechanism by which this occurs would be very beneficial to your argument for the importance of soil pH to soil erosion.

I appreciate the organization of your objectives (line 79). In the first objective (i), you say "status" when I think you mean "value", because the pH meter yields a value and not a status. Objectives ii and iii appear to be so similar that they could be combined into one objective. It may make more sense to state your first objective and then a hypothesis of what properties are influencing the values you find.
Experimental design: I'm familiar with the "nested sampling method", and I can imagine many ways it is appropriate for this study. However, you could state this explicitly in your methods.

There is insufficient detail in the soil sample collection section of the "Research method" section to reproduce the fieldwork and labwork. Which crops were harvested? Which "soil types" determined your sampling densities? Was the water used tap water or DI water? You have listed the specifications of the pH meter but not the specifications of the pH glass electrode, if that's what you've used. What other properties of the soil did you measure? Your objectives state you wish to determine which properties determine soil pH, but only soil pH was measured. Did you measure topographical data such as slope angle and aspect? These are required for your conclusions (line 283).
Validity of the findings: Your production of the semivariogram is very good and appropriate for assessing spatial variability of soil pH values.

Sections of your results entitled "Analysis of influential factors", "Topographic factors", and "Land use pattern" are not results of your study and belong in your discussion. Discussing potential factors does not add data to your study, and to infer that these are influential to soil pH in this study without collecting data regarding these variables is inappropriate.

I'm unsure the following result is supported by a data set absent slope data of each value of soil pH: "The pH of soil is highest on the downslope, followed by the middle slope, and is lowest on the upper slope." (line 258)

Your conclusions fit the results that are based specifically on your data, but the statement about "reducing the cost of the production cycle" (line 279) is unclear. Production cycle of semivariograms?

It is clear that "soil pH is affected by both structural and random factors" (line 283), but this conclusion can be drawn from a wide range of coefficients of variation, as listed in the assumption in this model from your methods.
Additional comments: Thank you for your work and effort to characterize soil pH in such a large and interesting region of China. Your work is important to the expansion of our knowledge of these soils, whose properties are worth studying in greater depth. I have included in my review of your manuscript a critique of your basic reporting, but it is not intended to detract from your message and review of the literature, both of which are valuable to the manuscript overall but somewhat lost in somewhat convoluted language I found hard to understand. The organization and causal inferences of this manuscript are its greatest weaknesses, but its novelty and value to the mapping of these soils are its greatest strengths.


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: SPATIAL VARIABILITY IN SOIL PH AND LAND USE AS THE MAIN INFLUENTIAL FACTOR IN THE RED BEDS OF THE NANXIONG BASIN, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is aimed to investigate the Spatial variability of soil pH and land use as the main influential factor in redbeds of the Nanxiong Basin, China.
The writing is clear enough, however, the manuscript contains a number of spelling and grammatical errors which should be carefully revised before acceptance. The literature is suitable for the purpose of the article.
Experimental design: no
Validity of the findings: no
Additional comments: Overall, the manuscript is publishable, but, needs a critical major revision first. The minor comments are mentioned in the attached PDF and the major comments are outlined below:

1.	The section of discussion is not clear. Please add the contents of comparative analysis and the disadvantage of this study in the manuscript:
2.	The authors explained too much in some parts of the manuscript. However, the influential factor is not clear enough. These sections should be rewritten
3.	The materials and methods should be explained properly in the Research method section.


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: SPATIAL VARIABILITY IN SOIL PH AND LAND USE AS THE MAIN INFLUENTIAL FACTOR IN THE RED BEDS OF THE NANXIONG BASIN, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Major revisions I suggested in my first review have been satisfactorily addressed.
Experimental design: Major revisions I suggested in my first review have been satisfactorily addressed.
Validity of the findings: Major revisions I suggested in my first review have been satisfactorily addressed.
Additional comments: Thank you for addressing my first review's 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: SPATIAL VARIABILITY IN SOIL PH AND LAND USE AS THE MAIN INFLUENTIAL FACTOR IN THE RED BEDS OF THE NANXIONG BASIN, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no
Experimental design: no
Validity of the findings: no
Additional comments: Most comments are well responsed.
But the authors mentioned the land use and land cover for many times, but there is no related support information. A map of land use/ cover classification in the study area would be helpful to understand the text.
This comment should be addressed properly as this is the base of the comparative analysis in 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: AGE-RELATED DIFFERENCES IN FLEXIBILITY IN SOCCER PLAYERS 8–19 YEARS OLD
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: GENERAL COMMENT
Considering the popularity of soccer, the importance of flexibility for health and the lack of studies on flexibility in soccer, this paper has practical relevance for coaches and trainers in soccer. I would recommend it for publication once the authors addressed a few minor issues.

SPECIFIC COMMENT
1.	Abstract: “Muscle flexibility is one of the basic components of fitness for sport performance”. It must be revised here and throughout the text that flexibility is a main component of HEALTH-related fitness and is component of SPORT-related fitness only for those sports where it correlates with performance.
2.	Abstract: Add basic characteristics of participants in methods.
3.	Abstract: Add some mean and SD scores in the results.
4.	Abstract: Add a sentence commenting on the evaluation of the flexibility in soccer compared to general population in results and conclusions.
5.	Introduction: “Muscle flexibility is also one of the basic components of fitness for sport performance” should change. Flexibility is not related with sport performance in soccer, but with health.
6.	Introduce: Before aims, add 1-2 leading sentences about general trend of changes in flexibility by age in the ages you plan to study.
7.	Discussion: Although the literature on soccer and flexibility (Malina, Ribeiro, 71 Aroso, & Cumming, 2007; Manning & Hudson, 2009; Nikolaïdis, 2012; Vaeyens et al., 2006) is mentioned in the introduction, surprisingly it is not discussed. These references are major, thus, they should be discussed in 1-2 paragraphs after the 1st paragraph of discussion.
8.	Discussion: Major literature is missing and should be discussed.
 Inter-individual Variability in Soccer Players of Different Age Groups Playing Different Positions. Nikolaidis P, Ziv G, Lidor R, Arnon M. J Hum Kinet. 2014 Apr 9;40:213-25. doi: 10.2478/hukin-2014-0023.
Age- and sex-related differences in the anthropometry and neuromuscular fitness of competitive taekwondo athletes. Nikolaidis PT, Buśko K, Clemente FM, Tasiopoulos I, Knechtle B. Open Access J Sports Med. 2016 Dec 7;7:177-186
9.	Fig 2 needs error bars.


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: AGE-RELATED DIFFERENCES IN FLEXIBILITY IN SOCCER PLAYERS 8–19 YEARS OLD
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear an unambiguous work

Literature well referenced & relevant.

Structure conforms to PeerJ standards, discipline norm

Figures are relevant, high quality, well labelled & described.(See details)
Experimental design: The investigation must have been conducted rigorously and to a high technical standard. The research must have been conducted in conformity with the prevailing ethical standards in the field.
Methods should be described with sufficient information to be reproducible by another investigator
Validity of the findings: Meaningful replication encouraged where rationale & benefit to literature is clearly
stated.

Data is robust, statistically sound, & controlled.

Speculation is welcome but should be identified in discusión (What is the reason through age, the decrease in flexibility ?)

Conclussion

There is a tendency to repeat results. Must be improved
Why is recommended reinforce the necessity of prescribing exercise?
Additional comments: 1.Abstract. In results define before the acronims

2.- Fig 2: recommended put he SD in every point


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: AGE-RELATED DIFFERENCES IN FLEXIBILITY IN SOCCER PLAYERS 8–19 YEARS OLD
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well written
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The aim of the study entitled “Age-related differences in flexibility in soccer players 8-19 years old” identify age-related differences in lower extremity flexibility in young soccer players. This manuscript contains confirmative/descriptive information.
The paper is well written. However there are same shortcomings in the statistical analysis, results and discussion sections that must be addressed in order to improve the quality of the paper.

Introduction
Line 50: The body composition is another important performance factor in soccer. Please provide an overview on why monitoring body composition is important for soccer players. The following recent citations should be added: (Milanese et al., 2015, Seasonal DXA-measured body composition changes in professional male soccer players; Carling and Orhant, 2010, Variation in body composition in professional soccer players: Interseasonal and intraseasonal changes and the effects of exposure time and player position).
Statistical analysis
How did you calculate the inter- and intra-operator variability in the measurement? The values of inter- and intra-operator variability must be reported.
Was the statistical power adequate? Please comment.
Results
In this section, the results about the ROM test for each leg were not reported in the text and in the table. Please clarify this statement.

Discussion
In this section, the results about the ROM test for each leg should be discussed.


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: AGE-RELATED DIFFERENCES IN FLEXIBILITY IN SOCCER PLAYERS 8–19 YEARS OLD
Review round: 2
Reviewer: 1

Output:
Basic reporting: It's OK.
Experimental design: It's OK.
Validity of the findings: It's OK.
Additional comments: Great work! The paper has been improved a lot. I have no further comment.


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

Input:
Title: AGE-RELATED DIFFERENCES IN FLEXIBILITY IN SOCCER PLAYERS 8–19 YEARS OLD
Review round: 2
Reviewer: 2

Output:
Basic reporting: All request has been done
Experimental design: All request has been done
Validity of the findings: All request has been done
Additional comments: All request has been done


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: AGE-RELATED DIFFERENCES IN FLEXIBILITY IN SOCCER PLAYERS 8–19 YEARS OLD
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: IMPROVING THE EFFICIENCY OF THE FUKUI TRAP AS A CAPTURE TOOL FOR THE INVASIVE EUROPEAN GREEN CRAB (CARCINUS MAENAS) IN NEWFOUNDLAND, CANADA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I agree with the 4 points
Experimental design: I agree with the 4 jpoints
Validity of the findings: This detailed study aimed at improving the Fukui trap is very pertinent and timely and would be of great interest to the many researchers and managers around the world are using Fukui traps to monitor and manage green crab abundance in their invaded habitats.
The manuscript is well written and easy to follow. The experimental design, field execution and data analysis are well described.  The reader gets a clear picture of which modification to the trap yielded the greatest improvement in catches.  I recommend that this study be published.

Below are a few points the authors may want to consider.

•	I was confused because the % increase in efficiency, as described in lines 226-235, does not always match a simple calculation of CPUE of modified traps to control trap as tabulated in Table 2.  See table below.  Am I missing something, or did the authors make a simple calculation error?

Trap Type	Mean CPUE	Table 2 -CPUE modification/control		Increased Efficiency lines 226-235
Control	26.3
Sinker	38.7	38.7/26.3	1.47	59%     - greater
Mesh	33.9	33.9/26.3	1.29	29%     - matches
Assist	45.9	45.9/26.3	1.74	81%     - greater
String	24.5	24.5/26.3	0.93	1.03    -- greater

•	It might be helpful to the reader if the authors were to made some rough estimate of how much time it would cost to make the modifications.  Sinker may be easy to attach. But they are not cheap and not that readily available in large quantities, while the mesh method, that requires sewing would be very time consuming.

•	Starting with line 321 – continual trapping tends to preferentially remove large males from the system, as has been demonstrated by many researchers.  But if it is done too well, it can have unintended consequences as Ted Grosholz found out in a closed system in Sea Drift Lagoon:
http://www.marinij.com/environment-and-nature/20170820/stinson-beach-green-crab-invaders-vex-scientists
In the absence of large males, young green crabs thrived, possibly indicating a lack of cannibalism and increased resources for the young crabs.  ( – no need to include this in the manuscript, just something to keep in mind. )
Additional comments: This detailed study aimed at improving the Fukui trap is very pertinent and timely and would be of great interest to the many researchers and managers around the world are using Fukui traps to monitor and manage green crab abundance in their invaded habitats.
The manuscript is well written and easy to follow. The experimental design, field execution and data analysis are well described.  The reader gets a clear picture of which modification to the trap yielded the greatest improvement in catches.  I recommend that this study be published.  Below are a few points the authors may want to consider.

•	I was confused because the % increase in efficiency, as described in lines 226-235, does not always match a simple calculation of CPUE of modified traps to control trap as tabulated in Table 2.  See table below.  Am I missing something, or did the authors make a simple calculation error?

Trap Type	Mean CPUE	Table 2 -CPUE modification/control		Increased Efficiency lines 226-235
Control	26.3
Sinker	38.7	38.7/26.3	1.47	59%     - greater
Mesh	33.9	33.9/26.3	1.29	29%     - matches
Assist	45.9	45.9/26.3	1.74	81%     - greater
String	24.5	24.5/26.3	0.93	1.03    -- greater

•	It might be helpful to the reader if the authors were to made some rough estimate of how much time it would cost to make the modifications.  Sinker may be easy to attach. But they are not cheap and not that readily available in large quantities, while the mesh method, that requires sewing would be very time consuming.

•	Starting with line 321 – continual trapping tends to preferentially remove large males from the system, as has been demonstrated by many researchers.  But if it is done too well, it can have unintended consequences as Ted Grosholz found out in a closed system in Sea Drift Lagoon:
http://www.marinij.com/environment-and-nature/20170820/stinson-beach-green-crab-invaders-vex-scientists
In the absence of large males, young green crabs thrived, possibly indicating a lack of cannibalism and increased resources for the young crabs.  ( – no need to include this in the manuscript, just something to keep in mind. )


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: IMPROVING THE EFFICIENCY OF THE FUKUI TRAP AS A CAPTURE TOOL FOR THE INVASIVE EUROPEAN GREEN CRAB (CARCINUS MAENAS) IN NEWFOUNDLAND, CANADA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No additional comments (see general comments).
Experimental design: No additional comments (see general comments).
Validity of the findings: No additional comments (see general comments).
Additional comments: PeerJ MS 29803

This is an interesting article addressing an important issue: the efficiency of traps used for green crab fishery/harvesting programs. Although the focus of the study is on a rather practical issue, it also offers a wealth of information for those interested on the ecology of these populations. The introduction and stated goals are very clear. The experimental design and the statistics used to assess the differences among traps are rigorous, and the results are clear and described to a good level of detail. Tables and Figures are informative and of good quality. Some of those results are frankly spectacular (80% increase of efficiency!), so the authors should be commended for conducting a well prepared study based on solid preliminary information. The Discussion highlights the main implications of the results and is not speculative. The practical implications of the results gathered by this study are obvious, and would be of great help for those working on the ecology of this invasive species or those trying to manage its invasions. I deemed the article a solid piece of research and I recommend acceptance with very minor changes (see below).

Specific comments

Lines 40-41: I suggest adding Pickering et al. 2017 (Mar Env Res) and Poirier et al. 2018 (Helgoland
Mar Res).
Line 43: I suggest adding Rossong et al. 2006 (JEMBE) and 2012 (Biol Inv).
Lines 93-96: If I understood the third modification correctly, please explain how tense that mesh was… in other words give us an idea about how “hard” or “stable” that floating “floor” was for the crabs crawling into the traps.
117-118: Briefly justify your choice of bait.
123-124: For the sake of the readers, briefly state whether you expect (or not) that spacing among traps to influence catch rates.
210-212: Provide a value (range?) of how many rock crabs were caught, to have a sense of how small the bycatch was.
289-291: It is not entirely clear what difference the sinker made (in comparison to the strings) to increase catch while reducing or maintaining escape rates low. Instead of altering escape rates, could the crabs use the sinkers somehow to help themselves to enter the traps and increase catch instead?
308-310: What about replacing that mesh with an acrylic or plastic plate or similar dimensions?
332-334: This statement is correct but is not justifiable given the results you report: you did not find larger females in the modified traps. I suggest you state “if future modifications also increase the size of females being caught… “
337-339 and 343-346: Although Poirier et al (2018; Fisheries Res) assessed a modification on a different gear (fyke net), I think is valuable to cite that work here as those results will make your results even more interesting (fukui bycatch is much lower).


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: IMPROVING THE EFFICIENCY OF THE FUKUI TRAP AS A CAPTURE TOOL FOR THE INVASIVE EUROPEAN GREEN CRAB (CARCINUS MAENAS) IN NEWFOUNDLAND, CANADA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The purpose of the present paper is to evaluate modifications to the Fukui multi-species trap for removal of invasive European green crab in the waters of Newfoundland. The European green crab is a global invasive species and the Fukui trap is standard gear used in removal efforts in North America and elsewhere. These results will be useful to managers in many locales. At the same time, it is precisely because managers are likely to be interested in this work that I believe the authors should tread carefully in their recommendations (see below). The manuscript is well-written and clear and will make a valuable contribution.

The introduction & background provide adequate context but additional information (or at least citations) covering the invasion of green crab would be appropriate.
Experimental design: The research question is well defined, relevant & meaningful. I note the authors suggest their goal is to increase capture efficiency. However, capture efficiency is typically defined as the number of individuals caught as a proportion of the total number present. This is typically unknown. I would suggest the actual goal here is to increase catch per unit effort (CPUE).

The methods are described with sufficient detail & information to replicate.
Validity of the findings: Data are robust and statistical analysis is done appropriately. Conclusions are well stated (but see my thoughts below about contextualizing the results). It is important to note that these results pertain to the waters of Newfoundland. Managers/researchers using these modifications may see different results within their jurisdiction, particularly where bycatch is a concern.
Additional comments: Line 293-294: The authors suggest the sinker method is durable but this likely depends on the longevity of the traps. I know several researchers who have used the same Fukui traps for more than 15 years. The openings tend to sag over time and I’m afraid any extra weight may accelerate the process. I think any statements about durability should specify the number of deployment hours over which the modification was tested.

Line 336-356: The authors suggest the modifications do not increase bycatch. Again, I think it’s important to point out the context here given that green crab are a worldwide invader. While bycatch may be low in the waters of Newfoundland, conditions will be different elsewhere within the invasive range of green crab. In fact, on the US West Coast, researchers commonly restrict the size of the trap opening to reduce bycatch of demersal fish, sharks, and mammals.

Line 321-333 I agree that removing large adult green crab may be ecologically beneficial. However, female crab have a larger per capita impact on population growth (given the size of an individual brood, possible sperm storage, etc) and should be targeted for removal. I think it would be good to test for differences in catch of male and female crab across the four modified trap types. Moreover, I think figure 4 would be more useful if it explicitly considered total number of crab of each sex captured in each of the modified trap types.


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: DESIGN, OPTIMIZATION AND VALIDATION OF GENES COMMONLY USED IN EXPRESSION STUDIES ON DMH/AOM RAT COLON CARCINOGENESIS MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Table 1: It would be important to include for every standard curve the lowest and the highest Cq.
Why every gene has been tested for different amount of cDNA? Does it mean that under or above that amount of cDNA the curve is not anymore linear?

Table 2: It would be important to write for every primer where it maps on the cDNA (which exon, CDS or UTRs)

It would be nice to include and example of the agarose gel ran to check RNA integrity (rRNA 18S and 28S)

Rows 171-172: It is described the qPCR stating that the amount of primers used varies from 0.2 to 0.8 ul, depending on the concentration needed, that is stated on table 1, for each primer: since the relevant data is obviously the final concentration of the primer and not the volume, it is a useless and confusing information. The sentence should be removed or changed in  “for every primer the concentration has been optimized, ranging from xxx uM to xxx uM, as indicated in Table 1”
Anyway, primers should have the same concentration.

Rows 286-289: it is not clear how the lowest standard curve was increased without increasing the minimum cDNA concentration

Figure S1 is far more detailed than what is needed. A simple table indicating the diet of the rats in the different groups would be enough. How it is, is just confusing

PCR reaction and PCR empirical validation should be in the same paragraph. Same for qPCR

the reference database for NCBI should be stated (Rnor_6.0), for future references

Row 133: “the nucleotide sequence was retrieved from the NCBI Nucleotide database”
(instead of PubMed Nucleotide database)
Experimental design: - The specificity of a pair of primers and absence of primer dimers is assessed in a more sensitive way using the melting curve in the qPCR reaction. The agarose gel is useful only to determine that the specific product has the attended molecular weight; this observation can be achieved running on the agarose gel the reaction after the qPCR. Thus, all the sections about PCR should be avoided.

- Row 209: This paragraph is a bit confused. It looks like the scope of the paper is to analyse the genes in rats with different dietary, while it is to assess the stability of reference gene USING rats on different dietary.

- Row 229: Why the samples were pooled for RNA extraction? To have more RNA? Moreover, it would be useful to know the total amount of RNA that has been extracted

- State in a clearer way how repeatability and reproducibility has been assessed (is the reproducibility of the triplicates in the same reaction? the same samples have been re-ran? Different retrotrascriptions from the same RNA? RNAs form different rats?)

- In FIG 2 some kind of test should be reported
Validity of the findings: - The first 4 primers sequences have been checked: primers of Actb, Apc and Aurka are designed on the same exon: using primers designed on the same exon enables to distinguish between cDNA and DNA, meaning that if DNA is a contaminant of RNA it will be amplified in the qPCR giving a product of the same length as cDNA. Ideally, primers should be designed either spanning to exons or on 2 different exons, in order to make sure that it is only cDNA that is being amplified. Otherwise, to avoid DNA contamination, one should consider to
1-	treat the RNA with DNAse after trizol extraction
2-	add another negative control in which RNA have been mixed with the RT mix NOT containing the enzyme (to assess that the RNA is not contaminated by DNA)

- Row 203: In the qPCR, together with a NTC negative control, negative controls for RT-PCR (no RNA) and for DNA (no enzyme) should be included

- The stability of the reference genes has been tested using rats that were following different dietary. The result is therefore very specific for this experiment and should be validated for any other treatment. This should be clearly stated
Additional comments: The publishing of primers for qPCR that can be used in rat model of colorectal cancer is surely useful and would save time to researchers working on the mentioned genes. However, it is very important to specify where the primers map on the mRNA. Additional negative controls should be included or suggested to be included. A part for a few paragraphs that I have indicated and that can be improved, the article is well written and clear. The conclusion that B2m is the best reference gene should be limited to the tested treatments.

I am not sure that it was necessary to sacrifice rats in order to test the efficiency of qPCR primers. It would have been sufficient to use a cell line. If the rats have been sacrificed for another project it should be stated.


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: DESIGN, OPTIMIZATION AND VALIDATION OF GENES COMMONLY USED IN EXPRESSION STUDIES ON DMH/AOM RAT COLON CARCINOGENESIS MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: - Please list a reference for the genes routinely studied in DMH/AOM CRC rat model and two reference genes commonly used in the carcinogenesis model (line 91-93).

- The paper lacks figure showing the results of the experiments performed.

- The authors should revise the “result” and "method" sections.
The method section should include a concise description of the used methods; the result section should include the description of the results, including also the figures of the experiments.

- The authors should include details regarding: how obtain the gene accession number, how to reach the Pubmed nucleotide database. Since the author is providing a standardized protocol, the paper would benefit of adding a table with the list of the useful website links and software cited.

- Please state how much RNA was used as starting material for the reverse transcriptase reaction and if the resulting material was diluted for the qPCR reaction.

-The authors explain that primers specificity was assessed by PCR followed by agarose gel electrophoresis in order to check that unique band with amplicon size was obtained (line164), please provide the data of this experiment. Did the author verify the product by sequencing?

- Please clarify the sentence “qPCR must be within a range of 90 to 110% ” (line 197).

- Please clarify the sentence line 199-200 “each point on the standard curve was performed in triplicate and covered all potential template concentrations that may be encountered in the future studies”.

- How the authors can quantify the cDNA used? Please explain (line 218).

- Figure S1 lacks a description, please modify accordingly.

- Line 227-229 state that the colon samples of 3 rats where pooled together prior the RNA extraction: the author should not refer to this experiment as triplicate.

- Line 260-263 the authors described that the unique pick of the primers tested, please show the data.

- Please clarify line 305-323: how the use of the software helped to define the stability of the reference genes? Which data were analyzed by using the listed software? Which data point/rat models were used? Please show clearly the data.
Experimental design: - The research question is well defined; methods are described with sufficient detail and information.

- The authors clearly describe the lack of reliable primers in previous literature, however it would be useful clarify lines 87-88. Based on what evidences the author states that “primer sequence is missing or contain mistakes, primers are not specific or the melting curve is too low”? (Lines 87-88)
Did the author tested the mentioned primers and verified the non-specificity? How the non-specificity was addressed? How the author defines a too low melting temperature?
Validity of the findings: - The authors describe nicely the process to design, optimize and validate reliable qPCR primers that will be used from future researcher in the field.
It is important include in the paper the figures describing the results of the performed experiment.
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: DESIGN, OPTIMIZATION AND VALIDATION OF GENES COMMONLY USED IN EXPRESSION STUDIES ON DMH/AOM RAT COLON CARCINOGENESIS MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The article is well written and clear in its scope and methods. It provides useful information for future studies in the field.
The authors answered to all my concerns and improved significantly the article.


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: DESIGN, OPTIMIZATION AND VALIDATION OF GENES COMMONLY USED IN EXPRESSION STUDIES ON DMH/AOM RAT COLON CARCINOGENESIS MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors fully addressed all the points raised during the revision.
Experimental design: The authors fully addressed all the points raised during the revision.
Validity of the findings: The authors fully addressed all the points raised during the revision.
Additional comments: The authors properly addressed all the points raised during the revision.

I think the manuscript improved in terms of general clarity and description of the methodology and data.

Definitively this paper will be useful for scientist who will perform studies employing the listed validate primers.

Additionally, given the clarity of the experimental details and the access to figures's results, this paper will be  helpful for scientist who will design new primers to study different genes non mentioned in the list.


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: DESIGNING ONLINE SPECIES IDENTIFICATION TOOLS FOR BIOLOGICAL RECORDING: THE IMPACT ON DATA QUALITY AND CITIZEN SCIENCE LEARNING
Review round: 1
Reviewer: 1

Output:
Basic reporting: A well written paper which highlights the multi-faceted nature of citizen science projects. The authors provide a succinct and insightful overview of some of the challenges in contemporary biological recording, and discuss practical elements that are sometimes overlooked in other literature.
Experimental design: Pleased to see the use of mixed methods which offer an insight into the decision making process. The study could be readily replicated, or repeated with variation (e.g. different taxa or participant knowledge base).
Validity of the findings: I believe the findings to be of interest and useful to a range of people involved in biological recording. The authors have drawn on a variety of research areas, demonstrating that accurate species identification can depend upon a number of factors, and make suggestions on how to aid the learning process.
Additional comments: An interesting paper that addresses important aspects of species identification in the context of the wider use of technology. It is also encouraging to see further application of knowledge from other disciplines to species identification. A few comments:
•	Line 62: Perhaps explain or reference the term ‘digital turn’? I’ve never come across the term and although I have found it in a web search, I wasn’t sure if it relates to a particular book or a wider concept that the book covers.
•	Line 167: not important, but expanding Stevenson et al. to include Haber & Morris would show the reader that you are referring to the same paper being referenced throughout.
•	Line 266: Should read Go Botany (not Botony).
•	Line 318: reads “A paper version of the NASA-TLX subjective workload questionnaire (see below) was filled out by the participant for the completed identification task”. Does the see below refer to the explanation in lines 331-2? If so, would it be useful to include as an appendix?
•	Line 343: should nasa read NASA?
•	Lines 379, 394 & 416: which post hoc analyses were used?
•	Participant consent (for the future): I would like to have seen another option for gender (e.g. other or prefer not to answer), especially as Aberdeen has an Athena Swan award.
•	Perhaps one more careful read to identify double spaces or no spaces (e.g. before references in line 68 and 149), although this may just on my version of PDF (which has happened before!)

The paper was a pleasure to read, and just needs clarification in some places given that PeerJ is cross discipline and some readers may not be familiar with some of the concepts. Good luck!


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: DESIGNING ONLINE SPECIES IDENTIFICATION TOOLS FOR BIOLOGICAL RECORDING: THE IMPACT ON DATA QUALITY AND CITIZEN SCIENCE LEARNING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Parts of the literature review can be confusing, with sentences being too big and vague, not necessarily connected with the aim of the study.
Data classification applications in Citizen Science are very common, both in online and offline forms, but it is not mentioned or explained enough in the text.
Experimental design: Given that this is a usability based research for a citizen science application, trying to identify pain-points and the differences in learning for specific features, it could be useful to replicate the study with participants that are not familiar with any type of identification, and participants who are not involved with any type of environmental research.
Many of the users who use said applications are lay people, not necessarily familiar with any of the processes, which could potentially create different results in the study than the one with the conservation students.
Validity of the findings: no comment
Additional comments: Very useful research, covers areas in citizen science data identification that are not explored in-depth yet.


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF LONG NON-CODING RNAS IN TOMATO PLANTS IRRADIATED BY NEUTRONS FOLLOWED BY INFECTION WITH TOMATO YELLOW LEAF CURL VIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing of this paper needs to be improved. Background information is lacking in the the introduction. Additional analysis or more discussion on the differentially expressed lncRNAs should be included.

The abstract is unclear on the rational of this work. It should be more structured into a) why studying long non-coding RNA in tomato irradiated by neutrons followed by yellow leaf curl virus infection; b) the experimental design and analyses; c) the major results and conclusion.

L 31: Are the length of the lncRNAs small or the number of lncRNAs small? Are they only smaller than what was found in one previous study? Was that a tomato study? Please indicate.

L 35: up- and down- regulated lncRNAs in TYLCV infected plants compared to what?

The introduction lacks important information on the background, such as why infect tomato with TYLCV after neutron irradiation, why this needs to be studied? How can lncRNA tell us some information that other study could not, such as mRNA? Are there any other published lncRNA study in tomato? They should be reviewed if there are. I suggest expanding introduction 3rd paragraph with such information. I suggest the last paragraph of introduction to describe the general experiment setup and aim(s) of this study.

L44: there are other types of non-coding RNAs, such as microRNA, please change “known as” to “including”.

L53: the information is limited in terms of what? Please indicate.

L53-L55: Are lncRNA involved in biotic and abiotic stresses? If yes, please include them in the introduction.
Experimental design: The study is interesting but the research question is not very well defined, the reason for why to combine neutron irradiation and TYLCV infection in one study should be included. Methods lacks some details and information to replicate.

Material and Methods are missing details on how different programs were used and parameters for different programs. I am also wondering why you didn’t do de-novo assembly using Trinity, many studies have shown that combining de-novo and reference-based assembly can help to identify more transcripts. Also, did you remove repeats with many more mapping positions on the reference genome? How did you do expression analysis is unclear.

L77: Why to include both dry and presoaked seeds? Please indicate.

L81-L82: Is green chamber also growth chamber? Please be consistent.

L110-L111: Did you use cuffmerge after cufflinks to merge separate assembly into one? As I understand, you generate one set of lncRNA at the end of the analysis instead of eight, where did you conduct merging? It is not reflected in figure 1. Cuffcompare can helps you to compare your assembly to known transcripts and annotate your assembly with difference code. Did you use any of those code to filter your assembly?

L123: I thought you filtered out transcripts from each step individually, as what you showed in figure1, this sentence seems contradictory to your pipeline in figure1. Also for CPC2, is there a threshold value you used? Please indicate if yes.

L125-L127: Please indicate the e-value you used for your BLASTN search.

L129-L131: BWA and BBMap are both alignment tools, I don’t understand why you used two alignment tools to get FPKM values. What’s the distribution of FPKM values look like? How many transcripts did you filter out using FPKM of 1? Could this threshold be too high? Because lncRNAs are known to have low expression.

L134: Did you predict lncRNA target? How? Please describe.

L133: Tomato genome has been published for a long time, there should be published GO term for tomato genes as well. I think you’d better use the GO term for tomato genes for GO enrichment analysis instead of Arabidopsis homolog GO terms.

L146-L147: I am unclear how you did the alignment and quantification of lncRNAs, align lncRNA to lncRNA? What tool did you use for expression analysis? I would suggest using edgeR or Deseq for expression analysis.
Validity of the findings: For the finding, additional analysis or discussion should be included for the differentially expressed lncRNAs in response to TYLCV.

Results are too abstract, lacks detailed description of the findings. I also want to know for the 19 and 11 up or down regulated lncRNAs, what genes are they derived from if they are associated with genes and what target do they have? Since they are TYLCV responsive lncRNAs, they should be examined and discussed in detail.

L173-L196: Did you compare to other studies in respect of the lncRNA length distribution? Please include in the discussion section.  “protein coding transcripts”, not “transcripts coding protein”.

L178: for the one that were derived from six and seven exons, I think you need to give some explanation in the discussion section.

L179: Instead of using “Otherwise”, I suggest change to “in comparison”.

L185: Why were five lncRNA not assigned to any chromosome? They were mapped to the reference genome but could not be located on any chromosome? Are they located on random scaffold? Please explain.

L188: There are multiple circles on the circus plot, please describe in detail. Also please describe the distribution pattern in the result section. Are there more lncRNA in certain genomic region?

L191-L196: What are those eight lncRNAs? Which samples did not express them? Did neutron irradiation with different duration of time or seed condition have effect on their expression? Did you do PCA or mds analysis to see the samples clustering pattern based on their expression level of lncRNAs? I am expecting to see same experiment conditions cluster together. Same for the RNA-seq data from other studies, I think you should do PCA analysis to check whether TYLCV treated and untreated samples cluster together. Please include the results in the supplementary material.

L201: Please rewrite this sentence, it is confusing. I think you mean the number of up-regulated lncRNAs was higher, with 19 upregulated whereas 10 down-regulated.

L203-L215: What do the different colors mean in Figure3 D-F?

L209: Could the enrichment of “ABA-activated signaling pathway” be caused by your dry or pre-soaked treatment?

L218-L219: Is this consistent with what was already known? Please describe in the discussion section.

L222-L227: What are the lncRNA mRNA targets? What biological function do they have? Why do some lncRNAs have multiple targets? What are those lncRNAs? What genes are they derived from? All of these should be discussed.

Discussion sections lacks enough discussion about the study findings.

L230-L232: you are dealing with two factors at the same time, why is it important to combine these two factors in one experiment and check lncRNA? Please discuss.

L233-L234: You are missing reference here.

L236-L238: Please give more details on how lncRNA is involved in fruit development and TYLCV infection.

L249-L252: How can you show the “correlation” between your lncRNA with the CLN2777A study? Could it be that neutron-irradiation caused mutation on the susceptible allele, so that TYLCV susceptibility was alleviated?

L252-L256: Was the same genotype used for both RNA-seq experiment? It is possible that different genotypes have different expression of lncRNAs. Is the difference too big compared to other studies? Since only 59% lncRNAs are expressed. Please discuss.
Additional comments: This study is interesting and the data analysis was done in a reasonable way, however, the writing should be improved, and a lot of necessary information is missing, such as background information, detailed description of the result, and enough discussion of the findings. I would also suggest have someone who is more professional in English writing proofread the manuscript.


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF LONG NON-CODING RNAS IN TOMATO PLANTS IRRADIATED BY NEUTRONS FOLLOWED BY INFECTION WITH TOMATO YELLOW LEAF CURL VIRUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: a. The article is well written with clear and professional English. However there are minor edits need to be done.
for example: line 43 should be re-worded so that it's easier to follow. Line 128 delete "to identify lncRNAs"
b. Other studies of lncRNA on TYLCV in tomato were not mentioned in the introduction. Therefore the knowledge gap filled by this study is not well defined.
Experimental design: a. The hypothesis of this study is not clear. A RNA-seq analysis on the susceptible cultivar infected by TYLCV would enhance the biological interest of this study.
b. The expression analysis of data from Wang, 2015 seemed to be a mere repeat of the original publication. It would be helpful to compare the genotypes used in your study and wang, 2015, and how your differential expression analysis added to the previous publication.
Validity of the findings: In the conclusion, The influence of mutagenesis on the transcription of lncRNAs was not supported by data.
Additional comments: a. Figure 2C needs to define category Overlapping. Figure 2E needs to describe the four tracks.
b. Figure 3D-F need more description.


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF LONG NON-CODING RNAS IN TOMATO PLANTS IRRADIATED BY NEUTRONS FOLLOWED BY INFECTION WITH TOMATO YELLOW LEAF CURL VIRUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised the manuscript, I am OK with most of the responses. However, there are several places that I feel need more revision.
Experimental design: 1) cuffcompare is not designed to do expression analysis, please check.

2) Comment 9 (L77): Why to include both dry and presoaked seeds? Please indicate.
Response: We have addressed the reason why we included both dry and presoaked seeds in our study as follows.
In the manuscript: Presoaked seeds contain higher portions of oxygen and hydrogen than dry seeds. In particular, it is known that oxygen can interact with neutrons during the irradiation process to produce reactive oxygen species (ROS). ROS play a pivotal role as signaling molecules in plants involved in pathogen defense (Apel and Hirt 2004). Thus, it will be interesting to examine the possible effects of ROS in the plant transcriptome.
Comment: OK, but I think since you didn’t do expression analysis for your specific samples, the real contribution of this design is to help you get lncRNA from different conditions, because you combined all samples for lncRNA identification.

3) Comment 11 (L110-111): Did you use cuffmerge after cufflinks to merge separate assembly into one? As I understand, you generate one set of lncRNA at the end of the analysis instead of eight, where did you conduct merging? It is not reflected in figure 1. Cuffcompare can helps you to compare your assembly to known transcripts and annotate your assembly with difference code. Did you use any of those code to filter your assembly?
Response: We mapped all raw sequence reads from eight samples on the reference genome using Tophat. After that, a single bam file was processed with Cufflinks. Finally, we obtained 39,067 transcripts by Cuffcompare. We did not use any code to filter our assembly. We have revised the relevant paragraph as follows.
In the manuscript: All raw data from the eight libraries were aligned using Tophat 2.1.1 to the tomato reference genome (ITAG2.4_genomic.fasta) from the International Tomato Genome Sequencing Project (Trapnell et al. 2012). In addition, the Gene Transfer Format (ITAG2.4_gene_models.gff3) file was used for alignment using Tophat, resulting in a single BAM file. Cufflinks 1.3.0 (Trapnell et al. 2010) was used to assemble the BAM file into transcripts. Finally, Cuffcompare was used to annotate lncRNAs (Trapnell et al. 2010).
Comment: OK. Then how did you use cuffcompare to annotate lncRNAs? What kind of insight did you gain from cuffcompare? Please include the details.

4) Comment 16 (L133): Tomato genome has been published for a long time, there should be published GO term for tomato genes as well. I think you’d better use the GO term for tomato genes for GO enrichment analysis instead of Arabidopsis homolog GO terms.
Response: Yes, it is desirable to use tomato genes for GO enrichment instead of Arabidopsis genes. However, there is not a program or database for tomato GO terms, and tomato gene annotation is less comprehensive than that of Arabidopsis. Thus, we conducted GO enrichment analysis using homologous Arabidopsis GO terms.
Comment: I am OK with this. But you could use R package “goseq” for GO enrichment analysis in any organism with published GO annotations, please check this site: http://jnmaloof.github.io/BIS180L_web/2018/05/24/RNAseq-Annotation/.
Validity of the findings: 5) Comment: please also include a detailed figure legend to part E of figure A. E.g. what does each track mean?

6) Comment 24 (L191-L196): What are those eight lncRNAs? Which samples did not express them? Did neutron irradiation with different duration of time or seed condition have effect on their expression? Did you do PCA or mds analysis to see the samples clustering pattern based on their expression level of lncRNAs? I am expecting to see same experiment conditions cluster together. Same for the RNA-seq data from other studies, I think you should do PCA analysis to check whether TYLCV treated and untreated samples cluster together. Please include the results in the supplementary material.
Response: As the reviewer suggested, we have conducted PCA analysis to cluster conditions based on gene expression of lncRNAs. Our result showed that seven samples, except the sample soaked for 30 min (30S1), were clustered together. In the RNA-Seq data from other studies, only two conditions (mock and TYLCV infected samples) were used. Instead of carrying out PCA analysis, we generated a heat map showing differentially expressed genes between mock and TYLCV infected samples (Figure 3D).
In the manuscript: Some lncRNAs were not expressed in the eight conditions.
Next, we conducted PCA analysis to cluster the eight conditions based on gene expression of lncRNAs. Our results showed that, except for the conditions where the sample was soaked for 30 min (30S1), seven conditions were clustered together (Figure 3B).
Comment: for the TYLCV infected and mock samples, I thought you had different samples for the same condition. No need to include the “heatmap” if there are only two samples. Also the figure 3 legend is incorrect.

7) Comment 30 (L230-L232): you are dealing with two factors at the same time, why is it important to combine these two factors in one experiment and check lncRNA? Please discuss.
Response: We have included a paragraph in the discussion as follows.
In the manuscript: The irradiated tomato plants showed no significant disease symptoms after TYLCV infection. This result indicates that the genomes of tomato plants were mutated by neutron irradiation. Thus, the transcriptional regulation in these tomato plants might be changed. We examined lncRNAs to confirm the change of transcriptome caused by neutron irradiation. Moreover, several previous studies identified lncRNAs in response to TYLCV infection, which facilitates comparison of the lncRNAs among different studies.
Comment: How did you “confirm the change of transcriptome caused by neutron irradiation”? Maybe because only ~50% lncRNAs identified from your study were expressed in the other study? Please discuss. It would be good if you had TYLCV uninfected sample included in your experiment design, so you would be able to compare the effect of neutron irradiation on TYLCV resistance. Please discuss.

8) Comment 33 (L249-L252): How can you show the “correlation” between your lncRNA with the CLN2777A study? Could it be that neutron-irradiation caused mutation on the susceptible allele, so that TYLCV susceptibility was alleviated?
Response: Yes, you are right. In our study, we used the tomato cultivar Seokwang, which was susceptible to TYLCV after neutron irradiation. The eight selected tomato mutants were resistant to TYLCV or showed weak disease symptoms in response to TYLCV infection. Of course, there were some deviations of TYLCV resistance among different mutants; however, all selected mutants showed alleviated TYLCV susceptibility compared to wild type tomato.
Comment: I still don’t understand how you found correlation. Correlation is a statistical term, you need data to support.
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF LONG NON-CODING RNAS IN TOMATO PLANTS IRRADIATED BY NEUTRONS FOLLOWED BY INFECTION WITH TOMATO YELLOW LEAF CURL VIRUS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clarity of the writing has been greatly improved.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: GASTRIC HELICOBACTER PYLORI INFECTION PERTURBS HUMAN ORAL MICROBIOTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: As understood from the paper, the authors have tried to understand the effect of H. pylori infection on the composition of the oral microbiota. I have the following comments on the manuscript:

The epidemiological data provided lacks details such as
a)	The study duration
b)	The timeframe for recruitment of the volunteers i.e were they recruited together or was there a difference in time for sample collection?
c)	Whether the volunteers suffered from any other comorbidities such as gastritis or previously undertook medications in the recent past.
d)	Since H. pylori is associated with ethnicity it would be important to know the same for study volunteers.

It is also not clear the why the authors reasoned that H. pylori infection might have an influence on the microbiota? Or should be reworded to mean that the aim of the study was to understand whether the oral microbiota differs between UBT positive vs UBT negative individuals.
It appears from the observations made that there could be differences in the microbiota of UBT positive versus UBT negative individuals that might be an indirect effect or due to comorbidities which is difficult to resolve given the size of the study.
The hypothesis laid down in the introduction was that H. pylori is present in the oral cavity, but in the samples studied the authors did not identify H. pylori in oral swabs. In addition, since the difference observed is only limited to the daytime samples only points to the fact that it's not a consistent phenomenon. Therefore, it would not be fair to associate H. pylori positivity to the observed changes and would need a carefully designed study with a greater number of samples.
The manuscript needs a careful rewording the project the findings supported by the observations made.


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

Input:
Title: GASTRIC HELICOBACTER PYLORI INFECTION PERTURBS HUMAN ORAL MICROBIOTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No concerns
Experimental design: No comment
Validity of the findings: Analyses should be done at the Family and/or Order level, since patient-to-patient variability at the genus level is so large that most differences aren’t meaningful.  It may be possible to find more meaningful differences at higher taxonomic levels.
Additional comments: Major comments:
1.	Lines 154 and 155:  The sentence states that 14 were H. pylori positive and 10 were negative.  This should be reversed, since 10 were positive.
2.	The demographic table doesn’t add up.  Under gender, it says that 9 were male and 2 were female.  This should add up to 14.

Minor comments:
1.	The rate of infection is much higher than expected for Australia.  Were many of the subjects foreign nationals from countries with higher infection rates?
2.	Roseomonas was only detected in 3 patients and only at 1 or 2 sequences per patient.  This may pass the statistical test, but its relevance seems questionable at best.  The situation with Solobacterium isn’t much better.
3.	Lines 235-236:  The authors state that it is difficult to explain why microbiota changes are seen during the day, but not at night.  There is abundant evidence that H. pylori colonizes the mouth to some degree.  Since H. pylori is an obligate aerobe, more H. pylori growth would occur during the day, thus changing nutrient availability, H. pylori proteins and metabolites, and possibly pH within the oral cavity.
4.	It would be useful to determine the prevalence of Streptococcus mutans.  Reductions in S. mutans could prevent dental caries.  Also, urease production by H. pylori could reduce oral acidity, helping to protect teeth.


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: GASTRIC HELICOBACTER PYLORI INFECTION PERTURBS HUMAN ORAL MICROBIOTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I agree with the authors reply on the previous comments. I have only one suggestion to include a paragraph detailing the limitations of the study clearly especially the small sample size.


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: GASTRIC HELICOBACTER PYLORI INFECTION PERTURBS HUMAN ORAL MICROBIOTA
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 responded appropriately to reviewer comments.

Minor comments
I do not understand why the authors have changed the color schemes in figures 1, 4, and 5.  It is much more difficult to differentiate between light green and dark green than between blue and red.  The schemes should be changed to improve contrast.


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 SCALABLE DISCRETE-TIME SURVIVAL MODEL FOR NEURAL NETWORKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is written in professional English.
The literature references are properly summarized.
The paper follows a professional article structure.
Experimental design: The problem studied is within the aims and scope of PeerJ.
Researcher questions are meaningful and well-defined.
The experiments are not well designed. The description of section results-simulated data is very unclear. The authors should describe how data is simulated, and what the corresponding results are. Instead, the authors describe a mixture of ML models together with unclear results. I have checked the part for a couple of times, and I still don't understand it very well. Moreover, for the MNIST part of the simulated data, would the authors be more clear about this statement: "The ten digits were condensed into five classes, with shorter survival for higher numbers"?
Validity of the findings: no comment
Additional comments: Besides the unclear experimental design, I have two other major comments.
1) In the second paragraph of the Introduction, the authors motivate their work based on three points. To me, the first two motivations are not valid at all, i.e.,
- Few of the authors have published source code
- only some of the models are implemented in modern frameworks such as TensorFlow and Keras
I believe that these problems can be easily solved by some not-very-complicated engineering work.
2) In the description of the experimental results, the authors mention that "which was slightly inferior to the neural network survival model", this means the proposed neural network survival model is not really advancing the state-of-the-art. In this way, I don't see how this paper can meet the standard of a PeerJ publication.


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

Input:
Title: A SCALABLE DISCRETE-TIME SURVIVAL MODEL FOR NEURAL NETWORKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript needs significant re-organization. A lot of wording should occur in the Methods section appears in the Results section. And some sections of Results read like manual/protocol, rather than research results (eg. line 230-246)

The Stanford Medical System metastatic cancer data do not appear to be public/shared.
Experimental design: As mentioned above, some data are not public to be replicable.

More comparisons with other neural network based methods, including deepSurv and cox-nnet, need to be demonstrated (for both accuracy and running time efficiency).
Validity of the findings: At various places, the statements need to be backed up by result demonstration.
For example, the authors stated that one must use fix time intervals (yet have similar numbers of events) vs. alternative approach where the number of events is fixed but the time interval length varies (line 186-189).
Additional comments: The reviewer appreciate the idea behind this manuscript. It is certainly appealing to supplement a NN based model when the data violates the proportional hazards assumption in the cox-PH model. It is also neat to have trained minibatch in each discrete time to enable fast training of the models. The authors did a good job at motivating this approach. However, there are various technical and organizational issues of this manuscript.

1. The data sets used in this manuscript are yet to demonstrate that the Cox-PH model's assumptions fail on them, suggest to provide the evidence in supplementary figures.

2. There is no comparison with other neural network/ deep learning based Cox-PH extension methods, including DeepSurv and Cox-nnet. The authors need to compare their method with these two methods, in terms of accuracy and speed.

3. The authors described 4 simulated data sets, however only show results on 1 data set, they need to show all 4 data (otherwise why mentioning them?)

4. Various places, the authors made claims without showing the evidence, please include evidence as supplementary figures. For example, what supports the statement on line 186-189?

5. How many time intervals exactly to have in order to have the best performing method? Rather than a range of 30-50? Any ways to optimize it for the reader, rather than being a user supplied number?

6. This manuscript needs significant re-organization. A lot of wording should occur in the Methods section appears in the Results section. And some sections of Results read like manual/protocol, rather than research results (eg. line 230-246).

7. The Stanford Medical System metastatic cancer data do not appear to be public/shared. This violates peerJ policy.


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: CONVERGENT ORIGIN OF THE NARROWLY LANCEOLATE LEAF IN THE GENUS ASTER—WITH SPECIAL REFERENCE TO AN UNEXPECTED DISCOVERY OF A NEW ASTER SPECIES FROM EAST CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work provides sufficient context for the reader to engage with the topic, and the references given are relevant and necessary. The hypothesis is linked to the methodology used and made the focus of the discussion and species description. The specimen photographs as well as the botanical diagram are helpful and with appropriate scale bars. The species description at the end contains necessary detail. Some proof reading would help pick up on errors and typos in the manuscript.
Experimental design: As my background is not molecular, I will focus on my area of expertise which is morphology. The use of rudimentary linear morphometric tools is sufficient to answer the questions posed. The analysis of leaf shape and habitat correlation was interesting although I wonder why the authors elected to use a technique that required categorisation of the leaf shape index? The authors clearly state that this is in line with the published knowledge but I think this paragraph (line 165) would benefit by a further explanatory statement on the use of a discrete method as opposed to a continuous. Further, is there a bias from the use of a ratio as opposed to inspecting covariance of the measured variables independently?
Validity of the findings: In the results presentation, the morphological species comparison would benefit for a comparison table, for example the findings in paragraph starting line 277 would be easier to follow in a table format.
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: CONVERGENT ORIGIN OF THE NARROWLY LANCEOLATE LEAF IN THE GENUS ASTER—WITH SPECIAL REFERENCE TO AN UNEXPECTED DISCOVERY OF A NEW ASTER SPECIES FROM EAST CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is generally well-written. Nonetheless, I made some suggestions on the manuscript to help improve the text.
The introduction provide a clear context for the study. The literature cited is relevant and appears thorough.
The structure of the paper is conform to the norms in the field.
The figures are pertinent and sufficient for the purpose of the paper. In particular, the photographs and line drawing of the new taxon are excellent.
I checked the authors' deposit statements and all seems in order. The GenBank data are already available and were properly deposited.
Experimental design: The research reported is novel and is well within the scope of the Journal. Nobody has reported on leaf character evolution in tribe Astereae (Asteraceae) before and the subject is interesting also in a wider context.
The research questions raised by the authors are pertinent and appropriately defined given context provided.
The methods reported for plant phylogenetic analysis are standard in the field and are well-known and proven. The taxonomic sampling appears adequate given the aims of the study.
The Pagel approach used for correlations is appropriate for the type of question raised by the authors.
Validity of the findings: The authors could have confined themselves to merely indicate that evolution of narrowly lanceolate leaves result from parallel evolution in Aster (a result readily discernible from the phylogeny presented), but they also investigated a possible driving force behind the evolution of that trail: its correlation with riparian habitats. They could not identify however, a precise order of changes. Based on their analyses, the authors suggest a most likely path, nonetheless. This appears reasonable.
Taxonomic novelty: the authors have conformed with PeerJ policies concerning the new species and meet ICBN standards.
The authors clearly demonstrate the distinction between their new taxon and the superficially similar A. dolichophyllus, both phylogenetically and micromorphologically.
The taxonomic description at the end of the paper is of high quality and thorough.
The overall conclusions are cogent.
Additional comments: This is an interesting paper that combined taxonomy and evolutionary biology, each supporting the other. It is infrequent that this is done in plant taxonomy and the authors are commanded for having done so.


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: CONVERGENT ORIGIN OF THE NARROWLY LANCEOLATE LEAF IN THE GENUS ASTER—WITH SPECIAL REFERENCE TO AN UNEXPECTED DISCOVERY OF A NEW ASTER SPECIES FROM EAST CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further amendments required.
Experimental design: No further amendments required.
Validity of the findings: No further amendments required.
Additional comments: The amended manuscript has greatly improved in both language and clarity, after incorporating the comments provided by the reviewers. The current text reads well, with sufficient explanatory information included. The added comparison table helps the reader compare the four taxa easily. I recommend this manuscript for publication.


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

Input:
Title: ISOLATION OF WHEAT BRAN-COLONIZING AND METABOLIZING SPECIES FROM THE HUMAN FECAL MICROBIOTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper by de Paepe et al. entitled “Isolation of wheat bran-colonising and metabolizing species from the human fecal microbiota” is well written and clearly set out. The methods section in particular is very detailed.
Experimental design: The use of non-autoclaved wheat bran is likely to have confounded the outcomes of the study. The study design and the approaches microbiology and molecular methods are well thought out.
Validity of the findings: The new bacterial isolates from wheat bran seem somewhat surprising given that wheat bran is considered to promote butyrate formation but the incubations mainly yielded acetate. This may be a feature of employing suboptimal conditions for the incubations.
Additional comments: Specific comments to the authors
Abstract. The abstract could be improved, for example line 43 Provide at least an example of “these” microorganisms. Lines 38-42 is essentially belong as discussion points rather than the final statement of the abstract.
Lines 46-49. Several of the references cited are not recent as dating back to 1980.
Line 55. Change to “.. pattern for..”.
Line 62-64. There are many different Bacteroides and Eubacterium species, and some of these may have a role in wheat bran metabolism whilst others are unlikely to do so, it would therefore be much better to state this at the species level.
Line 106-107. What was the reason for the reduced level of what bran?
Line 144. As the aim of the study was to isolate wheat bran degrading bacteria what was the rationale for using glucose as the added carbon source to the YCFA medium (given that the main sugars are arabinose and xylose)?
Line 168. Again what was the rationale for using glucose?
Line 195. Provide brief details of the DNA extraction method used here.
Line 300. I would suggest that using wheat bran that had not been autoclaved is likely to confound the study. Low level contaminants may have been missed. Would it have been feasible to irradiate the wheat as an alternative approach?
Line 313. Indicate the closest species, as certain bifid species may be expected to use wheat bran sugars whilst others would not.
Line 382. Change typo “Pediococus” to “Pediococcus”.


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: ISOLATION OF WHEAT BRAN-COLONIZING AND METABOLIZING SPECIES FROM THE HUMAN FECAL MICROBIOTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Isolation of wheat bran-colonizing and metabolizing species from the human fecal microbiota” by De Paepe et al report the enrichment and isolation of different bacterial taxa from human faeces using the insoluble substrate wheat bran as sole nutrient source. The manuscript is clearly written, in professional and unambiguous English.

The manuscript is descriptive but provides interesting insights about the presence of wheat bran-metabolizing bacteria in the gastrointestinal tract. The methodology used can profit other scientists in the field. Indeed, the isolation of yet unculturable gut bacteria is an essential first step to increase our understanding of the microbiome-host interaction.

The size of the core manuscript seems rather extensive compared to the amount of results presented. To make it more concise, I would recommend the authors to move at least Table 1, 2, 3, 4 into supplementary tables, as well as Figures 7, 8, 9, 10, 11, 12 into supplementary figures.

Additional specific corrections:
Line 52 (comma missing): Macfarlane et al. (1997,2006)=> Macfarlane et al. (1997, 2006)
Line 55-56: colonization pattern insoluble substrates => colonization pattern of insoluble substrates
Line 103 (comma missing): The chemical composition is displayed in Fig. 1.The medium […] => The chemical composition is displayed in Fig. 1. The medium […]
Line 106 (comma missing): 0.1M phosphate buffer => 0.1 M phosphate buffer
Line 212: (LGCGenomics, (Teddington, Middlesex, UK) => (LGCGenomics, (Teddington, Middlesex, UK))
Line 201: primerpair => primer pair
Line 279: 30mA => 30 mA
Line 282: As an alternative to SEM microscopy, samples were also visualised using a Jeol JSM 7100F 283 scanning electron microscope (JEOL Ltd, Tokyo, Japan) => This sentence is rather confusing since authors proposed SEM as an alternative to SEM. I believe they meant the following: As an alternative to SEM microscopy, samples were also visualised via cryo-SEM using a Jeol JSM 7100F 283 scanning electron microscope (JEOL Ltd, Tokyo, Japan)
Line 346 (comma missing): in donors 1,3 and 4 => in donors 1, 3 and 4
Line 134 & 155: Please delete the brand of the anaerobic workstation => (GP-Campus, Jacomex, TCPS NV, Rotselaar, Belgium). It was already stated line 96.
Line 232 & 260: Please delete the version of R => (version 3.4.2 (2017-09-28) (R Core Team 2016)). It was already stated line 181.
Line 470: aleurone through Scanning Electron Microscopy (SEM) (Fig. 8) => aleurone through SEM (Fig. 8)
Experimental design: Authors have made a good job at providing detailed information so that other investigators could reproduce these experiments. Few experiments remains however unclear:

Comments regarding the wheat-bran preparation:
Since wheat bran is the core nutrient for enriching and isolating bacteria, authors should be more explicit regarding the preparation (=pre-digestion) and chemical analysis of wheat-bran (line 101). It is currently slightly confusing:

Authors refer to the paper of De Paepe et al. (2017) which is a modification of the paper from Minekus et al., (2014).
In De Paepe et al (2017) the legend of Table 1 states: “The methods used for wheat bran micronization and to determine the wheat bran composition are described in the Supporting Information”. However, no supporting information is available in that regards. When reading the experimental procedure from De Paepe et al (2017), it is also unclear how and when the analysis of the chemical composition of wheat bran has been performed. It could be pre-digestion with this statement: “Commercial wheat bran with chemical composition displayed in Table 1”. Or post-digestion with this statement “Insoluble undigested material was centrifuged and lyophilized to preserve until chemical analysis or batch incubation in hungate tubes”.

Therefore, in regards to the current manuscript, the chemicals used and the different steps/reactions of the digestion steps could be more clearly stated (for instance within the section supplementary material). The method use for determining the chemical composition as well. In Figure 1, please clarify the “state of wheat bran” used. If available, it would be valuable to provide the readers with the chemical analysis of both before and after-digestion.

Line 367: Authors stated that the bacteria retrieved from the enrichment were unexpected due to their lack of enzymes for the degradation of cellulose and arabinoxylan polymers. Various enzymes + mucin were added during the digestion of wheat-bran, but their fate is unclear at the end of digestion. Could these biomolecules be responsible for the unexpected growth?

Comments on SEM and cryo-SEM:
Line 470: Authors state a preferential microbial colonization and degradation of aleurone based on SEM pictures. This statement is hard to verify with the pictures provided. The scale of Fig. 8A is greatly different than those of Fig. 8B-F. Bacteria might thus be hard to visualize.
Are there any reasons for authors to use Cryo-SEM (Fig. 7A) vs SEM (Fig. 7B) to compare the effect of digestion? Two different scales were also used.

Additional specific comments:
Line 394-396: Do authors refer to another article or to personal data for the statement regarding the presence of enzymatic activity after pre-digestion? Please clarify.
Line 400-401: Do authors refer to another article or to personal data regarding the capacity of F. prausnitzii to degrade wheat bran in pure culture? Please clarify
Validity of the findings: Comment on the isolation of wheat-bran metabolizing bacteria:
Authors have first used “wheat-bran agar media” to isolate colonies from fecal slurry. The colonies were then directly transferred onto a nutrient-rich agar medium (YCFAG). Are there any reasons for authors not to transfer colonies on the same “wheat-bran agar media" for a second or even a third time to ensure that growth was due to the utilization of wheat-bran, and not because of remaining nutrients from the fecal slurry?

Colonies from YCFAG agar were subsequently transferred in YCFAG broth. Have authors tried to grow the pure cultures from Table 5 in the “wheat-bran broth” to confirm their capacity to use wheat-bran as sole nutrient source?
Additional comments: A suggestion to authors: They stated (line 299) that wheat bran was not autoclaved to avoid structural modifications. This is biologically sounded, but it seems that bacteria have grown in the control. In the future, authors could try to UV the wheat bran for couple of minutes (with shaking to expose all surfaces) in an attempt to remove, or at least reduce, the risk of contamination. Short exposure to UV should not impact its composition.


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: PROGNOSTIC VALUES OF GMPS, PR, CD40, AND P21 IN OVARIAN CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see pdf
Experimental design: Please see pdf
Validity of the findings: Please see pdf
Additional comments: No additional comments