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: STOP SIGNALS DELAY SYNCHRONY MORE FOR FINGER TAPPING THAN VOCALIZATION: A DUAL MODALITY STUDY OF RHYTHMIC SYNCHRONIZATION IN THE STOP SIGNAL TASK
Review round: 3
Reviewer: 1

Output:
Basic reporting: There are several grammatical and spelling errors remaining in the text

The literature review is still rather one-sided and the discussion does not refer to competing theories.

The figures are still not transparent and hide many of the results that could potentially be interesting to others in the field, or persons who would like to replicate the results.
Experimental design: The experimental design is well thought-out. The calibration definitely adds to the design and reveals different results. I am still worried that the chosen method of calibration adds extra noise because it relies on the voice key. The method described in "General comments to the author" avoids this issue and should be implemented. I understand this is annoying but I hope the authors understand given that the previous calibration already revealed vastly different results. This would then satisfy the criterion "Rigorous investigation performed to a high technical & ethical standard"

Moreover, the calibration process should be included in an appendix, including a description, results, and figures to conform to the criterion that "Methods [are] described with sufficient detail & information to replicate."
Validity of the findings: As stated below, the findings are valid but alternative theories should be discussed.
Additional comments: General comments
The authors have done a great job of responding to the comments, particularly the calibration of the measurement tools. I recommend the calibration process be put in an Appendix with the full results and figures to make this clearer to the reader. I also recommend the authors consider the more robust calibration method of recording the audio (stimulus, vocalisation) and voltages (button press) simultaneously as the current method confounds the variability and latency of the vocal onset detection itself. Given the amount of noise in the system, the authors may find that some results that were previously not significant (due to noise) could be significant. This is necessary for considering the alternative hypotheses that are currently not discussed.

The statistics are not reported in full and the full descriptive statistics and pairwise comparisons are not provided. These are necessary for full transparency and so the reader can also interpret the results (significant or otherwise). This is particularly important because the results could be interpreted through other theories that are not discussed here. By only presenting the results that support the current theory, support for other theories cannot easily be assessed.

Much of the spelling and grammar require proofreading and a more thorough literature review is missing (e.g., line 87 “Research suggest[s]”). More care should also be taken when reporting the statistics as there appear to be errors regarding directionality (missing negative symbols) and the values used (or, at least, the description of how the means have been derived).

The term “proactive inhibition” is not defined in the abstract and its use is vague. Why not just inhibition or vigilance? If this term has a specific meaning then this should be provided in the abstract before its use. This term has a particular meaning but it appears to relate more to memory interference and motor transfer between tasks than cue-based stopping. The reference for this term is an unpublished thesis (Castro-Meneses et al., 2015; note the year is incorrect in the reference list and text) and not a peer-reviewed text that defines “proactive inhibition” in this manner. Has anyone else used the term in this fashion? If not, the rationale for the use of this term (and reactive inhibition) should be more detailed. Given that responding in the irrelevant stop signal condition can be more automatic than responding in the relevant stop signal condition, results could reflect differences in vigilance, automaticity (in line with Repp and Su, 2013), or even attention and cognitive load (see Maes et al., 2015). These alternative theories should also be discussed.

Specific comments
Line 217
“Synchronicity” is used whereas “synchrony” is the correct term

Line 260
The audacity reference and website should go in the references section.

Line 451
Additional  “fi” in F value

Line 482
These comparisons do not examine the three-way interaction – they only examine the main effect of stop block for all of the different conditions. You require all comparisons here to explore the interaction appropriately.

For results with negative values, do not use hyphens. Use “to” or a comma to separate the values (e.g., in CIs) as the minus symbol is sometimes missing (e.g., lines 501-502). Moreover, these statistics do not match up with those in Table 1.

Author responses to reviewer comments
> We have made a calibration procedure that yielded this null result as significant. Please see full
> comments next. In relation to the phase analysis, the used statistical analysis is valid because an
> ANOVA is a special case of linear mixed effect model (it is a linear model with only fixed effects).
An ANOVA is not a linear mixed-effects model because there are no mixed effects. The interactions must be examined and covariates should be accounted for if the authors choose to assert that some variables (e.g., phase) are not important.

>  There are also references on delayed feedback and modality (e.g., Sugano et al.,
> 2012, and others on tactile feedback) that are missing and should be included
The authors missed the “and others…” – I gave an example reference but the literature review needs to be more complete.

Aschersleben & Prinz (1995) reference
The terms “in the brain” and “in the world” are outdated, even if Aschersleben and Prinz used them in 1995. Neuroscience has come a long way since then and the term “in the brain” means something functionally different than it did previously. If you choose to use these terms as descriptors, then link them to the processes or functions that represent them in the modern literature and only use the old nomenclature once (to make the link with Aschersleben & Prinz). Alternatively, discuss the neuroimaging literature to support invoking “in the brain” although I do not think this is the direction in which the authors were going.

> We like synchronized vocalizations, we have had long debates and come with that consensus
Unfortunately I was not part of those discussions and the manuscript now uses an incorrect term. Either use “vocalization” or find another term that does not assume synchrony from the outset.

Regarding the “Paillard-Fraisse” hypothesis
> Good pick up. Thanks. This has been corrected throughout the manuscript.
The spelling is still incorrect. You now write “Fraissie” in some sections (e.g., abstract)

Regarding hypotheses
Changing the hypotheses to “will” statements still uses future tense. It should be a statement of something that *is*. For example, one of your hypotheses should be “Vocalizations to auditory pacing signals are hypothesized to exhibit reduced negative asynchronies compared to finger responses”.

Figure 3
All the levels and factors need to be described or a table with the full descriptive statistics should be included. Again, this is a matter of transparency and all effects should be discussed so the reader can assess the results. Moreover, the lines between the figures are not APA format (also, legends should be inside the figure, Figure 3C requires annotations to show that the range does not start at zero, significance levels usually do not go in the figure). Figure 3A only shows a two-way interaction.

Aschersleben 2002 quotation
There are APA formatting guidelines for citing a citation and these should be followed. The colloquial use of ‘hold back’ should be described in more concrete terms.

Calibration method
Please include the calibration in an Appendix so the reader can assess how these tests were performed and what can be ascertained from them. The full results of this test should be included because there are several sources of latency and variability. The current description in the response could refer to several different aspects of the setup and it would be good to assure the reader of the test validity and show the results for:
1.	Latency of auditory feedback following button press
2.	Latency of recorded onset times for vocalisations relative to stimuli
3.	Latency of recorded onset times button presses relative to stimuli

It would be easier if the full write-up were included because the brief description is difficult to parse without figures. I believe that the third calibration test has not been performed and the second test was performed using an indirect method. Both of these tests relied on the vocalisation recording and threshold. A better method would be to record the stimulus audio, the vocalisation audio, and voltage change from the accelerometer for the button response. This would show the timing difference between the recorded asynchronies and the veridical asynchronies. The current test is suboptimal as it confounds some latencies and variability (i.e., the vocal response measure is a weak point) but it is a good start.

On the topic of the button response, Accelerometers are not the best tool for measuring onsets as they contain inherent delays in ascertaining velocity and are triggered a while after the response has been made (much like the microphone, as the vibrations ramp). A force sensor (FSR) or pull-up is recommended instead as these provide a quicker and more accurate signal.

In your description, was the response device a “headphone” or “microphone”? It should be the latter, no?

References
Maes, P. J., Wanderley, M. M., & Palmer, C. (2015). The role of working memory in the temporal control of discrete and continuous movements. Experimental brain research, 233(1), 263-273.


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: STOP SIGNALS DELAY SYNCHRONY MORE FOR FINGER TAPPING THAN VOCALIZATION: A DUAL MODALITY STUDY OF RHYTHMIC SYNCHRONIZATION IN THE STOP SIGNAL TASK
Review round: 3
Reviewer: 2

Output:
Basic reporting: It seems as though the authors misinterpreted my suggestion to cite Repp (2005) and Repp and Su (2013). This was not meant to encourage a discussion of phase correction and period correction, but rather an overview of the alternative hypotheses put forth to explain NMA specifically. These papers provide a summary of the theories generated surrounding the NMA phenomenon, including the Paillard-Fraisse along with several others. This is still missing from the current manuscript.

Based on my understanding of the Paillard-Fraisse hypothesis it meant to explain NMA, but does not speak specifically to phase correction. I believe a discussion of phase correction vs. period correction is relevant to the current study, but the way it is currently incorporated is making things muddled.

The inclusion of references for work examining performance variability is helpful for situating the work within the broader relevant field As I noted in my previous review, it would also be nice to see a sentence or two describing how the outcomes of this work as well as work examining asynchronies have contributed to our current understanding of SMS. If you want to keep a mention of the theory on phase correction and period correction processes this would be a good place to do it.
Experimental design: In my last review, I asked the authors to provide details about the equipment used, the instructions to participants and the vocalization selected. This information in this revised manuscript is very helpful.

I suggested “in future uses of this paradigm it might also be good to survey participants afterward about whether they could hear their taps in order to be able to provide at least anecdotal information on this”. This was meant for the authors to keep in mind, I’m not sure mentioning it within the manuscript is necessary.
Validity of the findings: The authors have provided a measurement for the vocal onset threshold based on the settings for the software used, but no units corresponding to this measurement. Is this in decibels?

The additional information about showing participants how the presentation software detected their vocalizations is helpful and informative as is comparison of different methods for detecting vocal onset.

While I understand the general idea behind the calibration of responses it is somewhat unclear in the manuscript why this was unnecessary. What is PowerLab doing? How were button press and vocal response times being recorded before that they weren’t veridical? More information is needed here about this process.
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: STOP SIGNALS DELAY SYNCHRONY MORE FOR FINGER TAPPING THAN VOCALIZATION: A DUAL MODALITY STUDY OF RHYTHMIC SYNCHRONIZATION IN THE STOP SIGNAL TASK
Review round: 4
Reviewer: 1

Output:
Basic reporting: Several main theories should be discussed:
Modality Appropriateness (Hove & Schwartze, 2014)
Dynamic Attending Theory (Large & Jones, 1999)
Multimodal/crossmodal integration (specfically those on tactile and auditory synchronization)
Phase and period correction (more than simply including the reference; see comments below for specific comments)

Again, the term "in the brain" is not appropriate here and no results are provided to show whether there is "subjectively perceived" coincidence or any coincidence "in the brain". There is only behaviour whereby participants could be integrating different types of responses and/or modalities.
Experimental design: There was no misunderstanding about the design of the calibration from my side. The authors did not correct calibrate their equipment and most tests relied on the voice key recording resulting in a "loop" (as opposed to a proper calibration). The voice key should only be used for the vocal condition. All conditions (including the vocal condition) requires audio to be simultaneously recorded with the stimulus (output) and the timings be compared with those provided by presentation. Here is a picture where the | represents the onsets of the audio over time.

Test for Keyboard:
----------------------------------------------------Time---------------------------------------------->
Stimulus Audio:-----------------------|----------|----------|----------|----------|----------|----------|----------|
Keyboard sensor/audio:-------------|-------|---------|---------|---------|---------|-----------|----------|-----

Test for Vocalisations:
----------------------------------------------------Time---------------------------------------------->
Stimulus Audio:-------------|----------|----------|----------|----------|----------|----------|----------|
Vocal audio:------------------|---------|-----------|--------|-----------|---------|-----------|----------|

The authors will measure the onsets for the responses (keyboard/vocal) relative to the stimulus. They then compare these times to those given by Presentation and report the differences and variability. This is a correct method to measure the veridical latency of the device(s) and is required given that the direction (positive/negative) and magnitude of the mean asynchrony is under investigation here.
Validity of the findings: As stated above, the correct calibration method is required. I will review the results section more thoroughly once this has been performed.

Interpretations regarding perception and anything "in the brain" should be identified as speculation and only occur in the discussion. The neural data that is currently reported is not linked to the current study and should either be removed or show how these previous findings can explain the present results.

Currently proactive inhibition is not linked to any of the SMS literature. This is strange because it shows a nice dissociation between controlled and automatic processes (such as those discussed by Repp and others; see Mills et al., 2015 or other work by Keller). Moreover, the Paillard-Fraisse hypothesis does not offer an explanation for this process so it seems odd that it is the favoured theory here.
Additional comments: The authors did not satisfactorily address the comments from the previous reviews. I provide detailed comments here but the main problems are the calibration process, the interpretation and chosen theory (which does not adequately explain the data in the current stop signal paradigm), and the discussion of neural data to support the interpretation of results. The authors now repeat the term "In the brain" despite the previous comments that 1) there is no evidence to support neural responses to coincidence, and 2) there is no perception task to show what is perceived as "coincident". Currently all of the results could be explained by different strategies when using different effectors for different tasks (matching the action with the stimulus compared to matching the produced sound with the stimulus), different physical constraints, or differences between the number of modalities used (i.e., vocal uses two, tapping uses one). It is likely a combination of all of these but there is no evidence that this is due to differences in perceived coincidence presented here so one cannot assess whether the tapping responses were actually perceived as different with regard to coincidence.

Specific comments (lines refer to tracked changes document):

Line 136
“ … finger tapping try to minimize…”
Should be “tried”. Also the “finger tapping” is not “trying” to do anything. It is the human who is trying to minimize energy usage during a finger tapping task.

Line 141
“…also Pikovsky, Rosenblum, and Kurths (2003) who gives a more holistic…”
Should be “that gives” – the reference is to the article not the authors.

Lines 149-150
The evidence is in favour of multiple theories. Dynamic attending is the main theory that is missed here.

Lines 155-158
Page numbers and citation missing for terms “in the brain” and “in the world”.

Line 160
“This unmatched timing coincide…”
This is not correct grammar and does not make sense.

Lines 160-167
Missing reference for this time difference. Also, specifically state which tactile/kinaesthetic information takes longer and what it takes than (visual? Auditory?)

Lines 175-179
“This finding shows that participants tried to recalibrate their taps to a subjective experience ‘in the brain’ of temporal coincidence.”
These were not neural imaging studies so there is no evidence for “in the brain” temporal coincidence. Also, their subjective experience is not involved here as participants are often unable to report when there is a temporal delay unless it reaches a certain amount. This is simply a perceptual phenomenon and these findings have been interpreted as matching the tap feedback to the audio of the pacing signal (instead of matching the tap itself to the pacing signal). This is “recalibration” and “habituation” – not “in the brain” temporal coincidence. Unless you have iEEG evidence then this is purely speculative and also misrepresents the interpretations of the original authors. This belongs in the discussion and outlook sections – not the introduction.

Lines 181-185
Poor grammar in this sentence. Also, why is this paragraph separate from others? Evidence for different tactile afferent conduction times between the foot and hand? This could simply be different physical systems with different constraints.

Lines 189-191
This sentence does not make sense and is a fragment. What was meant by “was not different from the onset of the tactile pacing signal”? Did the authors mean “significantly different” because -8ms is different than 0ms?

Line 193
“Significantly negative”?  Should be “significantly less than zero”

Line 194
This “contention” has not been discussed. Also, evidence cannot support a “contention” – evidence can “resolve” a contention.

Lines 195-211
This rationale makes no sense and, again, requires iEEG to actually test. In every case, the subjective experience will be different from the objective world – that is why we have perception. An inter-/ intra-modal comparison does not change this fact. Not only that, the perceptual system itself has its own inherent temporal resolution and delay and humans have habituated to this delay to perceive synchrony (or lack thereof) so what happens in behavior might not reflect the temporal coincidence of events occurring in the brain. One can have a “perception” of temporal coincidence but this is neither temporal coincidence “in the brain” nor is it necessarily a “subjective experience” of coincidence. Moreover, the authors don’t test perception of temporal coincidence – they test synchronization with a pacing signal. A task is missing where participants state whether they were synchronous or not (or how synchronous they were).  Therefore, this line of reasoning should be removed and replaced by the original authors’ interpretations or an alternative explanation that can be supported with evidence from the current experiment.  Conjecture and speculation belongs in the discussion.

Lines 226-227
Incorrect tense

Line 231
State the author instead of using pronouns (“they”).

Lines 247-248
Incorrect tense (should be past tense).

Lines 248-249
Why “unlikely to be precise”?

Line 311
Remove “In the brain”

Line 323
“Synchronicity” is not the correct term here (as stated in the previous review)

Lines 787-788
Repetition of “sensory modalities for the”

Lines 848-858
This paragraph requires further clarification. Also, why didn’t the authors look at tap-variability in the present experiment? The data is there and this would make an interesting analysis considering it is a one-experiment paper.

Lines 851-855
Why was this moved here? It belongs in the introduction and should be expanded upon. The whole discussion about automatic and controlled processes in phase and period correction tie in very well with the results and “proactive inhibition” forces what is usually an automatic process to become more controlled.

Lines 884-886
“…musical training may require learning to delay the response so it can occur” – this is unclear and does not make sense given that musicians already show less negative mean asynchrony.

Lines 903-906
This paragraph seems out of place and is not explained with regard to the results of the study. The authors need to make the link between these studies and their own study especially to support any notion of “subjective coincidence in the brain”.

Line 931
“longer in the vocalizations relative” should be “longer for vocalizations”

Line 1180-1181
“…that have a playback delay…” should be “that has a playback delay”


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: STAPHYLOCOCCUS AUREUS VIEWED FROM THE PERSPECTIVE OF 40,000+ GENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The PeerJ has a strange web form I have to fill out, which is expecting every manuscript to be a scientific paper with clear aims > hypotheses > results > interpretation. This doesn’t make any sense in this case, as much of this manuscript is actually describing a web resource. There isn’t an experiment per se. It has also asked me to rank comments in order of priority (which I have ignored and opted to go line by line). The most important issues for me have "  ***** "  around them. Should be easy to find. Anyway, I will do what I can.

This manuscript presents Staphopia, an online web resource/ database for genotyping data of s. aureus. Data has been gleaned from ~ 40K sequencing datasets from public database and seems to be primarily available through an API.

First of all, I will applaud the authors for wrangling such a large dataset. There is an incredible amount of logistics involved with serving this kind of data.  There are some interesting ideas presented in the manuscript that I hope the authors can extend.

Line 18: Staphopia is such a catchy name.

*****Line 318. Are the data generated by staphoia submitted back to the main MLST database. This would be very useful. The true power of MLST comes from the standardisation and simple nomenclature it facilitates. (the same issue applies to the cgMLST data).*****

Line 58: “seven core genes alleles” doesn’t make any sense. Perhaps simply ‘seven genes?

Line 66: The authors may enjoy reading our paper:  https://doi.org/10.1371/journal.pgen.1007261

*****Line 66: Much of the functionality presented in staphopia is already available through BigsDB. BigsDB is open source, and has been used by third parties to serve genomic data (SNPs, cgMLST, MLST) e.g. Insitute Pasteur has used it for a number of organisms e.g. http://bigsdb.pasteur.fr/klebsiella/klebsiella.html .  BigsDB already has a S. aureus MLST database (https://pubmlst.org/saureus/ ), which is looking for curators. Perhaps something more like IRIDA http://www.irida.ca , could be used if BigsDB doesn’t suit. Anyway, this introduction gives a compelling argument for accessible web resources but fails to give the impetus for staphopia specifically. It is unclear how long the code base for staphopia will be supported for; many bioinformatic resources simply disappear as developers lose interest or funding. *****

Line 106: Prokka is usually rendered in title-case ‘Prokka’, not PROKKA. See Seemann et al bioinformatics 2014.

*****Line 139: there are a lot of citations here. There is no indications how these citations contribute to the SCCmec typing employed. The first one is for BLAST. The rest seem to be papers that described the primers? This should be separated out. Are all these citations necessary? Are all these primers presented in Kondo et al? Aren’t the sccmec types in sccmec.org, can you point to that ?*****

Line 139: This error occurs all through the manuscript. The authors have used BLAST+ (im guessing from the version number cited). They should cite https://doi.org/10.1186/1471-2105-10-421 . They should refer to BLAST as BLAST+ . There is a dramatic difference between BLAST from the 1990 Altschul paper and from the 2009 BLAST+ reboot.

Line 144: The English here is completely garbled and needs to be rewritten. “We also aligned proteins associated with SCCmec are also aligned against the assembled genome…the pFASTQ BWA” ??

Line 146: typo “to to”  . Please review lines 137 – 152. There may be other errors.

Line 155: Supp Fig 1. The width of this figure is huge! And Far too detailed! This might be better in documentation, and a simpler schematic could be presented here for a general audience.

*****Line 170: Is this an ongoing agreement to use the Cancer genomic cloud? This has been used to generate data up to November 2017? But what about after this date? Does Staphopia keep up to date with new sequence data? On the basis of lines 169 – 185, it seems that this is a one off. This would drastically lower the utility of this service if this was the case.*****

Line 207: The use of numbering #1, #2 #3 with no paragraph breaks is very confusing. A figure of a workflow would be useful? Or at least some line breaks in the text.

Line 210: This gist might be better off in the main github. There is value in Keeping everything in one place.

Line 216: The number of STs / genomes are not mentioned.

 Line 271: Does this really require a figure? The ranges are enough in my opinion.

*****Line 323: I am outraged with the presentation of the cgMLST data in this manuscript. Firstly and most importantly, there is NO CITATION OR DESCRIPTION  for the scheme that was used. Based on the number of loci reported, I assume the authors have used the scheme described in Leopold et al, J. Clin. Microbiol. 2014, 52. This scheme is available at http://www.cgmlst.org , which should also be mentioned. According to the manuscript, Sequence types  (cgMLST) were calculated for all genomes in staphopia. But the resulting data is does not appear to be available (no mention in the API documentation). Furthermore the final result in the manuscript simply states the number of resulting sequence types based on cgMLST and remarks that most genomes had their own unique cgMLST profile. This finding has already been presented many times before for cgMLST (see https://www.nature.com/articles/nmicrobiol2016185 for a good example). *****

Line 404-415: This appears to be methods. What does the Tree actually tell us?

Line 459: Typos. Project ID (i.e. the PRJN ID)

Line 460: ‘was much more manual’ ; clumsy.

Line 463: typo ID.

*****Line 484: There is no description of staphopia’s runtime and performance as it currently stands. *****

Line 488: We, along with many others, have found MLST, rMLST, cgMLST fairly useful for global, large scale comparisons. We only switch to SNPs for fine analysis (outbreak, clonal complex etc). I am surprised this not presented as an alternative here.

Line 501: clarify average quality (i.e. refer to average ‘base calling’ quality)

Line 512: Typo Cores.

*****Line 510: It would be good if these genomes were presented (as a table; genomes name, description, accession code) somewhere in the manuscript or website. *****

*****Line 512: It would be good if these genes were presented (as a table; gene name, description, locus tag) somewhere in the manuscript or website.*****

Line 590: Please italicize all binomials names in the references. Some gene names e.g. mec also need to be italics. As a tip, if youre using Endnote or something like that you can change the text formatting for stuff like this in the citation manager itself. Don’t try to convert the citations to plain text and then edit them. Trust me.

Line 675 & 696: Some citations have title case. Where Words Have An Upper Case Letter. Some do not. Please check journal style and make consistent. Again, make the changes in your citation manager.

Figure 5: You can actually infer everything in this figure from Figure 6. This figure is redundant.

Figure 6 & 7: I don’t think these need entire separate figure. It’s the same X axis just different Y. These might be easier as a composite figure where each graph is a subfigure (A, B, C…).

Figure 1: You can actually infer everything in this figure from Figure 8. This figure is redundant.

Figure 1: Why is 2018 included here? It’s only April. The graph implies only ~200 S. aureus were sequenced for all of 2018.

Figure legends: Some of these are very short. “Top ten STs” for figure 5 doesn’t explain anything. Consider that a lot of readers will go straight to the Figures and ignore the body text as first. Give them all the information they need to interpret the figure!
Experimental design: Line 80: I am impressed with the clarity of the methods section. Congratulations on publishing the pipeline on docker and the use of github. This is a step I wish more people would adopt.

Line 93: I agree with the use of subsampling here. citation?

Line 95: I am not a Staph person. Perhaps this is a well know type strain, but there is no explanation in this manuscript why N315 was chosen as the reference genomes for all read mapping and QC.

***** Line 108:  I am unsure about whether using a single reference genome will suffice. Often using a single reference will introduce a major bias. Generally, genomes more distantly related to the reference have less SNPs called simply because less of the reference genome can be mapped or aligned. Perhaps S. aureus is uniform enough to permit this but for most species a single reference genome is a bad idea.  Some effort has been made to control for this in the tree presented in Fig 10. But I do not believe this has been handled for general SNP data available through Staphopia.  *****

Line 220: In my experience, people are fairly clueless when it comes to APIs. I would suggest includes a simple static download (csv, Excel) of this representative genome set. Along with a tarball of the assemblies. This will greatly improve the usage of this service.

Line 231: The tree construction method seems sensible.

Line 260: Is there a citation for downsampling reads in this way?

*****Line 274: This is an interesting approach. Using MLST to determine whether something is S. aureus or not. However, this introduces circular logic in next section, where the authors talk about the genetic diversity using MLST. They should have already thrown out any divergent S. aureus by filtering out these untypable strains, so theres no surprise that the vast majority of genomes  in the next section (line 306) are typeable standard STs. If those 232 genomes aren’t S. aureus, then what are they? We’ve used Kraken (just on the assembled contigs) to search for contamination. *****

Line 333: The description of the method here seems very different to the methods section. The only citation is Larkin and Kondo. (see my previous point).
Validity of the findings: *****Line 167: According to the API documentation there is no way to search the entire database for a specific metadata field. There appear to be predefined search results (e.g all published strains, or all strains from 1/ST representative set) but all other access points seem to require a strain ID (which is specific to staphopia. How do I discover the data I want when I do not know the strain ID?

For instance, I want to fetch all strains from a particular BioProject (PRJEB2756). This is often the only  accession number given in a publication.  I would need to go through the metadata of the whole database i.e.
GET https://staphopia.emory.edu/api/sample/
Then I would need to grab all the strain IDs and then request again for the metadata. i.e.
 POST https://staphopia.emory.edu/api/metadata/bulk_by_sample/
I do not know how many ID I could get at a time but the likelihood is that all the IDs at once (40K) is not possible. But OK, perhaps I pull down a few hundred at a time, over and over again. For each set I’ve pulled down, I would then need to iterate through each record looking for the BioProject of my choice. *****

Line 202: I am impressed with the effort to locate publications for the sequencing data.

Line 281: There is no comment on mixed samples. We see about 3-5% in the data we’ve looked at.

***** Line 311: This is deeply distressing that the MLST calling methods are not consistent. In a recent overview we determined Ariba was one of the best MLST callers but here there is a number of discrepancies. Which method is at fault? Is there some of systematic problem that is causing this issue? *****

Line 320: Is there an indication as to why so few STs have been sequenced? Many of these STs are probably errors (someone messed up their ABI traces), some of them might be very rare STs.

Line 339: 5 % of genomes had inconsistencies with calling mecA. Again, is this an error with one of the methods? Is one method suffering from some kind of limitation?

Line 371: I like this. We need good metadata and links to publications to understand the sequencing data. Otherwise the sequencing data is completely useless. This work here shows how poorly we, as a community, are with being upfront about our science.
Additional comments: *****Other: I am unsure of the scalability of the API given that it is serving large chunky files like genome assemblies as JSON. I ran 10 requests in parallel and found the turn around time was 20secs for one genome assembly. I’m going to guess that on a busy day with multiple users something like 1,000 genomes would take 5.5 hours to download.  The best time I could get was about ~2sec / assembly. For someone to download all the assemblies (and people will try to do that), with no one else using the service, it will take about 22 hours. I wish I could suggest a solution, but this is a very real scalability problem. *****

Other: https://staphopia.emory.edu/api/sample/unique_st  (no slash at the end) gives no response. I would expect an error message.

Other: The link for MIMS on the frontpage is broken: http://gensc.org/gc_wiki/MIGS/MIMS


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: STAPHYLOCOCCUS AUREUS VIEWED FROM THE PERSPECTIVE OF 40,000+ GENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reviewed manuscript describes an integrated analysis pipeline, database and Application programming Interface that aims to facilitate large scale genomic analysis of the thousands S. aureus genomes that are publicly available. The authors assessed the quality of the sequenced sample and provide a curated list of 380 high quality genomes representative of S. aureus genetic diversity. The API, code and database made publicly available will form valuable tools for researcher interested S. aureus and microbial genomics. The manuscript is clearly written in unambiguous language with an appropriate structure and figures. The introduction provide sufficient background informations and relevant prior literature is appropriately referenced.
Experimental design: The work presented fits in the Biological sciences, Medical Sciences, and Health Sciences scope of the PeerJ journal. The research presented compile new informations on sequenced S. aureus isolates and provide valuable tool to mine new knowledge from thousands of available genome sequences. The method described are rigorous and accurately described. All the code necessary to replicate the findings is publicly available.
Validity of the findings: The analysis presented here shows that most of the misidentified S. aureus samples (corresponding to S. argenteus) still have an MLST allele in the saureus.mlst.net database. This strongly suggests that the total number of ST in saureus.mlst.net database is overestimated due to S. aureus misidentification. This comment doesn’t invalidate the conclusion statement of “a great concentration of effort on sequencing a small number of STs” as “10 Sts make up 70% of all genomes” but the statement “only 1,090 STs of 4,466 previously collected STs”  (Line 505) and "only a quarter of ST were represented" (L34) should be tempered or at least discussed in the result section.
Additional comments: Minor comments and suggestions to improve the manuscript:

Line 31: Could you specify in the abstract which ST the chosen reference strain belongs to?

Line 92: What about non illumina-reads samples, were they incorporated into Staphopia database?

Line 106: Would be good to use a report annotations from one “high quality” (manually curated) S. aureus genomes. This would improve annotation consistency between the different genomes.

Line 117: Does it mean that if the pFASTQ file was single end no antibiotic/virulence prediction were done? Please clarify.

line 123: “for each” is repeated twice.

line 144-148: Typing errors, please rephrase

line 217: Could the authors specify how gold silver and bronze genome quality were defined (or refers to the result section)?

Line 231: Could the authors specify how many 31-mer validated genes were kept for downstream analysis?

Line 271: I don’t think that the analysis of the 44,012 genomes took more than 4 years (44,012 * 52min)? Could the author clarify this point?

Line 323: Providing access to the 1861 loci used for cgMLST will be valuable for the research community. Could the authors provide the coordinate of these loci?

Line 413: The number of variant SNP positions is inconsistent with the figure 10 legend (60,191 versus 44,377).

Figure 7: The legend of the bottom plot (aminoglycosides) is missing.

Line 423: Would be good to clearly state which important attributes are searchable in Staphopia.

Line 502: Is the threshold for gold and silver quality “35 average quality score” or 30 as specified in method line 287?


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: SEARCHING FOR BEST LOWER DIMENSIONAL VISUALIZATION ANGLES FOR HIGH DIMENSIONAL RNA-SEQ DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Table 1: Add additional columns to provide a clear description of the study designs. For example, profiling platform, sample size, groups under comparison, etc.

Paragraph below Table 1: Please state the method for normalization and the method for differential expression, so that the readers don't have to refer to the Zhuo et al 2016 paper.
Experimental design: When fitting MclustDA model, how is the number of clusters chosen?

It has been recently reported in the literature (PMID: 27601553) that, when molecular data possess confounding handling effects and normalization is used to the data, cross-validation tends to under-estimate the error rate. As a result, the repeated CV method (that the authors use for estimating error rates to rank gene combinations) may be prone to this issue as well. Would there be a way to check its performance, or to use an alternative method that is more robust to handling effects or data normalization?
Validity of the findings: A method called 'top scoring pairs' (R package tspair) also looks at the problem of ranking gene pairs with regarding to the classification power.  How would the proposed method compare to this method, when the goal is to choose top gene pairs?

The proposed method is rather computationally involved and intensive. Is there an R package available for others to use the proposed method to reproduce the results and to apply it to other datasets?
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: SEARCHING FOR BEST LOWER DIMENSIONAL VISUALIZATION ANGLES FOR HIGH DIMENSIONAL RNA-SEQ DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Better visualization is important for biologists to interpret RNA-seq data. In this paper, the author proposes a method for selection sets of two, three, or four genes, on which samples can be best separated into pre-specified groups. The problem focused on is biologically meaningful, and I am expected a good method, like what the author has proposed, will be very helpful and widely adopted by biologists. The proposed method is intuitive, easy to understand, and statistically solid. The paper is well organized and clearly written. I have one major comment that the author needs to address, and two suggestions, which I think would further strengthen the paper, that the author may choose to adopt.

Comment:

1. The major method the author compared their method with is PCA (in sections 3.4 and 4.2), and indeed PCA does not use group labels and thus one would not expect it to work well. However, the author should also compare with Fisher’s LDA, which is based on a much simpler model but also computationally much quicker, and see how much benefit of performance (in the sense of finding better gene sets) the proposed method has brought.

Suggestions:

1. If the author can make the proposed method computationally faster, the biologists, who are likely to be the main users of this method, will favor the method more. The current method requires pre-selection of a subset of genes that are likely relevant based on known pathway information. Particularly, they choose no more than 100 genes for their real data examples. If this pre-selection step can be eliminated or loosened, the method will likely be more widely used. One possible way of accelerating the algorithm is to simplify the cross-validation step. The author has been cautiously enough to do K-fold CV with C repetitions, which is good in the sense of making the results stable. However, I guess the repetitions of CV, or even the whole CV, can be eliminated, at least in two cases: (1) when the number of samples (RNA-seq experiments) are not too small; (2) for sets of (two, three, or four) genes when a non-significance is clear. For the first case, the author may construct computational study and see what number of samples is not “too small”. For the second case, sets of genes that are obviously non-significant (by a single-round calculation without CV or by other intuitive methods) should be eliminated. Other ways to accelerate the algorithm is also possible. Further, I would suggest the author focus one finding sets of two genes, as multiple 3-D or 4-D scatterplots are rarely used anyway.

2. Currently, the method gives a GSI value for each identified set of genes. It will be helpful to also give some sort of estimated p-values or FDRs for each identified set. This estimation does not need to be accurate; even a rough estimate will be helpful to the users.


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: SEARCHING FOR BEST LOWER DIMENSIONAL VISUALIZATION ANGLES FOR HIGH DIMENSIONAL RNA-SEQ DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: The authors response to the second comment under 'Experimental design' is not satisfactory. They reasoned that they 'classify genes instead of biological samples'. However, the purpose of their gene-selection method is to classify samples, and uses CV for the optimization of the selection. The authors should, at the very least, discuss the potential caveat of using CV in molecular data that may possess confounding handling effects.
Validity of the findings: None
Additional comments: None


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: SEARCHING FOR BEST LOWER DIMENSIONAL VISUALIZATION ANGLES FOR HIGH DIMENSIONAL RNA-SEQ DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author has addressed all my concerns in full.


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: DO TRICEPS SURAE MUSCLE DYNAMICS GOVERN NON-UNIFORM ACHILLES TENDON DEFORMATIONS?
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: this is a very clever experimental design.
Validity of the findings: no comment
Additional comments: I would like to thank the editor and the authors for allowing me to review this very interesting manuscript. In this study, the authors tested the hypothesis that the non-uniform Achilles tendon displacement behavior could be governed by contractile dynamics of the gastrocnemius and soleus muscles. To test this hypothesis, the authors employed a very elaborate and novel experimental design to quantify the in-vivo behavior of the gastrocnemius/soleus simultaneously with Achilles tendon displacement patterns (using two time-synched ultrasound probes) during a series of isometric tasks. The final results/data provided good support that the independent contractile behavior of gastrocnemius and soleus muscles are related to the non-uniform displacement profile of the Achilles tendon. Overall, this is a very well-written manuscript and I believe that this manuscript makes an important contribution to the field. I have a few questions and comments for the next submission (ordered by the importance of the issue, see below).

1. It is not entirely clear from the Methods section how exactly the superficial and deep areas of the tendon were determined from the ultrasound image (from ‘Transducer 2’ on Figure 1). This distinction seems very important for this experiment, since the sub-tendon areas are assumed to be representative of the portions that are attributed to the gastrocnemius and soleus. This information may be described in a previous publication, but more detail in the Methods section regarding the identification of deep and superficial tendon may be helpful.

2. From the two ultrasound data, it appears that the main dependent variables are focused on the shortening of the muscle fascicle or the displacement of the Achilles tendon along the longitudinal axis of the ultrasound probe.  I wonder if there was any attempt to align the longitudinal axes of the two transducers, such that the displacement values from one transducer would match one-to-one with the displacement from the other transducer. My guess is that the ultrasound probe orientation from both transducers needed to be adjusted on a subject-by-subject basis to get a clear image of the muscle fascicle or tendon. If this was the case, was Transducer 1 ever obliquely oriented relative to Transducer 2? Was the orientation of the two ultrasound probes ever recorded? Perhaps, a 10-20 degree rotation (between transducers) may not be such a big deal, but 30-40 degree rotation could start to affect the data when the goal is to relate the displacement values from one transducer to another.

3. During an isometric contraction, I would expect that the length of the entire muscle-tendon unit would remain the same. That is, the shortening of the muscle fascicles would be offset by the elongation of the tendon. From Figure 2, it seems like the amount of gastrocnemius muscle shortening comes close to offsetting the displacement of the tendon (at least, to the naked eye). Though, for the soleus muscle, the amount of shortening appears more than the tendon displacement. Do you think this could be due to an overestimation of the muscle fascicle shortening, or underestimation of the tendon displacement (or some combination of the two)? I don’t necessarily expect the authors to be able to fully address/answer this question, but I would appreciate any thoughts on this.

4. The order of Figures 3 and 4 may have been switched in the PDF document that I received.


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: DO TRICEPS SURAE MUSCLE DYNAMICS GOVERN NON-UNIFORM ACHILLES TENDON DEFORMATIONS?
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous text: Generally speaking, this paper is well-written. However, at times, the highly specific and overly flowery language can be distracting. It is advised that the authors consider shortening some of the longer/less clear sentences (e.g. Lines 64-68, Lines 87-90, Line 121) to improve clarity.

Literature references: The authors do a nice job introducing the reader to the subject, and explaining prior work, however some missed references are troubling. The authors seemingly preferentially cite their own work, and are missing key references that could potentially call into question some of their claims. For example, a number of prior studies have collected synchronous ultrasound data from two probes to investigate Achilles tendon mechanics, of which the authors only mention one, late in the discussion, after claiming in the abstract, introduction and discussion that they are introducing a ‘novel’ technique. They should remove the word ‘novel’ as it is misleading to anyone unfamiliar with the literature, and is inappropriate here.

The authors also reference the work of Szaro which shows the sub-tendons of the Achilles, but it is not properly described. They claim this work shows that the deep tendon is composed of soleus sub-tendons whereas Fig. 6 in the paper suggests the lateral gastrocnemius is also contributing to the make-up of this part of the tendon. Further, Edama 2015 demonstrates the variability of Achilles tendon anatomy which is also not cited. In addition, the pre-conditioning presumably comes from the work of Hawkins et al., but again is not cited. Some of these key misses in the literature are troubling.

Professional article structure: The article is very well-organized and complete. The authors should be careful with words like “discovered” (Line 90); “observed” would be a better and more professional choice.

Self-contained with relevant results to hypotheses: Generally this is well-done, but in the abstract the hypothesis should be stated earlier on.
Experimental design: Fits with Aims and Scope: Very well.

Research question well defined, relevant and meaningful. Yes.

Rigorous investigation performed to a high technical and ethical standard. Yes.

Methods described with sufficient detail and information to replicate. Yes.
Validity of the findings: Data is robust, statistically sound & controlled: The authors should justify their sample size, and clarify if any subjects had prior injuries specific to the triceps surae complex. Tendinopathy is known to lead to long-term changes in tendon structure, which could alter sub-tendon capacity for sliding.

Conclusions are well-stated: The major concern with this paper in terms of methodology is the claim that the deep tissue corresponds to the soleus sub-tendon, and the superficial tissue corresponds with the medial gastrocnemius sub-tendon. As mentioned briefly above, this does not account for the twist in the Achilles and doesn’t even seem to be supported by the paper they are referencing. In addition, the Achilles tendon shows enormous subject-to-subject variability (O’Brien 1984) which detracts from the claims being made by the authors.
Additional comments: The authors present very interesting data to test an exciting hypothesis currently being tested in the field. The paper is very well-organized, with solid methodology, and interesting discussion. Major concerns are addressed above. Below, specific line-by-line feedback may be found below.

Abstract, Line 45. The authors express that their hypothesis was supported prior to identifying their hypothesis. The hypothesis should be described earlier.

Line 67. Shouldn’t the later-cited 2014 paper be cited here in addition?

Line 82. This line suggests that “direct ultrasound imaging techniques” were developed after the papers cited from 2005 and 2013, whereas the seminal work of Maganaris and Paul on measuring in vivo mechanical properties of tendon was published in 1999.

Line 107. Muscle and tendon have also been shown to exhibit altered mechanical properties at different ankle angles. How might this influence your hypothesis?

Line 118. How was this highly specific approach controlled? Did subjects get practice?

Line 121. The structure of this sentence is awkward.

Line 137. What I meant by “manually adjusted in transverse rotation”?

Line 144. How were ultrasound data synchronized at 1000 Hz if the frame rate was only 70 frames/s and 61 frames/s? How did

Line 178. Did the authors do any quality control on their data? Or were all collected trials included in analysis?

Line 197. The authors should clarify what is meant that they “qualitatively assessed” results.

It would be interesting if the authors could discuss how their results fit into the recent publication by Matijevich et al., who observed unexpected shortening of the Achilles tendon during ankle plantarflexion. Isn’t the assumption of a series connection between muscles and sub-tendons called into question by this recent publication?

Line 237. This line seems to contrast with Fig. 6 in the cited paper which clearly identifies the lateral gastrocnemius in the deep portion of the tendon. Edama 2015 also suggests that there are substantial variations in human Achilles tendon anatomy, with it appearing quite common that the deep tissue observed could arise in the lateral gastrocnemius.

Lines 272-283. How might the observations of changing tissue mechanical properties in both the gastrocnemius and Achilles tendon at different ankle angles relate to these findings?

Figure 1 is very nice and shows great clarity, however, again the authors should refrain from terming this approach as novel. Also, what about the twist in the Achilles? Based on the referenced Szaro paper, wouldn’t the deep portion of the tendon be part of the lateral gastrocnemius sub-tendon?


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: DO TRICEPS SURAE MUSCLE DYNAMICS GOVERN NON-UNIFORM ACHILLES TENDON DEFORMATIONS?
Review round: 1
Reviewer: 3

Output:
Basic reporting: Throughout the manuscript, please define GAS and SOL and then use abbreviation consistently. At present, there are some abbreviations and some full spellings and the authors tend to switch back and forth throughout.

The figures were not in the correct order within the version I received to review. It is unclear whether the authors uploaded incorrectly or this was a journal formatting issue. Please check on resubmission.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Simultaneous analysis of muscle and tendon behavior is imperative to understanding the functional behavior and force-generating ability of muscle-tendon units. This study introduced a novel dual-probe ultrasound imaging approach to simultaneously measure muscle fascicle behavior and tendinous displacements in the triceps surae muscle-tendon unit. The premise for the study is based on previous work from the senior author that suggests non-uniform tissue displacements within the Achilles tendon is a functionally important mechanism for human movement. The results display differential behavior between the medial gastrocnemius, and its associated subtendon and the soleus and its associated subtendon. The manuscript is well-written and the experiments were carefully executed. As discussed below, you will see that I have a few comments and suggestions that I would like to see addressed.

Major Comments:

On Line 76, the authors suggest (and provide reference) that differential forces are likely to be the best candidate to explain non-uniform AT strains. However they do not examine the influence of force in their results. They highlight (Line 341) that the forces transmitted via the AT are ‘heterogeneous and highly complex.’ Yet the authors measure ankle moment (and could presumably estimate moment arm given the authors previous expertise). Using a total ankle moment (or force, if subject specific moment arms are accessible) together with the estimated relative PCSAs of the SOL and GAS in relation to the total plantarflexor force-generating ability (perhaps assuming similar activation levels), the authors should be able to determine the effect of force on their results. At minimum, I would like to see the effect of torque from the dynamometer included within their repeated measures ANOVA.

The authors find an R2 of 0.63 for SOL muscle shortening with peak SOL tendon displacement, but then find an R2 of 0.65 for SOL muscle shortening with peak GAS tendon displacement. The stronger correlation between soleus fascicle behavior and gastroc sub-tendon displacement seems to be an important finding that is not adequately discussed within the manuscript. This finding suggests, in part, that the anatomical associations between the MTUs of the triceps surae group restrict independent actuation. The authors hypothesize that superficial versus deep differences in AT tissue displacements would be accompanied by, and correlate with, anatomically consistent difference in GAS vs SOL muscle shortening. In the discussion (Line 231-234) they suggest that the findings fully support this hypothesis. Their results do indeed support this BUT also suggest that muscle-level differences are even more strongly associated with sub-tendons that are NOT anatomically consistent. I would like to see this addressed.

Discussion Paragraph Line 261-268 – the rationale for assessing the relative contributions of muscle length change versus pennation angle change remain unclear. Further, the paragraph within the discussion should include and compare these findings to other studies that have measured simultaneous changes in fascicle length and pennation angle.

Minor Comments:

Abstract – please highlight that you are measuring features of the medial gastrocnemius specifically

Line 60 – How much is largely? Can you provide the amount of total lower limb power provided by the plantarflexors/ankle.

Line 97 – please state that this is medial gastrocnemius

Line 114 – please provide evidence for why pre-conditioning the MTUs prior to tests is necessary. It is currently unclear why this important?

Line 115 – Include ‘maximum’ before isometric voluntary contractions

Line 147 – it is unclear why a threshold of 5% peak moment was necessary.

Line 154 – was this fascicle chosen to be in the mid-region of the muscle belly? Please clarify

Line 130 – enable should be enabled to be consistent with past tense throughout methods.

Line 203 – please test this effect of moment or force (rather than just ankle angle). See major comment 2.

Line 214 – ‘moreover’ seems unnecessary here.

Results section – the figures should be cited in numerical order (ie Fig 1, 2, 3, 4) but currently Fig 4 comes before Fig 3.

Line 269 – sentence should start with ‘The’.

Line 275-276 – the authors suggest that there are relatively invariant tendon kinematics, however Figure 2 suggests that the tendon lengthens by approx. 30 % more at -10 deg as compared to +30 deg. The is quite a large difference.

Line 278 – the authors mention subject variability as a possible reason for why the correlations became insignificant with increasing ankle PF. Presumably they have looked at this data, and can definitively state whether the differences between subjects were greater at these specific angles. Could it be that at these ankles, some subjects tended to lift their heel off the foot plate more than others, and this was a major contributor to the subject differences?

Line 319 – the authors suggest that these dual probe methods could be employed during more dynamic tasks (like walking) to help provide mechanistic insight into earlier findings (ageing locomotion). Is this true? Would it be feasible to have the 2 probes secured during a more dynamic task, like walking?

Line 330- It would be helpful to include how the effect of a horizontal registered pennation angle (due to Ultratrak methods) may influence the results. For example, were the aponeuroses typically parallel with the imaging plane? Does this even matter in the context of your results where you calculate % changes in pennation rather than angular values?

Discussion– can the authors suggest how their findings might compare to tendon tracking based on the position of the MTJ and the fascicle, rather than the sub-tendon tracking algorithm used here and the B-mode fascicle tracking. Would they expect similar correlations and findings?


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: DO TRICEPS SURAE MUSCLE DYNAMICS GOVERN NON-UNIFORM ACHILLES TENDON DEFORMATIONS?
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In line 32 of the introduction, the gastrocnemius and soleus are first stated, but then not abbreviated and defined until the end of the intro. This is not the case for the Achilles tendon (AT) which is defined in the first line of the intro, with the abbreviation used following this. I ask the authors to update this to be consistent.


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: PATTERNS OF TRICLOSAN RESISTANCE IN VIBRIONACEAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes the resistance of Vibrios to triclosan. The paper is extremely well written and clear. Appropriate literature is cited and sufficient background and context is provided. The article has nice figures and tables and provides the raw data.

The supplementary .txt file would not open for me. When I opened it, it was gibberish. The authors should confirm it has the correct information in it.
Experimental design: The research questions are well designed and thoroughly tested using sound statistical methods. Appropriate controls are used. Methods are provided in sufficient detail.
Validity of the findings: The findings are robust and clear. The results are novel and useful.
Additional comments: This paper was very well written and the research was done well. I only have minor comments:

L102. Is there any reason to believe these concentrations are stable? Or do you think they may vary? I think its important to state whether these were the concentrations at the time of sampling or if they were at a different time and to note that they are likely to change given various periodic and aperiodic sources fate and transport mechanisms in the coastal system.

L156. What if the duplicates did not agree? Or did they always agree?


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: PATTERNS OF TRICLOSAN RESISTANCE IN VIBRIONACEAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript meets basic reporting requirements.  The current study measures levels of triclosan resistance in environmental Vibrionaceae isolates from three sampling locations in South Carolina and Florida. Triclosan is a widely-used contaminant of emerging concern that is poorly removed in wastewater treatment plants and thus can be detected at measurable concentrations in coastal waters around the US. A better understanding of how this antimicrobial compound impacts the composition and antibiotic resistance of coastal microbial communities is needed. This study provides some valuable information to close the knowledge gap, but some revisions are needed to ensure the findings are presented in a valid and supported manner (see below).
Experimental design: The use of Kruskal-Wallis one-way ANOVA assumes homoscedacity among the groups.  Was this assumption verified? I am fairly confident that the data violates this assumption and could lead to inflated false positives particularly with non-normal data and the small sample sizes used in many of the comparisons.

The methods section on Identification of Environmental Vibrionaceae Isolates doesn't actually describe how isolates were assigned to species and clades based on the hsp60 sequences.  Fully document how the assignment process was conducted (OTU clustering? Closest BLAST hit?).  I also request that the authors document the level of confidence in those assignments and discuss how the varying confidence among isolates could impact the differences observed among groups.  The statistical results are highly dependent on these categorizations and the groupings are likely to vary depending on which assignment method is applied.  Overall, the references cited are appropriate, but for taxonomic assignment, I would recommend using Dikow, R. B. (2011) Systematic relationships within the Vibrionaceae (Bacteria: Gammaproteobacteria): steps toward a phylogenetic taxonomy. Cladistics.

Why was a BLASTN search of the V. cholerae fabV allele considered an appropriate in silico experiment?  If the gene of interest has any degree of vertical gene transmission, searching for nucleotide similarity will certainly return more hits from closely related taxa compared to other bacteria.  For finding homologous genes in less closely related taxa, a search at the protein level may be more appropriate, but NCBI has better options available for searching proteins that have already been annotated as fabV.  For example, the first result in a search of protein clusters for fabV includes a cluster of 766 proteins from 450 organisms conserved across the domain bacteria.   Only 185 of these proteins are from 60 Vibrionaceae taxa indicating much broader representation outside the Vibrionaceae than suggested by the BLASTN search.  This in silico anaylsis is thus flawed and should be redone or removed.
Validity of the findings: The last paragraph of the introduction is vague and does not fully present the state of current knowledge versus the new information provided by the current study.  Provide a few references that summarize the data previously collected on triclosan resistance in Vibrionaceae.

The interpretations in the third paragraph of the discussion (Lines 237-242) are not supported by referenced sources.  DeLorenzo et al. 2016 found increased Vibrio relative abundance at 0.33 ppm and Lydon et al. 2017 only found a relative enrichment at >4 ppm, not at ~5 ppb triclosan levels.  Specifically, the assertion that Vibrio abundance increases in the parts per billion range is not justified. Given that measured environmental concentrations were 10,000 times lower than the dosage that experimentally resulted in Vibrio enrichment, the interpretations are overstated regarding potential enrichment in coastal waters under current concentrations. Moreover, the enrichment effect could be temporary, so more work is needed to understand how persistent exposure to low concentrations of triclosan could affect coastal microbial communities.

The authors hypothesize that triclosan resistance is an evolutionary inherited trait rather than due to environmental exposure (Lines 256, 266), but the results of this study are not directly targeted at that hypothesis. To more directly address this hypothesis, a different analysis such as using a Mantel test relating a distance matrix of triclosan MICs versus a second matrix based on phylogenetic distance (used to construct the tree in Figure 5).  Another advantage of this approach is that it avoids the need to classify the hsp60 sequences into clades and/or species, which is inherently difficult to do well for bacteria with high gene transfer rates.

I think it bears mentioning in the discussion that the lower MICs for the Cholerae clade could be complicated by the fact that five of the six organisms tested for this clade were previously characterized isolates rather than those collected from your sampling sites.  If the authors only consider the isolates characterized in this study, is a significant difference among clades still observed?  While Figure 4 shows no difference among isolates from the three sampling locations in the current study, previously characterized isolates are omitted from the comparison since they all differ is sampling location.
Additional comments: (I added line numbers to the manuscript and will reference them in my comments)

Line 27 (3rd line of abstract): The Vibrionaceae family is singular, but "among" refers to multiple subjects, so "across" may be a better choice.

Lines 33, 35: Since the authors are using non-parametric statistics, it makes sense to summarize group MICs using the median value, but in a few instances the authors switch to mean MICs, which is confusing since the data are not normally distributed.

Lines 40-42: The last sentence of the abstract is partially redundant and could be better used to highlight the potential implications.

Lines 81-82: the phrase "…given the rising health burden of this group." is vague and should be made more specific.

Line 105: year for Preheim et al. citation should be 2011 instead of 2005.

Line 173: "resistance triclosan" should be "triclosan resistance"

Line 175: > 100 ug/ml should be ≥ 100 ug/ml

Lines 223-224: what evidence supports that collected environmental isolates have "known variability in prior environmental triclosan exposure"?  The fact that environmental isolates were collected from sites containing different triclosan concentrations at time of isolation doesn't imply variability in past exposure.

Line 247: I believe the authors mean "plastic" (flexible content) rather than "elastic" (expanding and contracting genome size) genomes.

Lines 274-275: Should funding source(s) be included in the acknowledgements?

Figure 3:  Should V. coralliilyticus be included?

Figure 4: For consistency, format this figure in the same way as Figures 2 and 3.

Figure 5:  Branch support numbers are unreadable and should have larger text size. It would also be helpful to overlay taxonomic assignments (clades, species) onto this tree.


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: ECOLOGICAL AND MOLECULAR CHARACTERIZATION OF A CORAL BLACK BAND DISEASE OUTBREAK IN THE RED SEA DURING A BLEACHING EVENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: I can individuate two separate parts in this manuscript. The part related to microbial analyses seems solid with correct methods and analyses and clear results. The problem I see is that the question on this part is not well stated. It looks like the objective of the paper is quantifying the occurrence and then they also did a characterisation of the microbial fauna. I suggest to well state the objectives of the microbial fauna characterisation because it would give more value to the paper, in fact the comparison with microbial fauna associated to other BDD is interesting and worth to valorise.
The part related to community composition and incidence of the BBD is less solid . There are some problems in the experimental design. For example it is known that BDD is light, temperature and consequently depth dependent. Authors says that their transects vary between 6 and 25 meters (lines 113-114), which is a huge variation. Data on BDD occurrence should be associated with depth, at least, in order to understand if the variability encountered is due to the depth or to the characteristics of the site.
Regarding the results and discussion I have some doubts on the disease %: authors found a 0.04% infection, commenting that those results are similar to other regions, but I see that values in different paper are way higher. Johan et al 2016 (Diversity) and 2015 (Hayati Journal of Biosciences) report variable infection % with high differences between reef crest (12%) and reef slope (0.05%). Results of the present work seems more comparable with reef slope data than with reef crest ones, so definitively more precise data on the depth of the transects are needed. Also other authors (e.g. Sato et al 2009-, proceedings royal society B) report higher percentages. Looking at the literature, looks like the data of the present paper show a lower infection percentage than other sites.
Validity of the findings: Conclusions on the most affected taxa should take into account the frequency of the infected taxa. If a taxa is more present it have more probability to be infected, also if the infection is not selective. Daya on the frequency of the taxa should be compared to the frequency of infected colonies of that taxa in order to normalise the infection rate to the abundance of the taxa.
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: ECOLOGICAL AND MOLECULAR CHARACTERIZATION OF A CORAL BLACK BAND DISEASE OUTBREAK IN THE RED SEA DURING A BLEACHING EVENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Hadaidi et al. conducted a series of coral disease surveys along the coast of the Red Sea, where there are no published baseline levels of black band disease. In addition, the authors collected samples of the black band disease mat and characterized the bacterial community to compare with other samples reported around the world. The manuscript is very well written in clear and concise English and very little grammatical editing is needed.
The introduction covers a decent amount of background information setting the stage for the study. The references cited are relevant and well placed. There are a couple of references that could be included such as (Muller and van Woesik 2010 in JMBE and Muller et al. 2017 in PLoSONE). You may already be aware of these and left them out for relevance reasons, and that is fine, but they could be explored for inclusion relative to the environmental influence on BBD disease (2010 paper) and the microbial community (2017 paper).

The tables and figures are appropriate and well made, but need some adjustments (see general comments).

There are no explicit hypotheses stated, but there are identified objectives and the results are relevant to them.
Experimental design: When did these surveys actually take place? I can’t seem to find the dates in the text.
The research objective is clear and fulfills an identified research gap, providing baseline data for BBD within the Red Sea and helps to characterize the consortium of bacteria that creates BBD within this region. There are certain limitations to the experimental design, however. Site selection seems haphazard at best and there is no depth stratification within the sampling design.  Since there are survey replicates within each of the three regions, perhaps regional comparisons of the coral disease prevalence could be conducted?
The lack of replication of the BBD samples within species is an additional concern as well as the collection of samples from only one site. The authors obviously take care not to run stats or extrapolate information beyond what is possible within the limited samples, which I appreciate, but this also limits the amount of impactful science that can come from this publication.
Since the sites and transects varied by depth, it would be interesting to also explore the relationship between prevalence and depth. Also, how about regional comparisons of disease prevalence?
Validity of the findings: You mention this outbreak site that occurred during a bleaching event, but there is no bleaching severity information. Other sites bleached and had less levels of BBD or BBD absent, so how would bleaching be related to this particular site’s prevalence? Can you get bleaching severity data retroactively to explore whether this has any association with black band disease severity?
I am also wondering about the sewage outflow information. Any chance you could quantify that through nutrient data? Otherwise, I would simply discuss the fact that a bleaching event was occurring and this happened to be located near a sewage outflow in the discussion as additional information on the site characteristics where the outbreak occurred. These two factors are purely speculation in their relation to disease severity (since they were not directly measured) and this should be explicit within the discussion.
 Summary of thoughts:
1.	That data provides an identified data gap within the Red Sea
2.	The sampling design limits the impact of the science produced from this study
3.	Tables and figures need further clarification and additional information
4.	Speculation that bleaching may be associated with the outbreak site does not have data to support these results directly. Just needs to be placed in context better.
Additional comments: Line 68: change ‘may results in’ to ‘may result in’
Lines 107 to 109: report the actual permit numbers; identify what the acronym KAUST stands for.
Line 113: if area covered substantially differed among sites then this should be provided as supplemental info
Line 126: the sample collection calls these samples ‘disease lesions’. To me, the lesion would be the area of denuded skeleton caused by the BBD consortium, whereas the actual sampled BBD would simply be the cyanobacterial dominated mat that creates the lesion area. I recommend changing this for clarity.
Line 167: Can you provide more information on the negative control, what was actually used as the negative ‘sample’

Table 1. The ‘GPS’ label should be units; Lat, Long or decimal degrees, or UTMs…whatever these actually represent.
Table 2. I am a bit confused about the coral numbers used in the prevalence calculation. The total number of colonies was surveyed in a smaller area than the disease survey (25 x 1 m vs. 25 x 6 m) and the total number of corals counted within the coral surveyed area should be multiplied by 6 in order to get an estimated total number of corals, correct? However, the prevalence values calculated within this table seem to reflect the original number of corals within the 25 x 1 meter transect. Also, within this table the area of the surveys seems off. The coral survey area is reported as 20 m2, but should be 25 m2 and the disease survey area is reported as 49 m2, but should be 150 m2 (25 x 6).
Table 3. States there are sequencing statistics, but really you are just presenting qualitative information.
Table 4. OTU need to be defined as operational taxonomic unit. This is also where I am surprised that sequences from Muller et al. 2017 are not presented (i.e., did not pop up within your searches), and maybe this is an issue with accessibility within NCBI. Also, how did you pick which accession numbers to present? Was it top three best match to your sequence?
Figure 1. Where is the Jeddah site listed in Table 1?
Figure 2. How did you define a dominant taxonomic group? This should be described in the text of the paper as well. Also, what is the family identified as JTB215?
Figure 3. Could you identify sequences that come from the present study?


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

Input:
Title: ECOLOGICAL AND MOLECULAR CHARACTERIZATION OF A CORAL BLACK BAND DISEASE OUTBREAK IN THE RED SEA DURING A BLEACHING EVENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed all of my edits and suggestions, included clarifying text, added information for ease of interpretation, and qualified some conclusions. I have no other suggestions for the authors and recommend publication at this time.


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: MINIMIZING CONFOUNDERS AND INCREASING DATA QUALITY IN MURINE MODELS FOR STUDIES OF THE GUT MICROBIOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a nice article on a small, yet important, topic for the improvement of mouse studies with consideration to the microbiome as an experimental variable. This is an important contribution to the field where there is a lack of standards. Dirty bedding transfer is definitely an approach many use to minimize microbiome variability so it is highly relevant with data demonstrating the ability of the method to decrease variation. Going forward the field needs more rigorous investigation of the potential adverse effects of the method, as this is not thoroughly investigated here. Nice and clear figures.
Generally, very well-written, clear and concise language, easy to follow and understand and this is also supported by the figures.
I have three major concerns and some minor comments.

Major:
1.
Line 187-189:
How did you monitor adverse affects on animal behavior? Was it based subjective on feedback from the technicians or did you monitor in a controlled way and compare to previous data on injuries etc.?
The study lacks a more controlled way of monitoring the potential adverse effects and I would advise against concluding that there are no adverse effects of the method without proper data to back it up, e.g. corticosterone measurements (e.g. in feces), behavioral testing, infrared thermography or even just BW monitoring. I think these consideration and caveats should be described in the manuscript and reflected in the conclusions.
2.
Title + line 346 + figure 3: how can it be claimed that this method can improve reproducibility without testing this? You have observed the decreased variation. You show that the method with decreasing the variation can indeed lead to a big effect size, e.g. of a dietary intervention. But from what I understand you have not made repeated experiments, showing that this a reproducible method?
Title: Also, how was it concluded that data quality is maximized? The effect size seen in the dietary intervention was not compared against a control group without dirty bedding transfer?
3. See comment under Experimental Design

Minor:
Line 63-64: Hoy et al. did not define the “cage effect” or were the first ones to describe it. Thus, it is more correct to write something like “…., variation between cages can be observed, a phenomenon called the “cage effect”” and cite: Hildebrand et al, Genome Biol 2013., McCafferty et al 2013,  Hoy et al 2015.
Line 87-88: “…, appreciable differences in starting gut microbiota have been observed.” Can you provide a reference for that?
Line 97: I would use “namely” instead of “viz.” to make it easier to follow for a broad audience.
Line 125: How was bedding sampled? Aseptically?
Line 130: Write here time of day of the fecal samplings. Did you consider timing of sampling in relation to when the last cage change had happened? This can potentially introduce bias as recently published:  Rodriguez-Palacios A, Aladyshkina N, Ezeji JC, Erkkila HL, Conger M, Ward J, et al. “Cyclical Bias” in Microbiome Research Revealed by A Portable Germ-Free Housing System Using Nested Isolation. Sci Rep. 2018;8:3801. doi:10.1038/s41598-018-20742-1.
Line 216: I was once told by a native English reviewer that the term “gender” is for humans and “sex” should be used for animals. Just a suggestion, I know many papers use the term gender when referring to mice.
Line 216: There is a typo: transfer -> transfer
Line 216-230: It is interesting to see that there are strong sex differences despite exchanging of bedding between the sexes. That really underlines that the sex effect on the microbiome is quite strong.
Line 263-264: The sentence is US-centric. Consider a more generic verbiage. As an example, consideration of the 3Rs (to which reproducibility is inherent) has been mandated by EU regulations for years.
Line 272: Did you use the same batch of diet? For chow diets, there may be seasonal batch variation potentially affecting microbiome
Line 286: Is there a typo: co-house -> co-housed?
Line 285-287: I don’t fully understand the sentence. The male mice were single-housed, from what I understood?
Line 290: I would usually not refer to a figure in the discussion
Line 290-297: Isn’t is obvious that the microbiomes of the three cohorts would differ? They are not originating from the exact same source. I know they originally all came from Jax, but did they come from the same barrier there? And then there is of course the facility and room specific environmental impact. So not surprising that there is a room effect.
Line 298: Isn’t it more a limitation of the databases used than the software QIIME per se?
Line 306-316: Please comment on why you did not do the diet intervention in cohort 2 or 3. That would have been relevant to see if the effect size would actually be bigger in cohort 1.
Experimental design: The study design is clear to understand (both text and the figure).
Yet, the setup is somewhat flawed by the fact that cohort 1 and 2 differ in more aspects than the dirty bedding transfer protocol. Ideally, these two cohorts should have been obtained/bred exactly the same way and dirty bedding transfer only performed in one of the groups. This must be commented on and addressed in the manuscript.
Validity of the findings: No concerns, besides the comments I have already made. The statistics and methods used are appropriate.
Additional comments: Are there organisms known not to transfer via the dirty bedding? If some, potentially important species do not transfer this way the method may be less reliable for studies where these organisms are imperative.


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: MINIMIZING CONFOUNDERS AND INCREASING DATA QUALITY IN MURINE MODELS FOR STUDIES OF THE GUT MICROBIOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: The research addresses an important issue in the field: cage-to-cage variation fecal microbiota observed in mouse studies.  The comparisons between cohorts (results section 1) are especially relevant and will be useful in designing future studies.

In section 2 of the results, the impact of diet is assessed in cohort 1 before and after diet switch.  However, there was no control group of mice not receiving the new diet (AIN-76A), and one could argue that the observed changes were due to drift over time alone.  Are you able to point to earlier published results or make another argument in response to this possibility?
Validity of the findings: The purpose of the SOP presented in this paper is to reduce cage-to-cage variation prior to the start of the experiment.  However, you do not directly analyze the cage-to-cage variation.  Instead, the within-group distances are used as a surrogate measure: the dispersion within a cohort decreases due to increased cage-to-cage differences.

The conclusions would be much better supported by a statistical analysis of the cage-to-cage differences.  Based on UniFrac distances, I would expect to see no difference in within-cage similarity, but see a decrease in between-cage distances when comparing cohort 1 to cohort 3.

This applies to all sections of the results.  In section 2, did the dietary intervention alter between-cage/within-cage distances?  In section 3, how large were sex-based differences relative to differences between and within cages?  In section 4, did cage-to-cage differences increase over time as the mice aged?

The cages should also be marked on the figures for inspection (maybe in supplemental).  For example, in Figure 1C, do the samples in the upper right come from the same cage?

As a secondary issue, the authors should be careful to treat repeated measures properly in the statistical tests.  If the same mouse is measured twice, the values should not be considered as independent samples.  In the ANOSIM test, I believe that the "strata" keyword argument can be used to handle repeated measures (though I'm curious to see if people disagree with me on that).
Additional comments: Minor comments:

In section 3 of the results, did the sex-based differences match previous research?

In section 4 of the results, it seems to me that the analysis of PCoA axis 1 and 2 doesn't buy you much.  What is the advantage of showing a correlation with a specific axis?  Although repetitive, I would recommend sticking to the distance-based tests unless the correlation with axis 1/axis 2 has special importance.

Part of the motivation for researchers to adopt the SOP would be to increase power in mouse studies (by decreasing the within-group variance). To really drive this point home, I think you are in a position to estimate how much the SOP increased power in the experiments presented here.  That number would be very relevant to researchers planning future studies.

In section 1 of the results, it would increase clarity to say "significantly decreased within-group distances" rather than "significantly decreased distances."  I also discourage the "clustering" language unless you actually show clustering results.


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: EXERCISE CAN IMPROVE SLEEP QUALITY: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: None
Experimental design: Data collection and analysis
Usually two authors are required to do the screening the titles and abstracts. Why authors used 5 individual authors for screening?
Again in data extraction, only two authors are needed, why did you use 4?
Method: why authors used Random –effects model? While when we do not have heterogeneity more than 20%, using Fixed Effect is recommended.
Sensitivity analysis is when we have heterogeneity and in the forest plot or funnel plot we recognize one or two studies may cause the heterogeneity. In this case, we exclude one or two studies from analysis and run the meta- analysis.
The first line of meta-analysis is Fixed method and the second line is random effect, while authors did vice-versa.
Validity of the findings: Results:  Sleep quality: the heterogeneity is too much; please take advantages from sensitivity analysis for excluding study/is which cause heterogeneity.
From Table 1: it seems that authors should have considered sub-group analysis for postmenopausal women versus women in other age, or people who had cancer and insomnia should consider as a subgroup.
Additional comments: None


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: EXERCISE CAN IMPROVE SLEEP QUALITY: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The introduction part seems to be too short far a systematic review. Its highly recommended to add more relevant references especially regarding the effects of different types of exercise in insomnia with emphasis on sleep cycle. the article literature doesn't have sufficient background to provide the broader field of knowledge. relevant literature must be referenced appropriately.
why haven you used  PubMed as an important data base where you could find more relevant references. you have pointed out in the first line of abstract that "no systematic review have examined the effects of exercise on insomnia....", while in a very simple search, you can find something different as follows:
Effect of exercise on sleep quality and insomnia in middle-aged women: A systematic review and meta-analysis of randomized controlled trials.Maturitas. 2017 Jun;100:49-56. doi: 10.1016/j.maturitas.2017.04.003. Epub 2017 Apr 5.
its not possible to consider the insomnia while you haven't discussed the sleep process. in this regard, there are some more references helping your article to improve, so add them to your work as such:
1- The Effect of Sleep Deprivation on Choice Reaction Time and Anaerobic Power of College Student Athletes. Asian J Sports Med. 2012 Mar; 3(1): 15–20.
2- The Exercise-Induced Weight Loss Improves Self-Reported Quality of Sleep in Obese Elderly Women with Sleep Disorders. Sleep and Hypnosis: A Journal of Clinical Neurosciences and Psychopathology 2018;20(1):54-59
the rest parts including article structure, figs, tables and raw data shared seems to be appropriately prepared.
Experimental design: the original primary research is within Aims and Scope of the journal and research question has well been designed. all in all, this part is good
Validity of the findings: no more new technologies for insomnia monitoring has been considered in the research. the authors should focus more actigraphy results for the research question instead of PSQI scale. the following references can help well:
Utility of Actiwatch sleep monitor to assess waking movement behavior in older women. Med Sci Sports Exerc. 2014 Dec; 46(12): 2301–2307.
Measuring Sleep: Accuracy, Sensitivity, and Specificity of Wrist Actigraphy Compared to Polysomnography. Sleep. 2013 Nov 1; 36(11): 1747–1755.
Additional comments: Generally speaking, It is a good work, but needs some more relevant references focusing on exercise mechanisms as noticed above.
Good luck.


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

Input:
Title: EXERCISE CAN IMPROVE SLEEP QUALITY: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Nothing