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: RESAMPLING-BASED METHODS FOR BIOLOGISTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Overall this is a well-written paper on an important topic. However, there are some significant omissions in the presentation and lack of clarity on some of the methods/code that should be addressed.

Major issues

I think a significant limitation of the paper is staying so tied to R/Mosaic. I don’t find the R code intuitive enough (or well annotated enough). Other statistical applets freely available online (www.isi-stats.com/ and click through to the Applets page here: http://www.rossmanchance.com/ISIapplets.html , www.lock5stat.com and follow links to Statkey) have excellent visualizations that make explaining and understanding the concepts of resampling methods more accessible than reading R code. I think these are worth mentioning (or highlighting? Of course, these are methods I use personally and advocate for, so am somewhat biased here). Alternatively, I think that much more attention to explaining R functions, annotating and interpreting the code is needed.

Subsection on sampling distributions (pages 2 and 3). I find the presentation here confusing. I think that you need to provide (a) additional annotation of the R code and (b) additional annotation of the results. For example – a bit more explanation of what the functions means, and more statistical annotation (e.g., population mean vs. sampling means), that you are considering a ‘hypothetical’ situation in which you have all 164 values (the population) and are looking at what *could* have happened if you’d randomly sampled quadrats

Subsection on Central Limit Theorem (page 4). I think it would be helpful to reference back to the polar bears example here. Why not talk about how it ‘worked’ and using 2SE for the CI here, etc? Might also be worth pointing out that the discrete (and skewed) nature of the population in this case didn’t impact normality of the sampling distribution.
Bootstrap confidence intervals subsection (page 4). Maybe emphasize “many” a bit more to build intuition “many, many” or “infinite” . I would also point out that this ‘assumption’ that the sample is like the population (e.g., representative) is not a ‘new’ assumption that is being made. The term with replacement is used as if the reader knows what that means – I think I’d explain it, it’s a fairly technical term. I think this section doesn’t clearly enough state that you are sampling with replacement a sample the same size as the actual sample? Also, doesn’t clearly enough state that you compute the sample mean in that resampled sample. I wonder if a picture/visual would help here? Table/box that gives an overview of the method? Finally, of course, the beauty of the bootstrap is that you don’t have to use the mean! Median, other statistics, etc. are fine. You come back to this later, but not in a ‘single variable’ context – so maybe worth mentioning here?

R/code in this section is also similarly under-commented/explained to previous sections

Null distributions and p-values

It bothers me that the example you use here is not biological! Surely there is something more biological you could use?  Similarly, in a more ‘real’ context would you really want to do a one-sided test? Aren’t two-sided tests almost always conducted in practice?

Case studies

Could include something (or at least mention?) other experimental designs also? E.g., RCBD, or even just a novel statistic for some kind of case-control study (e.g., comparing genetic information between diseased/non-diseased folks)

Discussion

I think there are a number of additional points of discussion possible here
1.	There are actually lots and lots of kinds of bootstraps which can be better/worse in various situations
2.	You don’t really talk about permutation tests or when permutation tests are appropriate vs. bootstrap (e.g., it’s more about study design similarity than tests vs. CIs – you can use permutation test SEs to do CIs and bootstrap SEs to do tests)
3.	You don’t talk about benefits to bringing this into classes with students, grad students, etc. (or efforts to do that: e.g., www.causeweb.org/STUB, among others).
Additional comments: Potential typo: line 85 “…making use…”


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: RESAMPLING-BASED METHODS FOR BIOLOGISTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper aims to help students/biologists to understand statistics by popularizing resampling (bootstrap and permutation) by providing background of the basic procedures and three case studies. The text is well-written, well-structured and easy to follow. Nevertheless, I have a number of issues with the resampling procedures and associated claims. See General and Details below.

I missed reference to (Manly 2007) [fourth edition is almost there; Manly & Navarro Alberto 2020, I suppose] which also aims at biologists.
I missed proper treatment of permutation tests and of permutation tests for particular regression coefficient(s) in linear models (Y-permutation using the Freedman & Lane approach, see (Anderson & Robinson 2001) and X-permutation, see (Dekker, Krackhardt & Snijders 2007) for DSP (double semi-partialling) with general discussion and more references. In the context of hierarchical designs, see (Anderson & ter Braak 2003) and the R package permute. See also under Issues.
For references, see below.
Experimental design: This is a tutorial review.
Validity of the findings: In places, the authors are too optimistic in my view. The proposed procedures do not always deliver what is claimed, as laid out in a number of issues under General comments.
Additional comments: This paper aims to help students/biologists to understand statistics by popularizing resampling (bootstrap and permutation) by providing background of the basic procedures and three case studies. As such, the paper is neither original research nor a review. I will treat as a tutorial review.
The text is well-written, well-structured and easy to follow. Nevertheless, I have a number of issues with the resampling procedures and associated claims. Some of the issues boil down to the statement that the bootstrap and resampling procedures that the authors explain/introduce do not work in the situations where the bootstrap and resampling procedures are really needed.  The critical phrase in the paper is “If the bootstrap distribution is bell-shaped” [and, I add, unbiased] then simple procedures works and, I would add, arguably parametric procedures as well... [see issues 3-6]

Issues

1. My main issue is on line 260-263, which entails a permutation test on the effect of x2 in a linear regression of y on x1,x2 and x3. The procedure using the mosaic package is to shuffle the values of x2 across the sampling units “thus meeting the assumption of the null hypothesis” [line 263]. However, shuffling x2 does more than that. It not only nullifies the regression coefficient associated with x2 but also removes any correlation between x2 and (x1 and x3) in the data. Even, the approximate correctness of the procedure cannot be guaranteed if x2 is somewhat correlated to x1 and x3. There are two know solutions that lead to approximately valid permutation tests: Y-residual permutation, known as the Freedman-Lane procedure and X-residual permutation known as DSP. See (Dekker et al. 2007) for a relatively recent update and discussion on these procedures.
To help you a little on this. You have X-permutation after line 261.  If in
do(10000)*{lm(y˜x1 + shuffle(x2) + x3, data=dat)}
x2 is replaced by the lm-residual of x2 on x1 and x3 and the lm gives access to the t-ratio of the regression coefficient of  x2 (b2/se(b2)),  then you are in business. One should look at the null distribution of the t-value of b2 [instead of at b2 itself] because one needs an (asymptotically) pivotal statistic for such tests (both DSP and Freedman and Lane).

2.  In the section on Null distributions and p-values, code is given for a Monte Carlo permutation test. It looks to trivial and OK, but in detail there is something wrong: the resulting p-value could be 0 [OK, rarely so, with 10000 shuffles]. P-values should never be 0 (Phipson & Smyth 2010). See also (Hemerik & Goeman 2018)[difficult paper] and [easier] Box 1 of Appendix A4 in (ter Braak 2019) for how to correct this.

3. The introduction mentions the difficulty students may have with GLMMs and I share the concerns of the authors with this [see (Wang et al. 2012) and (ter Braak, Peres-Neto & Dray 2017) where specific resampling procedures are applied to GLM to make up for missing random terms, the need for which are shown in  (ter Braak 2019) with accompanying permutation tests on interaction in a GLMM]. However, despite optimistic claims elsewhere and in this paper, there are, in my opinion, no generally valid bootstrap or other resampling procedures for GLMMs. The case study is discussed below in more detail; in brief, the proposed analysis might work for the data at hand but is not a general solution. Specific solutions have been proposed for significance testing in specific balanced hierarchical designs, see the R-package permute and (Anderson & ter Braak 2003). These solutions should be mentioned.

4. The abstract and introduction mention three common assumptions in traditional parametric statistics: Normality, equal variance and independence. The paper should make clear which assumptions are then avoided/not-needed, when proceeding to resampling procedures. Such explicit statement is missing. Note that violation of independence (pseudo-replication (Hurlbert 1984)) is arguably a bigger issue than deviation from normality. See the line transect and time series options in the permute package for avoiding the independence assumption.

5. The ‘95% rule’ (Box 1 and line is a misnomer in my view. It is the 2SE rule, aiming to give 95% confidence intervals.

6. I miss an easy rationale for the bootstrap: the bootstrap sample relates to the sample as the sample relates to the population (sample : population ==  bootstrap sample : sample) as the sample is equal to the population from which bootstrap samples are drawn (Hall 1988). This rationale is essential for bias correction and studentized bootstrapping intervals. Note that the naive bootstrapping intervals and the mean + 2bootstapSE intervals do not work where you really need the bootstrap.

7. Case study I. The data at hand are nested: 5 stations within each of 9 beaches and the response is a count. The authors propose a simple regression and make up for all arguments against the simple regression by a cluster-level bootstrap (without a bootstrap of units within clusters). The issue with nested data is that there are two regressions possible, one between-beaches and the other within-beaches. The authors choose to resample beaches; so they focus on between-beach variability. That is OK if the predictor (sea level) does not very within beaches (or only a little), but would be nonsensical if sea level varies mostly within beaches. The reader does not know what is the case (neither do I as I did not search for the data...). Mixed models and glmms combine these two regressions in a supposedly optimal way; the combination that lm makes depends on the numbers within each beach (here equal numbers, so perhaps more or less ok, but suboptimal). The current description of the cluster-level bootstrap does not work for data that are unbalanced. All of this needs to be discussed, perhaps in a supplement. Moreover, mixed models and glmm are such an important advance that the example needs it, and an appropriate bootstrap (also, bootstrap stations of beaches sampled) applied to the glmm. Note that lme4 has the bootMer function.
Moreover, the proposal does not solve the issue mentioned under linearity (the negative values).

An area where a sort of cluster level bootstrap (bootstrapping ‘id’s with a series of observations) is applied, is multivariate analysis/ordination in ecology; see R packages vegan (functions anova.rda for example) and permute, both modelled after my commercial package Canoco (ter Braak & Šmilauer 2018) (ter Braak 1990)[the latter document is open-access], for example (van den Brink & ter Braak 1999) (Vendrig, Hemerik & ter Braak 2017). A (many)glm version is in R package mvabund.

Details

Line 85 making use [of] the
L133  and Box 1 “we can make many copies of our sample data and use the resulting data set as an estimate of the whole population”. I did not understand that you also merged the copies to get a ‘big’ population. The only estimate of the population that we have is the sample; why make copies? If you would like to stay with the idea of the copies I would suggest:  “we can make many copies of our sample data, merge them and their use the resulting data set as the estimated population”. Or perhaps mock population.
L 142 “If the bootstrap distribution is bell-shaped” is not even sufficient for the next to be true. There should be no bias either!
L274 Data and both R and Rmd scripts of Case Studies should, in my view, be supplied in a zip as Supplement, in addition to the current pdfs, to facilitate inspection, study and use by readers. I did not have those, and I did to try to reconstruct everything.
L287 “but for larger values of NAP we would predict Richness will eventually become negative, which is not possible.” You do not resolve this by resampling. A solution is to log transform (but see (Warton 2017) and (Warton et al. 2016)) and apply lme, because of issue 7 (Case study I). I do not see any objection to use GLM instead, and because of issue 7, GLMM with a full cluster bootstrap.

L381 As said in L388, they also need to communicate what is estimated and the estimate with uncertainty.

L391 I leave this statement with authors. I have a different opinion. Bootstraps for general GLMMs is an underdeveloped area, in particular because the semi-parametric bootstrap runs into the difficulty that random effects are the result of shrinkage estimators. See appendix A1 of ter Braak 2019. You may also wish to cite the PITtrap for count and binomial data (Warton, Thibaut & Wang 2017).

Box 1 Second bullet. Add: This alternative is termed the parametric bootstrap.

Appendices: Please add Conclusions: after a shown result. Now the unexperienced reader has to find out himself.

Appendix B. Please check for bias. Also, except for the quantile bootstrap, the scale on which is bootstrap estimate is formed matter. Here we have a ratio, so it would be wise considering the log and backtransform (as in the boot package).

Cajo ter Braak 29 October 2019

Anderson, M.J. & Robinson, J. (2001) Permutation tests for linear models. Australian & New Zealand Journal of Statistics, 43, 75-88.https://doi.org/10.1111/1467-842X.00156
Anderson, M.J. & ter Braak, C.J.F. (2003) Permutation tests for multi-factorial analysis of variance. Journal of statistical computation and simulation, 73, 85-113
Dekker, D., Krackhardt, D. & Snijders, T.A.B. (2007) Sensitivity of MRQAP Tests to Collinearity and Autocorrelation Conditions. Psychometrika, 72, 563-581.https://doi.org/10.1007/s11336-007-9016-1
Hall, P. (1988) Theoretical comparison of bootstrap confidence intervals (with discussion). Ann. Statist., 16, 927-981.https://projecteuclid.org/euclid.aos/1176350933
Hemerik, J. & Goeman, J. (2018) Exact testing with random permutations. Test, 27, 811-825.https://doi.org/10.1007/s11749-017-0571-1
Hurlbert, S.H. (1984) Pseudoreplication and the design of ecological field experiments. Ecological Monographs, 54, 187-211
Manly, B.F.J. (2007) Randomization, Bootstrap and Monte Carlo methods in biology, 3rd edition. Chapman and Hall, London
Phipson, B. & Smyth, G.K. (2010) Permutation P-values should never be zero: calculating exact P-values when permutations are randomly drawn. Statistical Applications in Genetics and Molecular Biology, 9, Article39.https://doi.org/10.2202/1544-6115.1585
ter Braak, C.J.F. (1990) Update notes: CANOCO version 3.1. Agricultural Mathematics Group. http://edepot.wur.nl/250652, Wageningen
ter Braak, C.J.F. (2019) New robust weighted averaging- and model-based methods for assessing trait-environment relationships. Methods in Ecology and Evolution, 0.https://doi.org/10.1111/2041-210X.13278
ter Braak, C.J.F., Peres-Neto, P. & Dray, S. (2017) A critical issue in model-based inference for studying trait-based community assembly and a solution. PeerJ, 5, e2885.https://doi.org/10.7717/peerj.2885
ter Braak, C.J.F. & Šmilauer, P. (2018) Canoco reference manual and user's guide: software for ordination (version 5.10). Microcomputer Power, Ithaca, USA
van den Brink, P.J. & ter Braak, C.J.F. (1999) Principal Response Curves: Analysis of time-dependent multivariate responses of a biological community to stress. Environmental Toxicology and Chemistry, 18, 138-148
Vendrig, N.J., Hemerik, L. & ter Braak, C.J.F. (2017) Response variable selection in principal response curves using permutation testing. Aquatic Ecology, 51, 131-143.http://dx.doi.org/10.1007/s10452-016-9604-1
Wang, Y., Naumann, U., Wright, S.T. & Warton, D.I. (2012) mvabund– an R package for model-based analysis of multivariate abundance data. Methods in Ecology and Evolution, 3, 471-474.http://dx.doi.org/10.1111/j.2041-210X.2012.00190.x
Warton, D.I. (2017) Why you cannot transform your way out of trouble for small counts. Biometrics,
Warton, D.I., Lyons, M., Stoklosa, J. & Ives, A.R. (2016) Three points to consider when choosing a LM or GLM test for count data. Methods in Ecology and Evolution, 7, 882-890.http://dx.doi.org/10.1111/2041-210X.12552
Warton, D.I., Thibaut, L. & Wang, Y.A. (2017) The PIT-trap—A “model-free” bootstrap procedure for inference about regression models with discrete, multivariate responses. Plos one, 12, e0181790.https://doi.org/10.1371/journal.pone.0181790


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: RESAMPLING-BASED METHODS FOR BIOLOGISTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good.
Experimental design: Good.
Validity of the findings: Good.
Additional comments: You have addressed my concerns.


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

Input:
Title: RESAMPLING-BASED METHODS FOR BIOLOGISTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This paper aims to help students/biologists to understand statistics by popularizing resampling (bootstrap and permutation) by providing background of the basic procedures and three case studies. The text is well-written, well-structured and easy to follow. The revision led to a much improved paper; my previous major concerns have been taken care of (two more minor ones left). See General and Details below.
Experimental design: N/A
Validity of the findings: The paper reflects, in my view, the state-of-the-art on resampling as possible in a tutorial review. On two points, the main text or supplement could be improved as detailed below in Comments:
1. the null hypothesis testing using bootstrap vs permutation when variances differ.
2. adjustments when only a small number of permutations can be taken e.g. because each run takes long, or interest in very small p-values, for example in the context of multiple testing.
This would make the text quite balanced and  complete.
Additional comments: The revision led that to a much improved paper; most of my previous concerns have been taken care of.

 Details

L99 add perhaps: “, throughout the paper in the format with first the R code and then its output”  [I suggest this as I did not understand this format, for example, that in p316 tally gives this kind of output automatically].

L115 On reading I liked the 75 to be mentioned earlier. “subset of size 75 from all quadrats”  or more simply/shorter, and in agreement with line 116-117 , “a random subset of 75 quadrats”.

L135. Perhaps replace by  “2 standard deviations is exact for 21 data points, 1.96 for many, many data points”

L320. Add “ given a p-value of 0.13” ?

L344 I found an older reference for residual X-permutation (please check). Please add in front of Dekker et al 2007: (Collins 1987): Collins MF. 1987. A permutation test for planar regression. Australian Journal of Statistics 29:303-308.

L593. “Models that use random effects to model correlation offer substantial advantages when interest lies in predictors that vary within a cluster (Muff et al., 2016).”  This statement is strictly true for binary response, but not for normal or Poisson distributions. For the latter, one can simply add a factor ‘cluster’ to the LM/GLM. For the statement to be true I would change to: “when interest lies in predictors that vary both within and between clusters”.

L600 “Alternatively, Warton et al. (2017) proposed bootstrapping probability integral transform (PIT-) residuals as a general method appropriate for non-independent data.”. I do not think so. Change “non-independent” to “non-normal”.

L255-392 On Fig. 4 and the null hypothesis and bootstrap vs permutation. The variances in Fig.4 differ greatly between groups. I missed something on the topic, namely that the null distribution using permutation comes from the null hypothesis that the two distributions are equal. The null hypothesis is thus, strictly speaking, not solely on mu. It is the test statistic that makes that the test has power for particular alternatives. Here the test statistic has power against differences in means, but it is might have inflated type I error if the variances differ (and the true means happen to be equal).  Compare the Behrens-Fisher problem and papers that show that differences in variance lead to liberal tests on the mean. This aspect works in favor of the bootstrap test in which only the mean is shifted. Please consider what you can communicate on this, in the main text or supplementary. Related to this: you make no difference between approximate and exact resampling tests; perhaps better to leave the paper in this respect as is.

In your answers to comment you wrote “We understand this point (i.e., the p-value should not be 0 since the original data represents a possible permutation and should be counted in the numerator and denominator; not doing so will cause the p-values to be off by approximately 1/10000).” I would prefer if this subtle point was mentions somewhere, best perhaps in a Supplement, e.g. in the supplement for the partial regression coefficient. Note that some models may take long to run so that it is not easy to do 10000 resamples, or interest is in very small p-values, e.g. in high-dimensional data context. With the adjustment of including the test statistic of the original data, the (Monte Carlo) test is exact in the long run, even for 19 permutation.

Cajo ter Braak 30 January 2020


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: EVALUATION OF A NOVEL SYNDROMIC SURVEILLANCE SYSTEM FOR THE DETECTION OF THE 2007 MELAMINE-RELATED NEPHROTOXICOSIS FOODBORNE OUTBREAK IN DOGS AND CATS IN THE UNITED STATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think this is a very valuable paper.  It is really well written article and is mostly very clear.  There were a few typographic errors and omitted words, I think, possibly cut-and-paste errors.  The article was well structured, though the sentence on lines 126-128 is about the benefits of your system rather than the method itself and could be moved elsewhere. You only need to define acronyms once in the abstract and once in the text, rather than in the intro and again in the discussion.

My major source of confusion was the acronym PPV, I presume, is positive predictive value, but PPV is never defined as an acronym.  It was sort of defined as an equation, but this was confusing to me:  "the time to alert and proportion of alerts generated during the reported outbreak period."
Experimental design: This was a data mining exercise using software to analyze a very large database to determine if specific clinical criteria recorded in medical records could have been used to identify a historic outbreak based on baseline data prior to the outbreak.  Though my epidemiology and statistics background is somewhat lacking, I found the study design to be innovative and I could understand the general concept.
Validity of the findings: My statistical background is somewhat lacking, but the tables and charts were easy to follow and very convincing.  The results made sense in the context of the historical outbreak in question.  Conclusions were well explained and solidly based on the results.
Additional comments: This paper will make a good contribution to the literature, and I am excited about the implications for this software to improve our ability to make timely patient care and diagnostic decisions in the short-term and the potential impact for regulatory agencies in the longer term.  My major confusion as a reader who is not an epidemiologist or statistician was on the definition/use of the PPV, which I don't think was well explained or described.

Here are a few specific comments:

Abstract:
L28:  What does PPV stand for?  Is this the positive predictive value?  This was a source of confusion for me throughout the text.  Is the PPV the proportion of alerts during the outbreak over all of the alerts?  This is unclear to me.

Introduction:
L49:  Fraudulently added and adulterated seems redundant in this sentence.
L50:  Worth mentioning that the reason a high nitrogen compound was used was to fool the methods used to estimate protein content.

M&M:
126-128:  This paragraph describes the benefits of the system, but probably doesn’t belong in the methods section.
L165:  A word is missing in this sentence “Because the 2007 outbreak affected across all the US States,”  Affected what?

Discussion:
L236 & 290:  PDOR and DOP: You don’t have to define acronyms again if you already did so in previous sections.
L278:  Garneted isn’t a word.  Typo for garnered?


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: EVALUATION OF A NOVEL SYNDROMIC SURVEILLANCE SYSTEM FOR THE DETECTION OF THE 2007 MELAMINE-RELATED NEPHROTOXICOSIS FOODBORNE OUTBREAK IN DOGS AND CATS IN THE UNITED STATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dates on Figures 4-7 are hard to read, and more-readable ticks could be generated twice a month while still allowing insights related to the key dates of the food intoxication

Line 278, should the word ‘garneted’ be replaced with ‘garnered’
Experimental design: This study elegantly expands on a surveillance system already tested on a simulated disease outbreak. The methodology is applied to a dataset containing an authentic outbreak which is an important step in validating the system.

In terms of replicating data, the author could clarify how records are used. As a reader, I assumed that records were processed on a per record basis not a per-animal basis. As a consequence, one animal might contribute multiple entries to the PDOR calculation. So, in Figure 1, Is one record a summary of all data collected about one animal on one day ?
Validity of the findings: The data suggests that the system could be used to detect food-borne disease outbreaks – as long as it is running in real time – is the system being used as part of an on-going surveillance system ?

Given that abnormal creatinine levels were the key driver in identifying the outbreak, can the authors comment whether a simpler monitoring of proportion of cases identified with high creatinine set against a simple threshold would have performed worse

The authors discuss the differing findings associated with age when assessing the creatinine-derived signal. The conclusion that this highlights the value of stratifying data by age to improve sensitivity. The data might also reflect the increased euthanasia of older patients presenting with high creatinine which would also decrease the signal if records are handled on a per-record basis (euthanased animals contributing fewer data points and therefore a shorter signal)

The authors do not comment on the potential biphasic nature of the data initial rise early January, second rise mid February – potentially highlighted by the older dog data – was there any information about batch-releases of contaminated foods that might corroborate this ?
Additional comments: This is really interesting work and helps to build the toolkit for veterinary health informatics research.


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-BASED DEVELOPMENT OF 15 MICROSATELLITE MARKERS IN FLUORESCENT MULTIPLEXES FOR PARENTAGE TESTING IN CAPTIVE TIGERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article titled, "Genome-based development of 15 microsatellite markers in fluorescent multiplexes for parentage testing in tigers" describes new genetic markers mined by the authors, screened, validated, combined into a multiplex, and then used in actual parentage scenarios.  The work done has been extensive and its impacts on the genetic diversity of the declining population of tigers broad.

The introduction starts with setting the historical landscape of why tigers have declined and how this has led to a loss in their genetic diversity through captive inbreeding.  In the introduction it is evident that the written English language is not mastered by the authors.  Sentence structure is of varied complexity and often lends to confusion of the purpose of the writing.  For example, lines 59-61, 66-68.  This confusion is increased in lines 58-89. I think the authors are trying to outline drawbacks of microsatellite detection methods, but this is not clear.  It is recommended to separate information into smaller paragraphs that have a central focus and purpose then provide helpful transitions between paragraphs. Other lines that need clarification through proper use of English: 181-183, 202-203, 215-216, 250-251, 325-326.

Figure 1 helps visualize an overview of the methods and work that the author's completed, however, this manuscript is not a methods paper.  Thus, it is in my opinion that dedicating a page and figure to the work flow is unnecessary as this information is gleaned from the methods in the paper.

Figure 2 is clear, and adds to the manuscript.  The locations of the markers used in the multiplex are easy to visualize with this figure.

Figure 3 is informative, however, is better suited as supplemental information.
Figure 4 is unnecessary as this information can be gleaned from the electrophoretic data in Figure 7.  In Figure 7, the loci are clearly labeled on the electropherograms and thus provides the information from Figure 4 and new information of Figure 7. Figure 4 should be eliminated as it is repetitive of information found in other figures.

The title and labeled for Figure 5 needs to be more descriptive.
Figure 8 is a creative way to show the concordance of sexing with this assay.

I think the information in Table 1 and 2 can be combined somehow to save space.  They display similar information and with detailed table title and legend can be combined.

Information in Tables 5, 6, 7, 8, and 9 is extensive and makes it evident the extensive work done in this manuscript. However, much of the information should move to the Supplemental section.  It is recommended that Tables 6, 8, and 9 as it shows data specific to this data set be moved to supplemental. The allele frequencies (Table 5) and population statistics (Table 7) are most needed as reference for a reader.
Experimental design: There has been extensive work done in preparation for this manuscript.  It is not clear to the reader if other microsatellites have been identified and developed for tigers.  The literature is not shared in this article.  Upon a quick search, the following that have identified microsatellites and made multiplexes were found:
Williamson, J. E., Huebinger, R. M., Sommer, J. A., Louis Jr, E. E., & Barber, R. C. (2002). Development and cross‐species amplification of 18 microsatellite markers in the Sumatran tiger (Panthera tigris sumatrae). Molecular Ecology Notes, 2(2), 110-112.

ZHANG, Z. H., ZHANG, W. P., YUE, B. S., SHEN, F. J., Zhang, L., Hou, R., & ZHU, M. Y. (2006). Twelve polymorphic microsatellite loci for the South China tiger Panthera tigris amoyensis. Molecular Ecology Notes, 6(1), 24-26.

Zhang, Y., Li, D., Rao, L., Xiao, Q., & Liu, D. (2003). Identification of polymorphic microsatellite DNA loci and paternity testing of Amur tigers. Dong wu xue bao.[Acta zoologica Sinica], 49(1), 118-123.

Singh, A., Gaur, A., Shailaja, K., Bala, B. S., & Singh, L. (2004). A novel microsatellite (STR) marker for forensic identification of big cats in India. Forensic science international, 141(2-3), 143-147.

In fact, the citation by Xu, et al. (2005) is used in the manuscript to show that they measured the population is suffering from inbreeding (line 52-53), so why is this work that is under consideration needed?  This begs to question, why re-mine the genome to find new microsatellites, why is the previous work not sufficient for determining paternity, how is this work  novel?  It appears to me that no research gap exists.

Methods sections 2.5, 2.9, and 2.10 are not sufficiently detailed to reproduce this work. For example, in 2.5, what kind of sequencing what performed? what is the depth of sequencing?  In 2.9 and 2.10 the methods are not clear.
Validity of the findings: The conclusions are very short for such extensive work.  The conclusions are concise and well stated, however, not linked back to the original research question or explain how it can address a need.
Additional comments: The English language and writing must be significantly improved throughout the manuscript. There are too many places to list lines.  It is recommended that someone of native English speaking review and edit this 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-BASED DEVELOPMENT OF 15 MICROSATELLITE MARKERS IN FLUORESCENT MULTIPLEXES FOR PARENTAGE TESTING IN CAPTIVE TIGERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: English needs to be reviewed. Several relevant papers not included, background loses focus. See author comments.
Experimental design: For the most part the ED is good, although there are some confusing aspects with regards to true sample numbers tested, who is included in the database and why domestic cat is tested.
Validity of the findings: See my comments below.
Additional comments: The paper describes the development of 15 STR markers for tiger (P. tigris). This research is needed, but the paper needs major revision prior to publication. The English needs to be reviewed as there are some inconsistencies as well as spelling and grammatical errors. I have pointed out some of these errors in my comments below.

The structure of the paper is incorrect with the introduction consisting of methods and the main point of the development of test not being fully described or explored. The paper is far too long with large amounts of repetition that needs to be removed. The low number of individuals for validation (31), and the fact that several of them seem to be related, means that most of the frequency and statistical results are not reliable. This is also confusing, you state that you have 55 tigers (still too few for a database), and what is tested and what is included in the frequency tables is not clear. I don’t understand the point of including domestic cats, and so few if they are required. That being said, I think the work is important and by publishing the primer sequences other groups could expand the frequency tables and make the whole assay more reliable. I think that with significant revision the paper will be suitable for publication.

There are too many figures and tables.
Figure 1 can be removed. I don’t think this is necessary at all.
Figure 2 can be removed and put in the supplementary section if authors want.
Figure 5 can be removed.
Figure 6 can be removed as this is described in text. This data does not display well in this format.
Figure 7 – Your female tiger should be XX not YY, your male tiger is correct in that it has an XY. You need to switch your labels as they are incorrect. Based on these results there seem to be a lot of microvariants.
Figure 8 can be removed.
Table 2 I’m not clear on why domestic cats are included in this study.
Table 4 can be supplementary.
Table 5 the observed size can be removed from this table to condense.
Table 6 Supplementary.
Table 8 Supplementary.
Table 9 supplementary.

Line 19: suggest “captive breeding programs”.

Lines 21-28: This is a confusing section. I suggest something similar to:

Using this pipeline, we developed a new multiplex panel named TPI-plex (Tiger pedigree identification). By sequencing the entire tiger genome, 139,967 STR loci were identified, and 12.76% of these displayed three or more alleles among multiple individuals. A total of 214 candidate STRs were identified and screened with 31 unrelated individuals. Of these, 15 loci were chosen for inclusion in the multiplex. The mean allele number and mean expected heterozigosity (


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-BASED DEVELOPMENT OF 15 MICROSATELLITE MARKERS IN FLUORESCENT MULTIPLEXES FOR PARENTAGE TESTING IN CAPTIVE TIGERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have described the need for the study for ex-situ conservation of tigers and undertaken the all required analysis for validating their. (Details are in attached report).
Experimental design: Required experimental design and analysis are quite rigorous to validate the objective of the paper. Method are clearly described all the relevant information. (Details attached).
Validity of the findings: Proposed TPI plex would be useful in managing captive tiger populations more scientifcally.
Additional comments: Kindly see required specific changes needed in attached file.


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-BASED DEVELOPMENT OF 15 MICROSATELLITE MARKERS IN FLUORESCENT MULTIPLEXES FOR PARENTAGE TESTING IN CAPTIVE TIGERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
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: GENOME-BASED DEVELOPMENT OF 15 MICROSATELLITE MARKERS IN FLUORESCENT MULTIPLEXES FOR PARENTAGE TESTING IN CAPTIVE TIGERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English in this version is improved.

The references need to be checked to ensure the proper sentence structure is presented (capitalization especially).
Experimental design: I still have issues with the unrelated individuals - see below.
Validity of the findings: Again - reservations due to the relationship status of the tigers.
Additional comments: 37-38 - IUCN stands for International Union for Conservation of Nature - please correct.

38-39 - please see Kitchener, Andrew C., et al. "A revised taxonomy of the Felidae: The final report of the Cat Classification Task Force of the IUCN Cat Specialist Group." Cat News (2017) for current internationally accepted classification for tiger subspecies. Note there are no long nine accepted subspecies, with balica and sumatrae included with sondaica. jacksoni is a nomen nudum and should not be used.

51-62 - Atkinson, Kirsty E., et al. "An assessment of the genetic diversity of the founders of the European captive population of Asian lion (Panthera leo leo), using microsatellite markers and studbook analysis." Mammalian Biology 88 (2018): 138-143 is a recent study demonstrating inbreeding effects in a related Panthera species.

81 - you developed the system, take out attempted.

93 - were sampled in this study

104 - Eppendorf.

130 - size ranges did not overlap

133-134 - even if you're not giving the final primer concentration for each primer, a range should be provided here.

139 - commas look strange.

150 - "The allele at each locus that was amplified from the homozygote was sent to Sangon Biotech.." - I don't understand this. Do you mean "A homozygote was selected at each locus and sent to..."

2.7 I'm still not clear on the unrelated individuals. Here you include 18 of the bengal tigers which you previously stated "96 - The 20 Bengal tigers from Changsha Ecological Zoo kept unclear relationships and 97 their partially known and probable genealogic information was provided by the zoo staff". If they are not clearly unrelated then they can't be included in these statistics.

242 - repeat numbers

260 - Take out the interestingly sentence - it is not interesting. You have 16 loci and 5-11 alleles, so SOME of your loci have to have the same number of alleles. It would be interesting if they all had different numbers of alleles.

306-307 - I have major issues here. You are using 31 tigers as your reference population that you claim are unrelated, however the numbers provided in this section as verified related, are also contained in the unrelated reference group. I think it would be fine if you removed T13 from your reference population - but this would require all frequencies and equations to be redone.


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: EVALUATING THE STRESS-RESPONSE OF DENTAL STUDENTS TO THE DENTAL SCHOOL ENVIRONMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The grammar was generally fine, but I found a few unclear and awkwardly written sentences. For example,

In the introduction and Objectives section, “Results show that the heart rate of more experienced students had lower values than that of their the juniors.”  Better, “Results show that the heart rate of more experienced students had was than that of their the juniors.”  Also line 222

“…in further stages of multidirectional project.”  Unclear what multidirectional project is. Also line 30

“…working with phantoms.” Should this be phantom teeth? Line 54

“… medical students experience the high levels of anxiety and…” Remove the word the. Line 64

“…have to master manual professional workshop…” Line 70 awkward

Intro should be revised, some redundancy Lines 71-72 is repeat of 64-66. 101-102 also similar

“Only one Senior student from UAG group was qualified to law activity compartment.” Line 276. What is “law activity compartment?”

The entire introduction, lines 53 – 112 is one paragraph.

“Thanks to this storage, which changes more dynamically and to a greater extent than the others.” Line 285. This is not a complete sentence and what is meant by “storage”?

“…software and sensors can be used to detect different level of activities in dental students…” Should this be stress-related activities? Line 341

The literature cited provided sufficient background, the structure of the article was appropriate, and the figures were relevant. I think the manuscript needs be edited for grammar.
Experimental design: The article is within the scope of PeerJ. The aim of the article was given as, “The main task of the researchers was to create an objective scale which could be implemented to determine the level of stress in further stages of multidirectional project.” I think goal would be a better word than task. It is the authors’ opinion that a method currently does not exist. It is unclear to me what a “multidirectional project” is?

It is my opinion that the study was ethical, and the materials section provided sufficient detail.

The authors repeat by saying, “The aim of this research was to find under medical simulation condition a way to standardize indicators useful for objective measurements of stress level.” Line 116. I am not sure this was accomplished. They thoroughly examined stress of students performing a dental simulation. However, I do not think they addressed how it was standardized. A number of measurements were taken, but did those measurements accurately measure stress?

A lot was written about the students’ results, but the results were not related to stress, the aim of the paper. This includes the marks given in Tables 1 and 2. Therefore, I do not think this is relevant. In addition, “Statistically important differences (t-student test) were obtained for the shape of prepared teeth” Line 206-9. Is this relevant?

“…differences were obtained in the shape of the phantom tooth worked upon by the junior,…”  It might be my unfamiliarity with the simulation, but this statement is unclear to me. Students were given a phantom tooth to work on, so how is there a statistically significant difference in the phantom teeth? Was the difference in the printed teeth? Also lines 39-40. Again, I do not think this is related to the aim of the study.
Validity of the findings: I believe that the authors provided all data they used for their conclusions.

Table 5 head rotation data, especially for the junior class has a very large sd. Hard to draw any conclusions from these data.

In the Conclusion, the authors state, “The obtained results are significant and suggest that it would be useful to create an objective scale for stress measurement.” I do not see how the conclusion that it would be useful to create an objective scale for stress measurement was demonstrated. The statistical analyses demonstrated differences in the study groups, but how does that relate to the study aim? Also lines 44-45.
Additional comments: The stresses attributed to dental students apply to most students in the healthcare field.

In the introduction the authors write, “These findings support the results demonstrated by Abu-Ghazaleh et al. that stress in dental students at the University of Jordan increased over the years of the clinical training. Specifically, fifth-year students showed high levels of psychological stress due to awareness of expectations and responsibilities.” Then why did freshmen in this study have higher stress than seniors? Lines 86-88. Authors should comment on this.

“The negative impact of depression on dental students continues after graduation [13]. Does this mean that students with stress in dental school continue to have stress due to dental school, or is the stress due to different causes? Line 89.

When graduates begin their practice, is stress lower in graduates from programs with better clinical education?  Lines 93-96

“Dental schools are known to have highly demanding and stressful learning environments and higher level of depression which often affects women [1] Higher compared to what? Line 305-6


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: EVALUATING THE STRESS-RESPONSE OF DENTAL STUDENTS TO THE DENTAL SCHOOL ENVIRONMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 54: change “, already when” to “; from when”
Line 54: change “and start working with phantoms and” to “working with phantoms”
Line 59: add comma after diagnostics
Line 70: add comma after direct contact with the patient
Line 109-112: move to conclusion
Line 115: delete “to find under medical simulation condition a way”
Line 118: reword
Line 138 add comma before “and”
Line 152 add period
Line 169: check spacing before and after period
Line 173: check extra spaces
Line 175-179: explain in plain English and then add code in an appendix
Line 181: do you mean a bitmap file?
Line 184-186: please explain in plain English and add code in an appendix.
Line 201-202: delete sentence “Results are presented…”
Line 203-204: delete sentence “A value of p<0.05”
Line 294: check for extra spaces
Line 295: delete “It has been shown that”
Line 298-300: Reword sentence.
Line 327-330: Reword. “Both their preliminary results and ours show that electronic tools with software and sensors can be used to detect stress level at any point of time during a procedure.”
Table 3: Format vertically so the Table fits on the page.
Table 5: Please format vertically so the Table fits on the page.
Experimental design: Line 133: clarify how the students were selected, “randomly”, “from a class”, “volunteer”
Line 167 please provide reference for the algorithm, the C# code in an appendix, or github link
Line 201-204: please describe how you are adjusting for multiple testing
Line 206-208: please justify why you are using a t-test to compare pairwise means without adjusting for multiple comparisons.
Table 6: Please provide mean and standard deviation
Validity of the findings: Line 232-233: Since you are performing 60 experiments, your family wise Type I error is very high. Please explain how you are adjusting for multiple comparisons.

Figure 13: There is a lot of noise in this graph. Does this affect your analysis? How does this impact your research?

Table 4: Please provide more than just p-values

Table 5: What impact does the signal to noise ratio play in your analysis? Have you considered standardizing your variables for this analysis?
Additional comments: This paper has improved from the first time submitted, however there are still major revisions that need to be made. Mainly around the issue of multiple testing. This statistical issue needs to be addressed prior to publication. The methods section on statistical analysis should be expanded. In particular the authors note "p< 0.05" leads to rejection. My main concern is that statistics is more than just about p-values, it is about the actual effect size and the impact that would make on patients. This is not addressed in the paper. In addition, the authors should go through another round of editing for clarity, grammar, and presentation.


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: EVALUATING THE STRESS-RESPONSE OF DENTAL STUDENTS TO THE DENTAL SCHOOL ENVIRONMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The main concern about this paper is on the selection of the students. I understand that the authors randomly selected the students and the only criterion for inclusion in the study groups is being a student. Are there any exclusion criteria which the authors believe that may affect the gathered results?

The abbreviations should be written in full version in their first use.


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: IDENTIFICATION OF KEY MIRNAS IN THE PROGRESSION OF HEPATOCELLULAR CARCINOMA USING AN INTEGRATED BIOINFORMATICS APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1,"At present, the potentially curative options for HCC patients are radiofrequency ablation, liver transplantation and tumor resection. However, these options are only effective for the early stage of HCC." please add your referred literature. as i know radiofrequency ablation and tumor resection were also recommended for advanced stage HCC of specific patients in the guideline of  HCC in 2017.
2, Please display the complete process of construction of the mRNA-TF-miRNA triple network. It's too general
3, where is the formula of calculating significance from? in which article, please add the literature; if created by yourself, please make some explanation to convince me.
Experimental design: how do you evaluate the reliability of the tool for prediction of miRNA-TF interaction?
Validity of the findings: I think that the miRNA-TF pairs that were confirmed in at least one database could not convinced me, I think it must be confirmed in both database.
Additional comments: please describe the Materials & Methods in details, especially mRNA-TF-miRNA triple network and Validation of mRNA-miRNA pairs.


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: IDENTIFICATION OF KEY MIRNAS IN THE PROGRESSION OF HEPATOCELLULAR CARCINOMA USING AN INTEGRATED BIOINFORMATICS APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors used clear and unambiguous English, literature references are cited properly.
The Discussion part of the article is questionable, because the first paragraph represents combined text from Introduction and Results.
Some figures should be improved. For example, fig.6 has unreadable format
Experimental design: The main weakness of the study is the absence of the experimental verification of results obtained. Nevertheless, such deep and comprehensive bioinformatic analysis of public datasets is valuable and let to reveal prognostic biomarkers for HCC.
The strength of the study is in analysis on two large datasets with subsequent verification the results in independent dataset. Another advantage is in included TFs. The ternary mRNA-miR-TF networks are of certain interest in the HCC diagnostic.
Validity of the findings: The results obtained are robust and statistically sound. However the interpretation might be more meaningful. For example, it remains unclear if has-miR-106b and has-miR-195 bind to conservative sites. You can also specify if they abberantly expressed in other types of cancer.
Additional comments: My major suggestion concerns:
fig.1- please, suggest this figure in the schematic view for the better comprehension
fig.6 - please, revise the scale of the figure, at the moment it is uninformative
lines 236-242 the text repeats the Introduction part. Please, revise this paragraph
lines 242-246 there is one more repeating of the Results, please write more interpretation
line 27: rewrite mRNA-TF-microRNA


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: IDENTIFICATION OF KEY MIRNAS IN THE PROGRESSION OF HEPATOCELLULAR CARCINOMA USING AN INTEGRATED BIOINFORMATICS APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all comments, which I gave in the first round of review. So, the manuscript meets the basic requirements
Experimental design: The authors made the necessary changes
Validity of the findings: The bioinformatic findings are valuable and let to reveal prognostic biomarkers for HCC. The subsequent experimental studies will verify the results obtained it this analysis.
Additional comments: The authors made the necessary changes in the revised version of 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: ANTHROPOCENE GEOCHEMISTRY OF METALS IN SEDIMENT CORES FROM THE LAURENTIAN GREAT LAKES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear, professional English is used throughout.
Experimental design: The major strength of this study is the extensive literature review.
Validity of the findings: no comment
Additional comments: The manuscript titled “Anthropocene geochemistry of metals in sediment cores from the Laurentian Great Lakes” by Meagan Aliff et al. reviews a set of sediment geochemical records from the Great Lakes to estimate anthropogenic and natural changes in the metal inputs to the Lakes over the last ~200 years. Using cluster analysis, the co-authors found five distinct clusters of metals related to distinct natural (e.g., whiting events) and anthropogenic processes (e.g., mining). In particular, the co-authors noted that certain anthropogenic contaminates, such as Pb, were observed to increase across all the basins, and was associated with the rise in the use of tetra-ethyl leaded gasoline. The co-authors’ findings are relevant to ongoing remediation efforts in the Lakes, and the extensive review of the available literature itself is a valuable contribution. I do have some suggests.

Lines 38-40: While the Native American influence on the Great Lakes was undoubtably smaller than Euro-American impacts, increasing concentrations of metals to the Great Lakes certainly did not begin with “…extensive European settlement around 1850…” Findings by Pompeani et al. (2015) indicate that extensive pre-historic copper mining on Isle Royale made a substantial, measurable impact to the delivery of Pb and Cu to the sediments in Lake Superior. I would assume that there are likely other pre-historic copper mining impacts to Lake Superior in the Michigan Copper Districts as well. I suggest that the co-authors choose their wording carefully to include the potential for Native American impacts, while maintaining that the Euro-American environmental impact after 1850 was likely larger.

Line 51: Pollutants are mobilized and deposited in lakes via the atmosphere, in addition to rivers. This is particularly important for Pb due to the burning of tetra-ethyl leaded gasoline and smelting. I would include atmospheric transport processes in this sentence as well.

Lines 71-75: Historical Pb concentrations, which likely reflect leaded gasoline combustion, have also been studied in Lake Superior by Pompeani et al. (2015). This citation should be included in this section as well.

Lines 187-190: It is not clear why the Barbieri citation is used here.  This EF equation was developed much earlier, which I think would be more relevant to cite.  For example, see Weiss et al. (1999) and citations therein. Also, why is the subscript labeled “soil” in the equation?  This study analyzes lake sediment, not soil. This needs to be changed or clarified in the text.

Line 240: I believe P2O5 is technically phosphorous pentoxide, not phosphate (which is PO4-3).

Lines 306-326: It is not clear why the study by Pompeani et al. (2015) is not included in this section, especially because they present Pb, K, Fe, and Cu concentrations from lake sediment cores in Lake Superior for >200 years.  This citation should be included in this section, relevant statistical analyses, and figures.

Line 531: To expand the distribution of sample cores, I would include the sediment core studied by Pompeani et al. (2015).

Pompeani, D. P., M. B. Abbott, D. J. Bain, S. DePasqual, and M. S. Finkenbinder
	2015	Copper mining on Isle Royale 6500-5400 years ago identified using sediment geochemistry from McCargoe Cove, Lake Superior. The Holocene 25(2):253-262.

Weiss, D.;, W.; Shotyk, P. G.; Appleby, J.D.; Kramers, and A.K. Cheburkin
	1999	Atmospheric Pb deposition since the industrial revolution recorded by five Swiss peat profiles: enrichment factors, fluxes, isotopic composition and sources. Environmental Science and Technology 33:1340-1352.


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: ANTHROPOCENE GEOCHEMISTRY OF METALS IN SEDIMENT CORES FROM THE LAURENTIAN GREAT LAKES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper reports on the analysis of 50 metals/metalloids and 10 metal oxides in 11 sediment cores collected from all ﬁve Great Lakes. It is clearly written and structured. The introduction provides reasonable context although studies of metal(loids) in dated sediment cores from outside of the Great Lakes are not cited. Tables and figures are informative although Figure 5-10 are difficult to read due to small size of each panel and 11 profiles in each one
Experimental design: The research questions are well defined in the introduction. The work appears to be performed to a high technical standard but analytical methods are described very briefly and no QA/QC information is given. There is a reference to Reavie et al 2020  in review (line 475) which may address this but it was not available as part of the Supplementary Info.
The manuscript also suffers from the lack of information on sediment core dating. It is unusual to not have it available within the same manuscript given the importance in linking to evidence for historical emissions of pollutants. Apparently it is in another manuscript but unavailable for review. There are some odd comments eg that 137Cs was determined only on one core (line 112) which is surprising given that it is usually determined alongside 210Pb and 214Pb by gamma spectrometry.
Validity of the findings: The study has many unique aspects starting with the large suite of trace metals, the analysis of the oxides in the same samples, and coverage of all lakes. Previous work on metals has generally not covered all Great Lakes in the same study (unlike the case for organic contaminants). There are also some novel results, for example, the observations on increasing sodium in the sediment and the relationship to increasing use of road salt is quite remarkable. It is also unusual to see measurements of the more soluble salts in sediment cores. In fact, on a quick search using Google Scholar I was unable to find anything specifically on that.
Given the large amount of data the approach of using cluster analysis and PCA to define groups of elements, and then discussing by group for each lake, is practical and appropriate. On the other hand the grouping means that a lot of elements are not discussed in any detail. An example is zinc which  shows large EFs (eg 4X in Lake Ontario), is combined with Cd, Sn, Sb, and Pb. Zn is the 4th most widely used metal globally and has important sources due to increased use for galvanizing steel etc, while Pb emissions have declined as the authors have noted. So the two differ greatly in the source function and yet are discussed together. Thus there is the potential for a series of papers where there is more emphasis on sources of individual elements.
While the historical concentration profiles are very interesting, their interpretation is confounded by changes in sedimentation rates (provided in metals3.csv – as “accum rate” – although with no units). In some cores the sedimentation rate has doubled (eg Erie Central) while in others (eg Ontario East) it has halved over the past 50 years. It would have been far better to report trends as fluxes to discuss historical trends and also ratios such as EF and IGeo in order to avoid obvious issues of dilution due to sedimentation of allochthonous or autochthonous particles.
Additional comments: In summary I think the authors should reconsider the way the manuscript is organized such as splitting it into multiple articles focusing on specific groups of elements while including dating information. Or perhaps they should wait until the Reavie et al article is published so that reviewers have more information on dating and methods.

Specific comments
line 15. This is not the case for organic contaminants - there have been numerous basis wide studies with dated sediment cores
line 112. Unfortunate that the dating information is not available because there can be difficulties interpreting the 210Pb data. Surprising that 137Cs would not be available given that it is determined with 210Pb by gamma spectrometry
Line 112 – Supplemental data. Are the results for total metals in the SI (metals3.csv) eg for Al and Ca, after subtracting the oxides? The digestion and ICP-MS give total?
line 125. Unclear how many elements were determined with this method. Authors should comment on why they did not use a total extraction (aqua-regia - HF) method. Also QA information such as use of certified reference materials is not provided. References should be given for the methods and rationale for their selection.
line 140. It's unclear how the oxides were actually determined or which metalswere done with ICP MS vs ICP-OES.  Also what about reference materials for the oxides?
line 143. This sentence should be deleted ie by simply stating that Hg was not analysed. Given the different emission sources and controls, which are fairly recent in the case of Hg, it is not appropriate to cite make a comparison with Pb based on a 27 year old paper
line 293. Why were Li and Ti not measured in all cores?
line 296. Should clarify that this is not a core from lakes on Isle Royale but simply a mid-lake core.
line 322 and elsewhere. Suggest using µg/g throughout since ppm can be misinterpreted
line 475. This could be corrected by using fluxes instead of concentrations
line 543. Duluth is actually lower population than Thunder Bay, but the metropolitan area including Superior WI has greater population than the Thunder Bay region
line 581. Forest fires could be very important for Lake Superior as well. This information is available for US and for Ontario
Table 1. Why were only some results Bonferroni corrected?
Figure 2. Why not use hectares or square km to be consistent with descriptions of farmland globally
Figure 5-10 are difficult to read due to small size of each panel and 11 profiles in each one


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: CRISPR SCREENING IDENTIFIES M1AP AS A NEW MYC REGULATOR WITH A PROMOTER-REPORTER SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: In this study the authors aim to study the regulators of MYC expression using pooled CRISPR activation screening approach on a reporter line. The approach the authors choose is a valid one, as genome-wide screens provide an unbiased approach to identify novel regulators.

Using a genome-wide screen, the authors uncover one new regulator of MYC expression. The study is a fairly straightforward pooled CRISPR screen but the screen itself has been performed to a poor technical standard. The authors could argue that as long as one regulator is identified, it does not matter how the screen was performed. But this is a missed potential of a technology and these points should be considered when performing such screens in the future.

o	The authors do not create a stable reporter line but rather first generate a genome-wide activation library and then transfect the reporter construct into the samples. This is bound to create huge variations. Was the transfection levels controlled? Could it be the case that some cells received multiple copies of the plasmid and other not? Reporter line screens have been reported in a number of studies (at least for CRISPR KO studies, see Breslow et al, 2018, Nature Genetics for example) and it is generally advisable that a stable reporter line is generated before performing pooled screens to avoid variations.

o	What was the multiplicity of infection while making the genome-wide libraries?

o	The number of cells selected with the phenotype is very very small. Normally from a genome-wide screen, one should expect redundant gRNAs enriched in the selected population. This is not the case in this screen, where the regulator is identified from a single clone.
Validity of the findings: Given the screen has not been performed to a high technical standard, the authors should tighten their study for the validation of M1AP as a regulator of MYC expression.

1.The authors should discuss in what context do they envision this regulation to take place. The authors have performed a very cursory analysis of the expression and only looked at hematopoietic cells. A quick search for expression profile in different cancer types shows that M1AP is up-regulated in a number of different cancer types (THYM,UCEC, OV, CESC (please see attached file). A more detailed study on expression on different cancer types is required.

2. Is there a possibility of knocking out M1AP in a hematopoietic cell line and studying how that affects MYC expression?

3. Based on CRISPR/Cas9- based essentiality screens, M1AP is not essential for any of the 676 cell lines that have been tested up to now (see https://depmap.org/portal/gene/M1AP?tab=overview&characterization=expression). How this does reconcile with its role as a regulator of MYC?
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: CRISPR SCREENING IDENTIFIES M1AP AS A NEW MYC REGULATOR WITH A PROMOTER-REPORTER SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is clearly written. The introduction and background are reasonable given the premise of the paper.

The paper generates the following kinds of conclusions:
1)A CRISPR activation library was used for the detection of new transcriptional activators of MYC;
2)They found that overexpression of M1AP can promote MYC expression;
3)They have developed a new tool to assess MYC activity and establish screening methods.

Because that the autofluorescence is highly problematic for collecting fluorescent cells in the screening. Using a photoconversion protein, Dendra2 for fluorescent screening is commendable. However, it is not clear how M1AP regulates MYC expression. The association between MYC and M1AP in the database has not been further verified experimentally.
Experimental design: In general the experimental design was clearly written. Some minor changes, additions,modifications would be suggested as follows:

1）pg11 ln:135: Why is the survival efficiency of cells so low, it is necessary to carry out parallel repeated experiments.
2）Fig. 1SB, what the sequences of sgRNAs for HPRT and MYC?
Validity of the findings: Most of the experimental results are reasonable, but there are still some problems.

1)pg13 ln:183-185: For this result, the corresponding experimental process description is unclear.
2)pg14 ln:192-194: There are lack of experimental data to support this result, “more than 23,430 different genes were randomly activated”.Usually,the sgRNA content should be verified in Lentivirus infected cells via NGS analysis.sgRNA representation should be measured at every key step in the process: amplification, viral production, or target-cell transduction.
3)pg15 ln:195: Inaccurate result description. Because the target plasmid was transfected with Lipofectamine3000 reagent, which is not a stably “pMYC-promotor-Dendra2”cell line. And considering that the transfection efficiency may not reach 100%, it is impossible that “pMYC-promotor-Dendra2 was transfected to each cell”.
Additional comments: Interesting paper definitely addresses a need of the scientific community. However, there are some problems with the inaccurate description of the experimental process and results, which need to be carefully modified by the author.


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: CRISPR SCREENING IDENTIFIES M1AP AS A NEW MYC REGULATOR WITH A PROMOTER-REPORTER SYSTEM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript entitled “CRISPR screening identifies M1AP as a new MYC 1 regulator with a promoter-reporter system” by Akiko Yamamoto et al. identified a novel MYC regulator called M1AP.
The manuscript is overall well-structured and focused. I think the major conclusions are well supported by experiments. The findings are interesting and will be utilized by the scientific community. I think the manuscript is suitable for the journal PeerJ and can be accepted after minor revision.

Please look for the grammatical errors present throughout the manuscript.
In some places the message that authors want to convey is not clear. For example:
Line 44-45: “The two cells were analyzed to identify containing guide RNAs (gRNAs)”. Please rephrase the sentence for clarity.
Line 109: “The was with Double-stranded DNA…”. Please re-write the sentence.
Experimental design: Experiments are well designed and executed.
Validity of the findings: Finding is novel and interesting
Additional comments: Please check carefully for the missing words in sentences.


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

Input:
Title: CRISPR SCREENING IDENTIFIES M1AP AS A NEW MYC REGULATOR WITH A PROMOTER-REPORTER SYSTEM
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: For my concerns on the way the screen was performed, they have now provided sufficient clarifications on the technical reasons on why it was conducted in the particular manner, while being mindful of how it can be improved in the future.
Validity of the findings: The authors have put significant effort in polishing up the paper by providing new analysis and interpretations. I appreciate that the authors have looked more into the context and the manner in which the regulation by M1AP is done and have provided a conclusion that is fitting to the supported results.
Additional comments: No additional comments


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

Input:
Title: CRISPR SCREENING IDENTIFIES M1AP AS A NEW MYC REGULATOR WITH A PROMOTER-REPORTER SYSTEM
Review round: 2
Reviewer: 2

Output:
Basic reporting: As presented, the writing is acceptable for the journal.
Experimental design: As presented, the writing is acceptable for the journal.
Validity of the findings: As presented, the writing is acceptable for the journal.
Additional comments: I would be very glad to re-review the paper in greater depth once it has been edited because the subject is interesting. The Submission has been greatly improved and is 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: CRISPR SCREENING IDENTIFIES M1AP AS A NEW MYC REGULATOR WITH A PROMOTER-REPORTER SYSTEM
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors have incorporated all the necessary suggestions in the revised manuscript. Revision looks satisfactory and has improved the quality of the manuscript.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The revised manuscript can be considered for publication in PeerJ.


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

Input:
Title: METALLIC ELEMENTS AND OXIDES AND THEIR RELEVANCE TO LAURENTIAN GREAT LAKES GEOCHEMISTRY
Review round: 1
Reviewer: 1

Output:
Basic reporting: A comprehensive review of trace metals in the Great Lakes, but useful as a general overview of trace metals as well.  I suspect this will be valuable to a number of other researchers and recommend publication.
Experimental design: All relevant studies have been cited and summarized (to my knowledge).
Validity of the findings: No comment
Additional comments: I have mostly minor comments to do with word choice and the like.  Some of the language is a little informal (e.g., line 100, 357, 590) so I suggest a careful review of that by the authors.
Line 31- Suggest rewording to “Metals are of particular interest because they record a history of pollution…”
Lines 40-41- Suggest briefly discussing why lakes that are not turbid are not ideal.
Line 132- “What this study shows…” Which study?  The study being summarized in the review here?  Or does this refer to the study Ouyang & Bartholic (2003)?
Lines 144-163- Organizationally, it makes more sense to me to put the natural sources and indicators of Li at the beginning of the section, followed by the anthropogenic ones.
Lines 236-238- Is it worth mentioning the impact of other nuclear events (e.g., Chernobyl, Fukishima)?
Line 292- Suggest rewording this sentence as initially I read it as stable concentrations of Ca up to 1955 (ppm), not 1955 AD.
Lines 340-342- These two sentences indicate very different spatial patterns of Ba pollution- worth explicitly pointing out I think.
Line 375- Suggest changing this unit from μg/m3 to ppb to be consistent with other units mentioned in the text.
Line 390- What is “plc”?
Line 558- Change “high densities” to “high concentrations”
Lines 558-559- Run-on sentence
Line 604- “Yes, but unclear”- this requires some further explanation but I don’t see it expanded upon in the following paragraph.
Lines 669-670- Disagreement in verb tenses.
Line 851- Worth briefly mentioning how Al influences the cycling of P and C
Lines 908-909- Suggest rewording as the structure of the sentence is awkward.
Line 936- Clarify that this is more intense weathering of silicates.
Table 2- Why does the average for sedimentary iron ores have a question mark after it?  Also how are these “range usually reported” values calculated?  Is this something the authors have personally observed based on some number of references (which need to be cited) or is this range based on someone else’s work (which needs to be cited)?  I see that subscript “a” is a reference to Onishi (1969) and Boyle and Jonasson (1973) but I can’t find subscript “a” in the actual table.


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: METALLIC ELEMENTS AND OXIDES AND THEIR RELEVANCE TO LAURENTIAN GREAT LAKES GEOCHEMISTRY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The major strength of the study is the exhaustive literature review.
Validity of the findings: no comment
Additional comments: The manuscript by Aliff et al. entitled “Metallic elements and oxides and their relevance to Laurentian Great Lakes geochemistry” reviews available published literature regarding metal geochemistry in the Laurentian lakes. The outstanding strength of this paper is the exhaustive literature review, which is a valuable contribution. I do have some suggestions. Broadly speaking, the introduction needs improvement in the flow of the writing (see specific comments). Given the extremely long review of each element, the conclusion section seems very brief. I would suggest adding more to this section, including more implications, findings, or future directions of research. However, I do realize this is one paper of a two-part study. Perhaps these are found in the companion paper, but this needs to be specifically mentioned in this text (what exactly is in the contents of the companion manuscript and how does it relate to this study). Additionally, some discussions found in the geochemistry review do not seem to be related to lake geochemistry at all. For example, “[Mo] is generally toxicologically harmless in humans and other animals except ruminants, in which it causes a Cu deficit, which is reversible by taking Cu supplements (line 610).” It is unclear how this is relevant to lake geochemistry. I suggest either clarifying the relationship to lakes or removing all discussion unrelated to lake geochemistry here and other places in the text. More specific comments are below:

Line 21: “We expect that this review will serve as a key reference for…”. This wording is awkward. How can a future outcome be anticipated in a scientific paper? I would suggest deleting this sentence and moving down the sentence (line 14) to replace it, “We summarized available information on metals and their roles as geochemical indicators in the Great Lakes.”

Line 25: Remove “…on…” Change to “…because it can be used to estimate natural baseline, remediation targets…”

Line 26: I think to you need a comma after “epoch.”

Line 29: The sentence “Sediments can be collected as chronological profiles and by analyzing these sediments geochemical histories can be revealed” is way too complicated.  How about the “Sediment cores can be collected and dated to reveal local geochemical histories.”

Line 31: “…hundreds of years…” Based on citations in this manuscript, this is most likely thousands of years.

Line 64: “…with both an eye to the complex… etc.” This is awkward wording for a scientific manuscript.  Why not delete everything after “…extensively studied…”?

Lines 81-86: Why the quotes for such small portions of another text?  You are citing the relevant work, right? Then they are not needed. Plus, quoting two sentences in one paragraph should not happen.

Line 113: Perhaps I am missing something, but it is not clear why only arsenic concentrations are shown in Table 2.

Line 137: Sometimes?  When did you not use your best judgement? Did you always use your best judgement? If so, then say it (this is a long way of saying delete “sometimes”).

Line 197: I don’t believe Pompeani 2013 discussed K leaching into rivers from fertilizer applications. I would add an additional citation that specifically mentions the fertilizer mechanism for K.

Line 922: Broadly speaking, natural Pb in all lakes is derived from weathering geologic materials in the watershed (bedrock, soils, tills, etc.), along with relatively small amounts delivered in air (loess, wildfire, etc.). There is no need to specify fluvial or shoreline bluff erosion for Lake Erie. That is obvious. This is like saying “Lake Erie contains natural Pb because it is underlain with bedrock that weathers through time.”

Line 1049: Two uses of fertilizer in a row.

Line 1084: The end of the conclusions has to be re-written to not sound like the co-authors are anticipating an outcome from their research.  For example, “But, as the first comprehensive summary of Great Lakes-relevant metals, we hope that this review will be helpful as a reference for future paleolimnological research. This summary was a natural first step in developing background information for a partner study on applied metals geochemistry throughout the Great Lakes.”

Why not change to something like this?

“This comprehensive summary of Great Lakes-relevant metals, serves as a key reference for the companion manuscript that details the implications of this review.”


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: METALLIC ELEMENTS AND OXIDES AND THEIR RELEVANCE TO LAURENTIAN GREAT LAKES GEOCHEMISTRY
Review round: 1
Reviewer: 3

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: Dear Editor and Authors,

Thank you for you submission. Clearly a lot of work has gone into collating a very large amount of information from very many texts. On this occasion I have decided to reject this article for publication, but I would strongly encourage the authors to resubmit the article once they have undertaken some changes.

Instead of going into fine detail I have included herein some broader changes, comments and suggestions which I believe would improve this manuscript to the point where it can be resubmitted for peer review. I hope what I say here is helpful and if the authors have any questions regarding the manuscript I am more than happy to answer them directly.

Generally the English is of a good standard, but does contain some errors, and I would encourage the authors to review this carefully before resubmission. Specific examples include: Line 117 ‘Compare that to the 1 to 2,900 ppm range shown in the table.’ this sentence doesn’t stand alone and examples of similar sentences appear throughout the text. In this case I would merge it with the sentence before in order to make it flow better; Lines 120 – 122 is another example of where I feel merging sentences would be beneficial; Line 194 ‘earth’s’ should have a capitol E and this mistake is repeated throughout the manuscript.

A more visual approach
It is always a problem to present large volumes of data in a concise way, this said I feel the authors approach can be made more concise by taking information out of the text and presenting in tabular form. This is especially true to information that is repeated for each individual element, for example background information about the element (abundance in the Earth’s crust), general environmental effects and or common health effects. Equally, including information such as the amount of sites and or studies a contaminant appears in and the age range, or maximum concertation would help to give the reader an immediate sense of the contamination scale.

In the same vein, presenting the data in more figures would help the reader and show case the author’s hard work. Other solutions may well be better, but, I could suggest perhaps presenting elements of concern (i.e. As, Hg, Pb) on a map per site and colour coding for abundance. The authors could also brake this down further by showing changes through time, i.e. showing abundances on a map for set times or periods, for example 1700, 1750, 1800 and so on. An example of this sort of figure but for pollen is fig. 2 in Williams et al, (2004) Late‐Quaternary vegetation dynamics in North America: scaling from taxa to biomes. Ecological Monographs, 74(2), 309-334, fig. 6 in Woodbridge et al. "Pan‐Mediterranean Holocene vegetation and land‐cover dynamics from synthesized pollen data." Journal of Biogeography 45, no. 9 (2018): 2159-2174. Fig. 2 in Moorhouse, et al. "Regional versus local drivers of water quality in the Windermere catchment, Lake District, United Kingdom: The dominant influence of wastewater pollution over the past 200 years." Global change biology 24, no. 9 (2018): 4009-4022 shows this for pollution in lakes across a catchment.

I would also like to see major industrial sources and other point sources of pollution on map, this is especially important for people who are not familiar with the great lakes area.

Added extra
I appreciate that it is beyond the scope of your original paper and that some of this may fall in the sister manuscript, however, I believe that by broadening the scope of the manuscript to include the following the authors will add more ‘value for money’ to the study. I also believe that it will help to concentrate the way in which the data is presented and help the manuscript to be more stand alone.

Statistical approach: Having done all the hard work to collect all this data I think it would a shame not to put it to work. I would like to see the authors statistically analyse elements distribution through time and space (different river catchments, lakes or geological areas), and if at all possible statistically evaluate a cause and effect relationship. To evaluate the first parameters I would suggest turning to the statistics of ecology and palaeopollen data which regularly evaluate the distribution of species in space and time. Test such as the 2 way ANOVA or general linear modelling can be good places to start. To analyse a cause and effect relationship the authors could also compare population increase for the area or agricultural produce with specific elemental abundance at different sites using a correspondence analysis for example PCA.

Comparison with environmental standards: There are various environmental sediment quality standards or threshold concentrations of specific pollutants above which public or ecological health is detrimentally effected. The authors already mention some of these in the text but I would like to see this highlighted further. This could for example be done in figure or tabular format, and show which sites exceed sediment quality standards and for which elements.

Thank you again for your submission and I look forward to seeing further from you.

Best Regards,
James Fielding, University of Southampton.


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: METALLIC ELEMENTS AND OXIDES AND THEIR RELEVANCE TO LAURENTIAN GREAT LAKES GEOCHEMISTRY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Nice work!


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

Input:
Title: IDENTIFICATION OF KEY GENES ASSOCIATED WITH PROGRESSION AND PROGNOSIS FOR LUNG SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The basic quality is OK.
Experimental design: The design is generally OK. Some methods not so reasonable (see below).
Validity of the findings: The PPI network is on protein level, whereas differential expression is based on mRNA level. It is not reasonable to put differential genes onto the PPI network to identify hub proteins.
Additional comments: 1) People rarely say "first and post progression survival", please explain.
2) Need to validate the discovery with the TCGA LUSC dataset.


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: IDENTIFICATION OF KEY GENES ASSOCIATED WITH PROGRESSION AND PROGNOSIS FOR LUNG SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript” Identification of key candidate prognostic genes associated with lung squamous cell carcinoma by bioinformatics analysis” authors have used three datasets and identified the differentially expressed genes.  Authors have also identified the PPI network and the prognostic capability of the selected genes.

The manuscript is written in good english.
More references can be provided.
Experimental design: The experiments design for differential expression analysis is okay. However, for the prognostic analysis, a better method can be applied.
Validity of the findings: Authors should have used more data for example TCGA to perform better analysis.
Additional comments: Following are my comments-
1.	Figure 1 and Figure 2 are describing the same experiment. Please merge the figure.
2.	In figure 2, many more genes are specific to the data set compared to common genes.  Authors should comment on this. Is there a possibility that origin of samples, method of analysis etc play a more important role than the disease itself?
3.	Authors have suggested that p53 signalling pathway is enriched in the GO analysis. However, it’s not shown in figure 3. Also, these enriched pathways are generally present in the majority of cancer type. Author should discuss in detail how these pathways are different regulated in LUSC compared to other tumor types.
4.	Are these significant 10 genes specific to LUSC? I think that the role of these genes is already reported in many cancer types.
5.	Authors should also explain how these 10 genes significant for LUSC pathology?
6.	figure 6 shows that 10 selected genes are overexpressed in LUSC compared to normal. I think it’s an expected result from this study.  Also, these genes are reported to play a critical role in carcinogenesis of many tumor types.
7.	Based on my experience, the cox regression is a better method of finding the prognostic markers. Also, why did authors have used only 10 genes for prognostic identification? The genes without differential expression in normal and tumour can be a good prognostic marker. I would suggest authors to perform the analysis with all the genes.
8.	For prognostic analysis, which sample set was used and how many patients were used for the analysis.
9.	TCGA has around 500 patient’s samples which may provide better understating with above analysis.


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

Input:
Title: IDENTIFICATION OF KEY GENES ASSOCIATED WITH PROGRESSION AND PROGNOSIS FOR LUNG SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic reporting of the manuscript is overall flawless. But I recommend the authors to check for typos one final time.
1. Typo in line 90: Date pre-processing --> Data pre-processing
Experimental design: 1. Cox regression for potential prognostic factors
I recommend the authors perform a Cox multivariate survival analysis for the potential prognostic factors they have found and discuss about the results. Although the Kaplan-Meier plot results truely suggest that overexpression of either of the 5 candidate genes associate with poor prognosis, survival analysis with known prognostic factors should be given to state the prognostic and/or treatment potential of the candidate genes. But I do not insist that the authors should exclude the candidates that are either correlated with themselves or turn out not to be significant by the Cox regression; presenting the data will be enough for the readers to figure out whether the candidate genes have additional prognostic values.

2. Stating the difference or similarity of the array platforms.
Since the authors used data from three studies spanning two array platforms. I suggest that the authors show the expression histogram of the three respective studies used for data collection as a supplementary figure. Also, the authors should state how different the data they used are, and/or why it does not matter to use the pooled data.
Validity of the findings: Findings of the manuscript seem valid and robust.
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: IDENTIFICATION OF KEY GENES ASSOCIATED WITH PROGRESSION AND PROGNOSIS FOR LUNG SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: Valid
Validity of the findings: Valid
Additional comments: All my previous questions have been addressed.