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: PUBLIC PERCEPTION OF THE VEGETATIVE STATE/UNRESPONSIVE WAKEFULNESS SYNDROME: A CROWDSOURCED STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This study evaluated the different perception of “vegetative state” and “unresponsive wakefulness syndrome” among 1297 participants recruited with an online crowdsourcing platform. The paper is interesting and well-written and its results may help the physicians in their complex interaction with caregivers and relatives of patients with VS/UWS. I don’t have any major criticism but only the following suggestion to bring to the authors’ attention. The perception of the “cognitive motor dissociation” (CMD) was not explicitly evaluated in this study. However, both the abstract (discussion) and the discussion (conclusions) discuss on CMD as a direct finding of the study. My suggestion is to revise these parts of the paper, focusing only on your direct results.

Minor
Line 168: It is not clear what "total n = 404" refers to.


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: PUBLIC PERCEPTION OF THE VEGETATIVE STATE/UNRESPONSIVE WAKEFULNESS SYNDROME: A CROWDSOURCED STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Using an online crowdsourced tool, the authors explored the public perception of patients according to the terminology used to describe their state of consciousness: “vegetative state” OR “unresponsive wakefulness syndrome”.

Using a significantly large sample, they showed that:
•	participants had more frequently heard about “vegetative state” (92%) than “unresponsive wakefulness syndrome” (19%).
•	most participants preferred the term “unresponsive wakefulness syndrome”.
•	the term “vegetative state” did not imply a higher agreement rate for withdrawal of life sustaining therapies.
•	participants were slightly more likely to consider the idea that a patient can retain some conscious cognition (cognitive motor dissociation) when presented as being in an “unresponsive wakefulness syndrome” than “vegetative state” (43.3% vs 38%, respectively).

The manuscript is clear, professional English is used throughout. Background/context and literature references can be slightly improved (see General comments for the author). Structure of the article, figures and tables match the required standard. Raw data are shared and well described. The manuscript is self-contained with relevant results to hypotheses.
Experimental design: The primary research is within the Aims and Scope of the journal. The research question is well defined, relevant & meaningful. It is stated how this research fills an identified knowledge gap. Investigation was performed with a validated online tool with a high technical & ethical standards. The methods are well described with detail & information to replicate.
Validity of the findings: The data is robust, well described statistically sound, & controlled. Conclusions are well stated, linked to original research question & limited to supporting results but could be slightly improved (see General comments for the author).
Additional comments: Comment #1:
I think the statement that “most experts prefer UWS” in the abstract is over rated and should be downgraded. For instance, in the recent AAN Guidelines (Giacino Neurology 2018): “While this term has no special merit or mandate for use in clinical practice, it is included here because of its wide acceptance in Europe. ». But in Naccache Brain 2018 (from Europe): “I do not consider this proposal as a satisfactory solution, mostly because the adjective unresponsive is open to many ambiguous meanings ranging from intentional responses to reflex or automatic behavioural responses. Using the adjective ‘unresponsive’ may confuse families and relatives who often observe behavioural responses in these patients (even though only reflexive)…“.

Comment #2
I think the chosen term “treatment withdrawal” is problematic for 2 reasons:

1) It is technically the life sustaining therapies that are withdrawn (e.g. ventilator support in the acute setting and, feeding and hydration in the chronic setting). In both acute and chronic cases, treatments are delivered until death (e.g. comfort care, that can include sedation and pain management).

2) Since this “treatment withdrawal” terminology was used in the questionnaire, without further details, authors should discuss a potential misconception of what this implies (framing effect again). One could argue that a more specific framing such as for instance: “Following detailed discussions with the relatives of a patient in the vegetative state, it is morally acceptable to end the patient’s life by withdrawing treatment (including artificial feeding and hydration) if there is no medical hope for recovery.” could have led to a lower rate of acceptance for WLST.

Regarding this issue I think it is important to better clarify what kind of “treatment withdrawal” is concerned in this study. Since the clinical vignette relates a patient in a VS/UWS for 2 years, I think it only concerns the chronic setting (so mainly the artificial feeding and hydration).

Comment #3
This study mostly refers to the well-known “framing effect”, classical bias in social sciences and psychology. I think this point deserves a better discussion. Authors refer to “subliminal effect” which is a very misleading term here (especially in the field of consciousness when it mostly refers to visual masking techniques). I think this term should be removed from the entire manuscript.

Comment #4
If I properly understood, the vast majority of the participants that had heard about UWS also have heard about VS. If this is the case, the sentence “Some respondents had heard of both terms (17.33%)” is misleading and should be rephrased. This implies another bias that should be addressed: people that have heard about UWS could also most likely have heard about CMD (this should be added in the discussion).

Another related issue that could be better addressed is whether the term wakefulness in UWS could increase the odd to associate the VS/UWS entity with awareness. Indeed, “wakefulness” and “awareness” frequently are misinterpreted (even among caregivers, see for instance Hermann et all bioRxiv 2018). Similarly, the term “unresponsive” of UWS has been suggested to critics (see Comment #1and Naccache Brain 2018 “… Adopting the ‘unresponsive wakefulness’ label may even suggest to some families, relatives and caregivers of such a patient that he/she is very similar to a conscious but paralyzed patient (like for instance conceiving the patient as being in the locked-in syndrome): a conscious but unresponsive person.").

Finally, since the estimated prevalence of CMD among VS/UWS currently is 15%; authors should discuss the potential implications of such an overestimation (~40%) by lay people as well as the potential impact of the spread of the UWS terminology.

Comment #5
Please discuss potential recruitment bias and generalizability (e.g. religiosity which seems lower than average in this sample [73% “not important”]).

Minor comments:
•	Abstract:
o	It is not clear in the abstract that participants are lay people, please clarify
o	Result: according to the Question (Table 1) I would reframe the presentation of the result as for instance: “CMD was thought to be less plausible … rather than “was less common »
•	Introduction:
some references could be added for covert consciousness (e.g. Monti et al NEJM 2010, Curley et al. Brain 2018)
•	Dicussion:
authors should cite and put results in perspective with previous work such as: Gray K, Anne Knickman T, Wegner DM. More dead than dead: Perceptions of persons in the persistent vegetative state. Cognition 2011;121(2):275–80.
•	Line 44 & 144: provide the full html link.
•	Line 103:  the note “A In the text, we use “vegetative state” and “unresponsive wakefulness syndrome” (in italics) when referring to semantics, and “VS” and “UWS” when referring to the medical condition” is misleading. I would recommend to use VS/UWS for the medical condition throughout the MS.
•	Line 105: “unresponsive patient” should be replaced by the unresponsive wakefulness syndrome italic notation described above (please make sure this is constant throughout the MS.
•	Line 114: Add that a language filter (only English) was implicitly used. Also consider providing additional data about the online survey to help further researcher to set a similar design: how much time was required to fill in the questionnaire? What was the cost, the funding?
•	Line 156: please provide the full formula used for searches on PubMed and Google Trends.
•	Line 170: consider using headers for each question: e.g. Attitude towards WLST:….
•	Figure 1 : color code overlap between B and D  is misleading. B legend can be improved.


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: PUBLIC PERCEPTION OF THE VEGETATIVE STATE/UNRESPONSIVE WAKEFULNESS SYNDROME: A CROWDSOURCED STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: Very interesting research, I endorse its publication.


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

Input:
Title: PUBLIC PERCEPTION OF THE VEGETATIVE STATE/UNRESPONSIVE WAKEFULNESS SYNDROME: A CROWDSOURCED STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Using an online crowdsourced tool, the authors explored the public perception of patients according to the terminology used to describe their state of consciousness: “vegetative state” OR “unresponsive wakefulness syndrome”.

Using a significantly large sample, they showed that:
•	participants had more frequently heard about “vegetative state” (92%) than “unresponsive wakefulness syndrome” (19%).
•	most participants preferred the term “unresponsive wakefulness syndrome”.
•	the term “vegetative state” did not imply a higher agreement rate for withdrawal of life sustaining therapies.
•	participants were slightly more likely to consider the idea that a patient can retain some conscious cognition (cognitive motor dissociation) when presented as being in an “unresponsive wakefulness syndrome” than “vegetative state” (43.3% vs 38%, respectively).

The manuscript is clear, professional English is used throughout. Background/context and literature references can be slightly improved (see General comments for the author). Structure of the article, figures and tables match the required standard. Raw data are shared and well described. The manuscript is self-contained with relevant results to hypotheses.

The authors have correctly addressed all my comments. I think their manuscript has been significantly improved and is now suitable for publication.

This is a very elegant and original study that will definitely add to the field.
Experimental design: The primary research is within the Aims and Scope of the journal. The research question is well defined, relevant & meaningful. It is stated how this research fills an identified knowledge gap. Investigation was performed with a validated online tool with a high technical & ethical standards. The methods are well described with detail & information to replicate.
Validity of the findings: The data is robust, well described statistically sound, & controlled. Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: The authors have correctly addressed all my previous comments. I think their manuscript has been significantly improved and is now suitable for publication.
This is a very elegant and original study that will definitely add to the field.

Best,
Dr. Benjamin Rohaut


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

Input:
Title: GENOME-WIDE ANALYSIS OF LONG NON-CODING RNA EXPRESSION PROFILE IN PORCINE CIRCOVIRUS 2-INFECTED INTESTINAL PORCINE EPITHELIAL CELL LINE BY RNA SEQUENCING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Tha paper seems  overall well written, although a minor  review in the grammar might be appropriate. The references seem to cover all aspects of the subject. The overal structure of the article is well displayed, as are the figures.
It seems an interesting and quite worthy paper to be published.
Experimental design: I am not qualified to examine the  methodology employed in this paper. The overal feeling is that it seems quite good, though the opinion of someone  who is more familiar with such methodology would be welcome.
Validity of the findings: The results seem quite interesting and the subject is highly innovative. Data seems robust and open the oportunity for a number of other studies which might use this  approach.
I would suggest that this paper  be sent to someone more familiar with this field than I.
Additional comments: The results seem quite interesting and the subject is highly innovative. Data seems robust and opens the oportunity for a number of other studies which might use this  approach. However, I would suggest that this paper  be sent to someone more familiar with this field than I.


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

Input:
Title: GENOME-WIDE ANALYSIS OF LONG NON-CODING RNA EXPRESSION PROFILE IN PORCINE CIRCOVIRUS 2-INFECTED INTESTINAL PORCINE EPITHELIAL CELL LINE BY RNA SEQUENCING
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript describes genome-wide profiling of long noncoding RNA in porcine epithelial cell lines (normal/infected by PVC2) by RNA-seq; with the goal of providing insights into the biological changes regulated by lncRNAs. Since no one has revealed the lncRNA changes in this aspect, the authors’ results are interesting and might have inspirations to researchers on the studies of porcine circovirus-associated disease (PCVAD). However, there are some clarifications that are required for this manuscript to be suitable for publication. My specific comments are indicated below. There are still numbers of grammatical errors throughout that need to be addressed to provide clarity.

1.	Lines 20-21: How “100 million raw reads” was calculated, for each sequencing library or all sequencing libraries. Otherwise, the “100 million” was not representative for your six libraries, and according to TableS1, we could easily fond the largest library contains nearly 150 million raw reads (148.7 million). The author should revise appropriately.
2.	Lines 51-52: The provenance or article for the lncRNA definition should be cited clearly.
3.	Lines 122-123: How you defined a read was low quality read? Did you allow mismatches when discarding reads which containing adapters? And what the percentage of “N” in your ploy-N sequences filtered?
4.	Lines 127-129: What were the parameters of TopHat? Did you use the default settings? If not, the parameters should be given. And the genome (suscrofa 10.2) used while mapping should be stated here—the first time it appears or used in your article. I also wondered why you chose suscrofa 10.2 instead of suscrofa 11.1. Otherwise you should cite the article of TopHat.
5.	Lines 131-133: Again, the parameters were not provided for StringTie and Cufflinks as well as the version of StringTie. And to my opinion, the articles you cited here were not proper. For example, in Shao & Kingsford 2017 they introduced an assembly software (Scallop) and compared the accurate while applied to assembly work with other softwares.
6.	Lines 135-140: The versions and parameters of CPC and CNCI should be provided as well as citing proper articles. Moreover, I wonder the steps before coding potential analysis. So, I think it’s better to described a lncRNA identification method rather than coding potential analysis.
7.	Line 143: How to classify you lncRNAs into three types (lincRNA, intronic lncRNA and anti-sense lncRNA)? And the definition of different types should be defined.
8.	Lines 149-153: Did you only keep coding genes between 10-100K upstream or downstream a lncRNA? Why don’t you consider the genes within 10K? How did you identify trans interacted genes with expression level? Pearson correlations or Spearman correlations? The method should be stated in detail.
9.	Lines 155-158: Parameters should be provided either.
10.	Lines 165-166: I guess the author misused the words of “less than”.
11.	Lines 172 and Table 1: The word “Products length” is much better for the fourth column of Table 1.
12.	Lines 178-182: Please checking how to cite an R package. And please find and cite the correct article for KOBAS.
13.	Lines 227-228: Again, how did the number of three different types calculated?
14.	Line 235 and Fig.S2: Figures’ legend should be described clearly. Take Fig.S2 for example, we couldn’t know the colors of different circles stand for. Even, we don’t know the units for the numbers beside each chromosome, single base or kilobase? Other figures should be checked either.
15.	Lines 252-253: Please clarify the cluster method of the heatmap, what’s the color bar stands for? Were the FPMK value transformed by a formula?
16.	Lines 265-266: Of the 373 DEGs, 362 were lncRNA trans-regulated target genes. Did you mean these 362 DEGs have related lncRNAs within 10-100Kb upstream or downstream?
17.	Lines 270-271: The lncRNA-mRNA regulatory network was shown in Fig 6, not Fig 5. Please illustrate the version for Cytoscape software.
18.	Lines 276-287: This paragraph was described according Fig 5. Low P values and large numbers of genes were un-clarified, the P value and gene number should be demonstrated clearly.
19.	For supplementary tables, suitable tittles with few words or annotations in it that are better for the readers’ understanding.
20.	Sometimes, the authors' discussion appears to be a repeat of the data presented and enrichment analysis in the results section such as lines 323-327 and lines 338-341. It is suggested that the authors provide an expansion of detail in the discussion section as well as a comparison to other studies.
21.	The nine lncRNAs in lines 253-255 were picked for qRT-PCR validation and lncRNA-mRNA regulatory network construction, and even the author found MSTRG.31692.1 was 20-fold-changes down-regulated. Are there one or more genes regulated by MSTRG.31692.1 or others? Whether the regulated genes were reported previously? If so, the correlations and locations should be provided.


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

Input:
Title: GENOME-WIDE ANALYSIS OF LONG NON-CODING RNA EXPRESSION PROFILE IN PORCINE CIRCOVIRUS 2-INFECTED INTESTINAL PORCINE EPITHELIAL CELL LINE BY RNA SEQUENCING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Professional English used in the present study.
The literature references is fit.
The article structure, figs, and tables are fit.
Experimental design: The experimental design is fit.
Validity of the findings: This study provides new insight into the mechanism underlying of PCVAD.
Conclusion are well stated.
Additional comments: 1. Line 113-118: The results from NanoDrop ND-1000 and Agilent 2100 Bioanalyzer should be added in the present study. The RNA amount and purity are very important to construct the cDNA libraries. In addition, I suggest that the preparation for library and sequencing should be listed in this section, but using the references. Then, authors should add the company name which provided the service for constructing cDNA libraries and sequencing.
2. Line 128: Authors should add the version of the porcine reference genome in the present study.
3. In the Materials and Methods section, it is not so clear how the authors screened and identified the putative lncRNAs. Then, It would be helpful if they illustrate a figure to clarify this process as DOI:10.1095/biolreprod.115.136911 and DOI: 10.1016/j.ygeno.2017.07.001.
4. Line 256-257: Fig. S3 should be showed in the main manuscript but in the additional files.
5. English must be edited again. There are some grammatical errors in the manuscript. Such as line 97 CO2, Line 102 mL, Line 345 differentially, etc.
6. In the present study, 13,520 lncRNAs were identified. I suggest that authors predicted the protein coding potential using CPC, CNCI, Pfamscan, and phyloCSF. It will decrease the false positive in identifying novel lncRNAs using four software in this section.
7. 13,520 lncRNAs were identified in the present study. How many pig known lncRNA were identified? How many pig novel lncRNA were identified?


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

Input:
Title: GENOME-WIDE ANALYSIS OF LONG NON-CODING RNA EXPRESSION PROFILE IN PORCINE CIRCOVIRUS 2-INFECTED INTESTINAL PORCINE EPITHELIAL CELL LINE BY RNA SEQUENCING
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the revised manuscript the authors have addressed many, but there are still some issues that should be addressed.
1. Although, the authors have provided parameters of almost all software’s and illustrated that “the preparation for library and sequencing were performed according to previous studies (Wang et al. 2016)”, still, the library types (FR, RF, PE or SE etc.) should be added for better understanding of the data analysis.
2. Throughout the tracked changes manuscripts, there are still many grammatical errors, a modification by some one of English-native could improve the paper for clearly understanding.
3. The author only provided an expansion of lncRNA characters-comparison, but we readers prefer lncRNA researches on PCV2. Otherwise, the repeat parts that I mentioned in my last review are not revised.
4. In the fig.5C, please clarify what’s the value of color bar stands for and what’s the method of Hcluster heatmap? If the author did Hcluster heatmap with log2-transformed FPKM value?
4. Figures and legends should be checked and revised again, for example, the words “Fig.3” were in the topleft of “peerj-31169-FigS2_Distribution_of_lncRNAs_along_each_chromosome.png”.
5. The tables in the supplemental files seems to be processed files, many redundant information was un-necessary. For example, the class code, chromosome, start and end were repeated in several tables.
6. In the Figure.1, the author filtered transcripts with CPC-scroe < 0, but in the method part the author wrote “All transcripts with CPC scores ≤-1 were removed”. Please clarify it.
7. For the softwares, R packages, not only the versions were needed but also the cited papers.
8. In the discussion part, the author explained too much of using PCV2-infected IPEC-J2 cells in this study. It should be stated simplified and described in the introduction part.


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

Input:
Title: GENOME-WIDE ANALYSIS OF LONG NON-CODING RNA EXPRESSION PROFILE IN PORCINE CIRCOVIRUS 2-INFECTED INTESTINAL PORCINE EPITHELIAL CELL LINE BY RNA SEQUENCING
Review round: 2
Reviewer: 2

Output:
Basic reporting: In my review, the professional English used throughout in the manuscript. The manuscript included sufficient introduction and background  to demonstrate the long non-coding RNA
expression profile in porcine circovirus 2-infected intestinal porcine epithelial cell line. The structure of the article is conformtable to an acceptable format of ‘standard sections’.  In my opinion, this manuscript is very fit to publish in Peer J.
Experimental design: The research question in the manuscript is relavant and meaningful. The research have been conducted in conformation with the prevailing ethical standards in the field. In addition, The methods in the manuscript were described with suffient information.
Validity of the findings: The manuscript reported that 13,520 lncRNAs were identified from intestinal porcine epithelial cell line after infection of Porcine circovirus 2. These results provides new insight into the
mechanism underlying of PCVAD. The RNA-seq data and other data can support the conclusions in the manuscript, and the conclusions were appropriately stated.
Additional comments: Thank you to the authors for making the requested corrections and I think the manuscript is well-organized  and fit for publication.


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

Input:
Title: ELECTRIC RESISTANCE TOMOGRAPHY AND STRESS WAVE TOMOGRAPHY FOR DECAY DETECTION IN TREES—A COMPARISON STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing of this paper is neither clear nor unambiguous.  It falls so far short of being understandable that this reviewer feels unable to even begin to review the validity of the science.
The references are not properly cited in the text, nor accurately listed in the bibliography.

The authors would be advised to seek professional help in rendering this paper into "clear and unambiguous, professional English."  Alternatively, perhaps the paper's third author, based in the United States, could take the lead on this.
Experimental design: It is unclear why the authors, in choosing to use the Argus TreeTronic equipment for Electrical Resistance Tomography, did not use the company's PiCUS equipment for sonic tomography, their version of the Rinn Stress Wave Tomography.  This does not appear to be discussed in the paper, nor referenced, even though there are numerous publications demonstrating its use.
Validity of the findings: Given the inscrutable writing of this paper, this reviewer feels unable to properly evaluate the paper.  That said, the experiment approach, while interesting, appears flawed, in using wood cores, which provide data in a single dimension.
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: ELECTRIC RESISTANCE TOMOGRAPHY AND STRESS WAVE TOMOGRAPHY FOR DECAY DETECTION IN TREES—A COMPARISON STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: A significant proportion of the literature cited is a bit outdated. Citations to more recent articles should be increased.
Line 246 must be corrected: (Van der Wal et al, 2015)
Figures 1 and 2 do not contribute anything to the understanding of the article.
Figures 3, 5, 6 and 7 should be summarized in a single figure that synthesizes the information provided.
Appendix 1 should not be an integral part of the article and be part of a complementary file.
Experimental design: The methods are described in detail and work could be replicated.
The sample size of the experiment is adequate.
Validity of the findings: It is an article that does not present a high of novelty neither in its methodology nor in its obtained results. Nevertheless, the main finding is the detection of a threshold of 30% in the true severity of the tree decay that allows one or another technique to become more effective.
This conclusion is robust according to the results.
Additional comments: I recommend this article for publication, subject to an increase in the relevance of the figures and an improvement in the proportion of relevant citations of the last 5 years.


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: ELECTRIC RESISTANCE TOMOGRAPHY AND STRESS WAVE TOMOGRAPHY FOR DECAY DETECTION IN TREES—A COMPARISON STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article is clear and unambiguous with well understand of English usage.
The references used were from a multiple sources.
The article structure, figures and table are well positioned significantly.
Experimental design: The experimental design had been well explained with sufficient information.
Validity of the findings: The findings presenting in this article show the scientific merit and contribution to the knowledge.
Additional comments: The manuscript had been well written with easy understanding and clear explanation for the reader. Anyhow, in my point of view there is a comment/suggestion in the article which can be improved to enhance the quality of this paper.


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: ELECTRIC RESISTANCE TOMOGRAPHY AND STRESS WAVE TOMOGRAPHY FOR DECAY DETECTION IN TREES—A COMPARISON STUDY
Review round: 1
Reviewer: 4

Output:
Basic reporting: The structure, aims, description and result reporting are good quality, suitable for publication. Literature review is thorough and adequate.

English usage is, albeit not flawless, mostly adequate, easy to follow and understand. There are some exceptions to this that are marked in the review material. These problems should be corrected.

Figures are relevant and mostly easy to understand. All three trendlines should be included in figures 8 and 9 to make the analysis easier to follow for the reader.

Results are self-contained and relevant, although the analysis could have a somewhat broader outlook to the application of NDT methods (see note at conclusions.)
Experimental design: Paper contains significant and relevant primary research in the area of biological research. The aim of the research is well-defined, relevant, and fills an identified knowledge gap. Research is carried out rigorously and yielded trustworthy results. Methods are described in sufficient detail; the description of the calculation should be improved as noted.
Validity of the findings: Result analysis is thorough, statistically sound and relevant. Conclusions are well stated, but a note regarding the wider applications of the discussed tomographic methods should be included, see note in the review material.
Additional comments: Minor notation mistakes are also indicated in the text.


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

Input:
Title: ELECTRIC RESISTANCE TOMOGRAPHY AND STRESS WAVE TOMOGRAPHY FOR DECAY DETECTION IN TREES—A COMPARISON STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is clear, Literature references are adequate.
Experimental design: The methods are described in detail and work could be replicated. The sample size of the experiment is adequate.
Validity of the findings: The results are a contribution to a field where new scientific communications do not usually bring novelties.
Additional comments: Homogenize the precision in table 1


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: EFFECT OF THE FLAVONOID BAICALIN ON THE PROLIFERATIVE CAPACITY OF BOVINE MAMMARY CELLS AND THEIR ABILITY TO REGULATE OXIDATIVE STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear, unambiguous, professional English language used throughout.
Intro & background to show context. Literature well referenced & relevant.
I suggest you to specify that the metabolic diseases “milk fever, ketosis” at line 45 are examples. I recommend (e.g., milk fever and ketosis).
Citations from the line 54 (Miller et al., 1993, Harrison et al., 1984, Allison and Laven, 2000) does not meet the editing requirements of the journal, please consider: (Miller et al., 1993; Harrison et al., 1984; Allison and Laven, 2000). Also, Miller et al. 1993, Harrison et al., 1984, and Allison and Laven, 2000 are missing in references list.
The name of Scutellaria at line 64 must be written in italics.
The reference (Robert et al., 2014) at line 79 must be written (Robert, Leboeuf & Dupuis, 2014)
The reference (Yu et al, 2015) at line 270 must be written (Yu, Pei & Li, 2015)
The reference Lin et al. at the line 282 don’t have the year in brackets.
The reference Loor et al. (2018) at line 305 is missing in references list.
The reference (Kensler et al. (2007) at lines 310-311 is missing in references list.
The reference (Wiese et al., 1995) at line 323 must be written (Wiese, Pacifici & Davies, 1995)
The reference (Jin et al., 2016) at lines 334-335 is missing in references list.
The Structure of References section is not conforms to PeerJ standards.
Structure conforms to PeerJ standards, discipline norm, or improved for clarity.
Figures are relevant, high quality, well labelled & described.
Raw data supplied.
Experimental design: Original primary research within Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
Rigorous investigation performed to a high technical & ethical standard.
Methods described with sufficient detail & information to replicate.
Validity of the findings: Data is robust, statistically sound, & controlled.
Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: Many references in the text are missing in the References section.
This scientific article can be accepted but after revision


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

Input:
Title: EFFECT OF THE FLAVONOID BAICALIN ON THE PROLIFERATIVE CAPACITY OF BOVINE MAMMARY CELLS AND THEIR ABILITY TO REGULATE OXIDATIVE STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Hello! Please pay attention to the modifications!


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: EFFECT OF THE FLAVONOID BAICALIN ON THE PROLIFERATIVE CAPACITY OF BOVINE MAMMARY CELLS AND THEIR ABILITY TO REGULATE OXIDATIVE STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Briefly, the paper is scientifically valid, well written and clear. References are OK. Professional English Language is used and it is fluent.
Suggestions are below.

Fig 1 Legend, please be more concise for panel A;
“The reaction product was quantified by measuring the absorbance at 370 nm with a reference wavelength of 492 nm.”, this sentence could be removed.
“Minus Fluorescence Intensity (MFI)”, I guess the authors wanted to write “mean” or “median” instead of minus. Did they?

Fig 3 Legend, Random numbers are present in the text starting from this Figure, e.g.  “…6 hydroxyl radicals”, “…7 dichlorofluorescein (DCF”)”, etc
“The fluorescence was measured with a multi-detection microplate reader using an 8 excitation and emission light at 485 nm and 535 nm, respectively.”, this sentence could be removed

Fig 4 Title, please correct H2O2 to H2O2, please remove the “3” before “production.

Line 131  “XX” hours. Please add the right time or rephrase
283 “in vivo” it should be italic
302 “H2O2”,
317 “in vitro”, it should be italic
343 “in vivo”, it should be italic
Experimental design: Material are very detailed and methodology are accurate and appropriate. The experiments seem clear and reproducible.

Labelling used for statistical significance is unordinary and may confuse the reader. I recommend to display the differences between the values in a different way and use asterisks for significance level.
Validity of the findings: The manuscript provides novel insights into modulation of mammalian antioxidant defences by baicalin. The research question is confined while sufficiently supported by the results. The discussion is well stated and coincise.

In the abstract the authors wrote “Baicalin prevented apoptosis at low concentrations (10 μg/mL)…”, but I suggest the authors to use a different verb.
Additional comments: Fig 1 panel C seems a bit uncomplete or uninformative as it stands. The filed is evolving and interesting. Improved in vitro models will help your future research and following impact.


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: CAPTURING COMPLEXITY: FIELD-TESTING THE USE OF ‘STRUCTURE FROM MOTION’ DERIVED VIRTUAL MODELS TO REPLICATE STANDARD MEASURES OF REEF PHYSICAL STRUCTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Congratulations on a very clear, concise, well written and referenced paper.

It would be good to add a site overview figure showing the location of the reef complex and the six survey sites.

A few minor areas in need of re-wording/fixing are listed below:

Fix the following “Error! Reference source not found” messages: Line 110

Reword line 156 to remove slightly awkward phrasing “less good agreement”. Perhaps replace with: There was, however, less agreement between the methods within more complex habitats…

Reword lines 163-164. I think know what you mean by “virtual RI increases more quickly than in situ RI” but it is not technically correct as it implies some sort of speed component. I think it is a case of the virtual method measuring higher RIs than the in situ method particularly above a certain threshold.

Fix grammar in line 174.

Throughout the text alternate terminology is used for the same thing (e.g. virtual, digital, SfM etc… are used interchangeably; likewise, traditional, in situ, tape and chain are also used interchangeably). Be consistent as much as possible to make it easy for the reader.

Line 206 is a bit confusing. What is meant by “rugosity techniques if it is not SfM? Reword this sentence.

Line 209 change awkward wording “…in in situ…”

Line 219 change technological to technology.

Please explain cluster computer further and what a “node” (referred to in Table 1) is.
Experimental design: The methods are well-designed, clearly explained and replicable.

The following additions would be useful:
-	In the data collection paragraph (lines 112-129) can you add an approximation of: how large the footprint of one photo was, how far apart the “lawnmower” tracks were, what the proportion of overlap (or overlap and sidelap) between adjacent images was?
-	Please expand on lines 128-129. What exactly do you mean by “multiple known dimensions in xyz space”? Was z depth? Did you record depth at each marker? Did x and y dimensions come from the known size of the marker and/or the distance between markers? How was the model accuracy of <5 mm calculated?
-	In lines 168-170 you say that dominant substrate type was included as a factor and increased the predictive ability of the regression. Please explain how dominant substrate type was included. Was each transect conducted over only one substrate type?
Validity of the findings: While the method presented is no longer novel the authors identify and fill an important niche and gap in our knowledge regarding how to articulate between traditional and SfM measures of rugosity to allow for comparability between surveys undertaken using the different methods. They also differentiate how their work differs from, and builds upon, previously published comparisons.

With respect to the discussion, has any other literature compared visual complexity assessment with SfM and tape and chain methods? If so, compare their findings in lines 205-216. If not, cool, make clear this is a novel aspect of your work.

Make sure you add to the conclusion your key finding which is that it is possible, using the regression relationships you found, to translate historical tape and chain RI measures into modern SfM measures, thus allowing long term monitoring studies to continue using the new method. It would also be good to add to the discussion whether your findings suggest that to do this a researcher should perform both methods at the same time (and if so how many replicates) to come up with a site-specific regression relationship or whether it may be possible for the same dominant substrate type to use a published regression relationship.
Additional comments: Congratulations on a nice paper, I look forward to reading the updated, published version!


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: CAPTURING COMPLEXITY: FIELD-TESTING THE USE OF ‘STRUCTURE FROM MOTION’ DERIVED VIRTUAL MODELS TO REPLICATE STANDARD MEASURES OF REEF PHYSICAL STRUCTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is simple, but employs sound logic to address an important question: Are rugosity measures derived from photogrammetry compatible with those derived from chain-and-tape and visual estimate methods?

There is an opportunity to provide explicit and very useful management advice at the end of the discussion around how to validate and convert long-term monitoring data to photogrammetric records. An estimate of conversion factors to relate visual and chain-based complexity measures to future digital complexity measures (and variability around those factors) would add to the utility of this manuscript. Would reliably converting between digital and chain-based rugosity be possible? If not, which measures are responsible for the excess variability? This is not necessary but would add to the manuscript.

Inconsistency of terms (rugosity/RI/complexity and SfM/Photogrammetry/digital methods) creates confusion. This should be addressed throughout.

20. “Stereo-view” is a specific subset of photogrammetry (one that is not used in this paper). Probably omit. Also need to choose either SfM or photogrammetry as the term for the whole paper. Make clear that they are roughly equivalent, and then just use one term throughout.

28. This paper only deals with coral habitats. Not safe to imply that sfm = chain and tape in ALL situations. A brief discussion, backed up by literature, of how sfm is likely to function in rocky and other reef types would be useful here. This would allow broader inference to be drawn later.

61. LiDAR, sonar and satellite-based techniques are not more detailed (1 - 10m pixels). They ARE more data dense, archivable and cover greater extent.

62. LiDAR, not Lidar.

89. “However, there has been limited research into whether these new digital methods of assessment can provide measurements that are directly transferable from current standard monitoring protocols, meaning it is not yet known whether ongoing surveys can transition to this new technique without risking the loss of comparability with older survey data.” This is true, and this study does a good job of addressing this question (in coral habitats). But it feels deceptive to intentionally withhold the Ferrari 2016b reference (which does address this question) until after this statement of the core question.

110. Reference error.

119-121. Just specify resolution. By saying that settings and equipment was the same, you pre-empt the possibility of them effecting the resulting model.

134. “to account for any fine-scale movement of the in situ chain”. Unclear what this means. The chain is not modelled, correct? Why is fine-scale movement of chain be important?

188. Dense cloud, or sparse cloud? Which was filtered?

206. Unclear which rugosity. Digital or chain?

207-210. Confused sentence. Re-word to make clear. Start with what the heading says. “Visual estimates of complexity were more closely aligned with digital RI (have you introduced this term??) than with in-situ RI measurements. This indicates that digitally derived RI measurements better match how our eye naturally assesses complexity.”
Experimental design: The experimental design is simple, but dappropriate to the question. Replication is sufficient.

I feel that the novelty of the paper is perhaps over-stated as this question has been addressed previously (as acknowledged by the authors at the end of the introduction). Additionally, a key improvement of this paper over previous work on this topic is the increased replication, and repetition in “a range of reef habitats” (though this paper still only deals with coral-dominated substratum). This statement begs the question about rocky reefs, soft substratum, artificial substratum and the many other environments where rugosity is used. This should be reflected in the manuscript.

124-128. I recommend a table laying out the model building steps (align, dense cloud, mesh, etc), the settings chosen (high accuracy, medium density, aggressive filtering, etc), and the range of resulting features (1 million points, 790k faces, linear precision of <5 mm)

128. Although this study uses ratios (reducing the importance of scaling) scaling method should still be explained clearly. How were the markers used for scaling? I.e placed at known distances… And what were the scale features?

129. Many people will interpret this as model accuracy in 3D space, which it is not. Accuracy in XYZ implies that any point on the model is +/- # from other points in the model as related to the real world (see Figueira et al 2015 and Bryson et al 2017 for examples). Best to say scaling accuracy of <5mm. Also, how many used per model?

131. Make clear if each digital measurement was taken in the EXACT same line of reef as the physical chain, or if each is nearby and assumed to be representative. Either is fine, but unclear which was done.
Validity of the findings: This study meaningfully expands on some early exploration of digital rugosity's backward compatibility. This is an important question, which this work adds to. There is still much to be done in terms of different spatial extents and resolutions (of chain, and of digital reconstructions). Most importantly, it should be made clear that this work is on coral habitat only. Extrapolation to rocky and other reef types is probably valid, with some caveats. But it is not safe to imply that digital RI/chain RI relationships at all substrates are now known. The sample size, design and range of habitats covered here are not exhaustive.

193. This is true, but I wonder if sfm would capture these features either. Especially with limited resolution, any moving object is not likely to be reconstructed by sfm as it will not be in the same place in multiple images and will be stitched out by the Photoscan algorithms.

193-197. Confusing sentence. Good to speculate which method is closer to the real-world complexity. But the logic and sentence structure of reason #3 here needs improving. I also disagree with the logic. Sfm misses huge amounts of complexity in a coral reef as it does not model areas much beyond vertical. So all overhangs are lost or under-estimated. A well-deployed chain and tape method should capture these crevices as long as they are large enough to fit links of the chain into… irrespective of gravity.

204. Photogrammetry is a rapidly expanding tool in ecology and many people are interested in exploring it. If the authors have any guidance on specific limits (i.e. the authors found that having visibility at least 2x the distance from cameras to the substrate was sufficient to build accurate models) that would add to the usefulness of this manuscript. If not, it is not necessary.
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: CAPTURING COMPLEXITY: FIELD-TESTING THE USE OF ‘STRUCTURE FROM MOTION’ DERIVED VIRTUAL MODELS TO REPLICATE STANDARD MEASURES OF REEF PHYSICAL STRUCTURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Line 91 Previous studies that compared chain-and-tape rugosity to virtual rugosity include Ferrari 2016b and Young 2017.

Line 110 “Error! Reference Source not found.”

Line 156, improve writing by replacing “less good agreement” with “worse agreement”

Line 206 “SfM matches very well to visual estimates of structural complexity” Qualify this statement statistically.

Line 219 “very recently” is meaningless phrase here. Do you mean only in the past five years?

Sentence starting on line 259: strange use of semicolon. Use colons and parallel sentence structure. E.g., “… the SfM method is quantitative, less biased by the recorder, and replicable. It allows detailed spatially explicit…”

Line 263: Why is reproducible science in quotes?

Table 1: (A) Processing time is meaningless unless you know the computing power. These are approximate, so you could add in caption something like “Processing time is approximate for a standard laptop with 8Gigs Ram, etc.” (B) Consider removing the cost column. Many SfM software options are free. Visual surveys have the dive time and equipment as a cost, as do these other methods. Why’d you decide to remove that cost? How does a chain and tape cost £100? What areas are these covering? At the very least fix capitalization of Software/Kit Cost title, although kit doesn’t not include dive kit, so again I’d just delete this column to avoid confusion and because it doesn’t add much value to your main findings. (C) What does archival mean in this context? I think the word “Repeatable” covers what you mean here. You can archive the data recorded in Visual assessments by recording your measurements; so I don't think archive captures what you mean here.

Caption Figure 1: Capitalize sentence in part (A). Spell-out numbers that start a sentence (in part C).

Figure 1: Great figure, but please add scale. How many meters long is the linear distance? The scale in part (B) is too small to read. Do the colors mean anything in (C)? If not, I suggest removing colors.
Experimental design: Thanks for providing the raw data, but data labels are not clear. What is SR vs LR vs RR? How did you define the dominant substrate?

Methods: Please describe how you determined dominant substrate.
Validity of the findings: Nice work.
Additional comments: This is a nice paper that adds to a growing body of work. I recommend for publishing with minor comments detailed above.


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

Input:
Title: A NEW METHOD OF GALL MITE MANAGEMENT: APPLICATION OF ARTIFICIAL DEFOLIATION TO CONTROL ACERIA PALLIDA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper gives new information to the eriophyid mites control.
Experimental design: is okay
Validity of the findings: are original;
Additional comments: No additional comments


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

Input:
Title: A NEW METHOD OF GALL MITE MANAGEMENT: APPLICATION OF ARTIFICIAL DEFOLIATION TO CONTROL ACERIA PALLIDA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript reports on a new and unique method of managing eriophyid mites; namely, defoliating the mite galls between natural fruit production/harvest periods to prevent the re-infestation of new plant growth.  The structure of the manuscript is clear and unambiguous; methods, tables and figures need improvement.  The Western world is familiar with wolfberry as goji berries; perhaps this could be included in the Introduction.  Additionally from the appearance of the plots in Figure 3, you should refer to the plants as bushes.

Specific problems with the Introduction:
1.  Line 42  By definition mites do not have wings; delete this.
2.  L 48 LI et al. should be Li et al.
3.  Throughout the manuscript delete the 's' from damages; i.e. damage caused by gall mites.
4.  L 52 should be 'Furthermore'.
5.  L 55  The citation of Greenberger et al., 2004 is not appropriate and doesn't support your hypothesis.  Boll weevils are not attached or encased in cotton leaves; therefore, defoliating cotton leaves per se will not be effective management.  In the cited manuscript, beetles were affected through direct contact spraying and in some combinations there was a synergistic effect between defoliant(s) and insecticide(s).  In your methods, to be parallel to the Greenberger publication, you should have tested the direct effect of the defoliate on the mites.  In contrast, Bemisia tabaci nymphs are attached to the leaves, so defoliating the leaves also defoliates the nymphs, which subsequently died due to lack of food.
Experimental design: The area/dimensions/number of bushes of each experimental treatment was not clearly stated.  The area of the experimental plot (L 97) was given as 2600 sq m.  There were five replicates of each pesticide treatment and defoliant with about 60 bushes as a buffer between treatment areas.  The number of treated bushes is not given (L101-111).

L 112 states that two bushes per replicate and four branches were selected for gall counting.  Two bushes out of how many?  How many leaves per branch?

There is insufficient information, and no raw data to ascertain what was done.
Validity of the findings: 1.  The validity of the findings are impossible to know as the number of treated plants was not given.  Additionally the degrees of freedom were also not given, therefore the number of treated plants could not be ascertained, even indirectly.
2.  On both figures there are only two lines - 'defoliant' which was the same over the 3 years of the trials, and 'pesticides' where were different combinations for each spray date over 3 years; and counts were made twice a month.  We should see these results for each treatment.
3.  Figure 2 axes are:  Number of ... per branch.  However there were four branches per plant for each of two plants, so I expect to see +/- S.D. which is not shown on the graphs.  Additionally was there a difference among the cardinal directions?
4.  The 'raw data' files are not present, just the pooled data for making the graphs, so it is impossible to know what the data were.
Additional comments: The idea of defoliating is new and novel, and probably worked just fine.  However, the data are not presented well and so conclusions are???


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

Input:
Title: A NEW METHOD OF GALL MITE MANAGEMENT: APPLICATION OF ARTIFICIAL DEFOLIATION TO CONTROL ACERIA PALLIDA
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an original research. The investigation is relevant due to the economic impact of gall mite on the production of wolfberry, a tree of a great importance in traditional Chinese medicine and food.
Professional English was used but several errors in syntaxes were noticed. Particularly, Experimental design and Results phrasing makes comprehension difficult as well as the interpretation of data. Please ask for support in English writing.
The discussion seems quite weak with few references.
Experimental design: Research question is well defined. However, some important questions regarding experimental design emerged during the reading. Methods are not well described to replicate. Please see the .pdf file with comments.
Validity of the findings: No comment
Additional comments: Please check carefully the comments on manuscript. Thanks.


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

Input:
Title: A NEW METHOD OF GALL MITE MANAGEMENT: APPLICATION OF ARTIFICIAL DEFOLIATION TO CONTROL ACERIA PALLIDA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript is much improved over the previous version.  There are some minor English editorial changes which, I'm sure, the production staff will catch and correct.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Line 53, line298 - what is correct date 2013 or 2003?

Line 122 - Tukey's HSD test not Turkey

The first paragraph of the Discussion is largely a repeat of the Introduction, and should be significantly reduced.  The Discussion is for the results not repeating the introduction.

I see that the authors used only one type of defoliant year after year.  I think something should be added in the discussion about practices to avoid the development of resistance to the defoliants, similar to IRM.

Line 205 Wiley et al. not in references

Line 249 Rhodiola rosea should be in italics
L 264 Oikos in italics

Figure 1 adult A. pallida observed using a scanning electron microscope

Figure 2  The arrows are actually white with a black outline.  Maybe make them solid black to see them more easily.

D, E, and F, I believe are average number of gall/day


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

Input:
Title: A NEW METHOD OF GALL MITE MANAGEMENT: APPLICATION OF ARTIFICIAL DEFOLIATION TO CONTROL ACERIA PALLIDA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Professional Englished used. Sufficient background provided.
Experimental design: Methods described with sufficient detail & information to replicate.
Validity of the findings: No comment
Additional comments: Authors made an effort to support the methodology/results as asked in the first revision.Authors demonstrated a qualified revision of the language and improved the manuscript.. I am totally satisfied with the corrections. As a consequence, I accept the publication in Peer J.


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: AMORPHOUS SILICA NANOPARTICLES INDUCE TUMORIGENESIS VIA REGULATING ATP5H/SOD1-RELATED OXIDATIVE STRESS, OXIDATIVE PHOSPHORYLATION AND EIF4G2/PABPC1-ASSOCIATED TRANSLATIONAL INITIATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: Experimental design is well designed .
Validity of the findings: The finding is novel.
Additional comments: Manuscript number: #32867

Title: Amorphous silica nanoparticles induce tumorigenesis via regulating miR-4734 and miR-4521 mediated oxidative stress, innate immune response and translational initiation
Comments to the Author

     In this manuscript author investigated the underlying toxic mechanisms of amorphous SiNPs on human lung epithelial cells model by using microarray data. Here, they demonstrated that a total of 323 genes were identified as DEGs from Beas-2B exposed to amorphous SiNPs, including 280 upregulated (containing 12 miRNAs) and 43 downregulated genes (containing 29 miRNAs). Furthermore, author demonstrated that these genes were involved in translational initiation (i.e. RPL24, PABPC1), innate immune response in mucosa (i.e. HIST1H2BK) response to reactive oxygen species (i.e. TXN) and oxidative phosphorylation (i.e. ATP5H) and these five were proven to be hub genes after PPI-module analyses [RPL24 (degree = 16), HIST1H2BK (degree = 15), PABPC1 (degree = 15), TXN (degree = 11) and ATP5H (degree = 11)] and they were overlapped with the target genes of differentially expressed miR-4734 and miR-4521.
     With these results, author suggests that amorphous SiNPs may induce tumorigenesis via regulating miR-4734 and miR-4521 mediated oxidative stress, innate immune response and translational initiation.
     In general, the manuscript is well written. However, data did not support their conclusion. I have some comments that I believe might help the authors in increasing the impact of this manuscript.

Comments
1.	Author should confirm whether amorphous SiNPs regulate above protein expression.
2.	Author should demonstrate whether miR-4734 and miR-4521 directly regulate these genes in this cell lines
3.	There some typos. Collect all.


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: AMORPHOUS SILICA NANOPARTICLES INDUCE TUMORIGENESIS VIA REGULATING ATP5H/SOD1-RELATED OXIDATIVE STRESS, OXIDATIVE PHOSPHORYLATION AND EIF4G2/PABPC1-ASSOCIATED TRANSLATIONAL INITIATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no novelty in the experiment design.
Validity of the findings: I checked the published work by Li et al, 2017, the origin of the raw data. In this paper, the results were somekind opposite to the raw data. Please recheck the data analysis and thus conclusions.
Additional comments: This work was done based on raw data from Gene Expression Omnibus database. The author analyzed the data, and got their conclusion, but without any certification. I think the downstream detection to vertify their conclusion is very necessary. More important to say, the author should recheck their data analysis. For instance, the result of the differently expressed genes was somekind opposite to the published work by Caixia Guo and Yanbo Li, 2017.


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: AMORPHOUS SILICA NANOPARTICLES INDUCE TUMORIGENESIS VIA REGULATING ATP5H/SOD1-RELATED OXIDATIVE STRESS, OXIDATIVE PHOSPHORYLATION AND EIF4G2/PABPC1-ASSOCIATED TRANSLATIONAL INITIATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clearly written.  The numerous abbreviations do make it confusing at times.  The reviewer suggests providing a clear definition for each abbreviation used, even if it appears obvious.

More background if needed from the original manuscript and dataset (Guo et al. Nanotoxicology 2017. PMID: 29164963).  Specifically, what are the differences/benefits of the authors’ analyses as compared to the published primary analyses?  What were the number of differentially expressed genes in both analyses?  Why were certain genes excluded?  What bioinformatics analyses were performed in both?  Compare and contrast the two studies.

The figures are not well labeled.  What are the units for the heat map in Figure 1?  What is the x-axis in Figure 2B?  The interaction networks are not clear.  What do they illustrate and how is it relevant?
Experimental design: The authors present new analyses of a published dataset from the Gene Expression Omnibus database (GSE82062).  This dataset contains triplicate samples profiled on Affymetrix Human Transcriptome Array 2.0 platform.  It appears that the authors used a 4-fold change (|logFC| > 2) instead of a 2-fold change, as originally published, to identify differentially expressed genes.  It is not clear what benefits this provides.

It is important to point out that the transcripts labeled as miRNAs in this dataset are not the mature, functional miRNA form but are the transcribed primary (pri-miRNA) or preprocessed hairpin (pre-miRNA).  As an example, MIR4734, with transcript ID TC17001428.hg.1 contains 30 probes of varying lengths and sequences that recognize the transcripts from chr17:36858515-36858583 (-).  To interrogate miRNA expression, a miRNA-specific platform, such as the Affymetrix GeneChip miRNA v4.

While the authors do provide the packages used for data analysis, the versions are omitted in some cases.  In addition, the reviewer suggests including the R scripts used as a supplement.
Validity of the findings: As presented, it is unclear to the reviewer how these analyses compare to and improve upon the primary manuscript (Guo et al. 2017).

Of note, the published primary analyses (Guo et al. 2017) found 821 differentially expressed genes with 5 upregulated and 816 downregulated.  However, in the analyses presented here, the authors identified 280 upregulated genes and 43 downregulated genes.  Please address this discrepancy.

What is the biological relevance of the protein-protein interaction networks?

As stated in 2. Experimental design, the reviewer is concerned that miRNA-based conclusions are overstated.  Since only the pri-miRNA or pre-miRNA are interrogated, no information is available for the mature, functional miRNA.  Although the mature miRNA may be differentially expressed following silica nanoparticle treatment, this cannot be garnered from the current expression data.
Additional comments: In addition to clarifying those items mentioned in 1. Basic reporting, 2. Experimental design, and 3. Validity of the findings, the reviewers has the following suggestions:

Show all differentially expressed genes in the heat map of Figure 1.  Identify clusters and perform bioinformatics analyses on those genes within each cluster.  A table of these expression data, including replicate expression, averaged expression, fold change, and p-value, would benefit as a supplement.  The currently presented Table 1 only shows logFC and P-values for the top 25 up or down genes (based on p-value).  This is also incorrectly labeled as top 10.

Relatedly, the data in tables 2, 3, and 4 appears to be on a subset of the entire analysis results.  What was the rationale in selecting data for each table?  The reviewer suggests including the complete DAVID results as a supplement.

To improve the current manuscript, the reviewer suggests some bench experiments.  What are the characteristics of normal lung bronchial cells following incubation with silica nanoparticles?  Such phenotypic assays are proliferation, apoptosis, migration, and invasion.  Perform qPCR validation of mature miRNA expression levels for those miRNAs of interested in lung bronchial cells +/- nanoparticles.

The reviewer is curious about the composition of the the SiNPs used.  How do these specific silica nanoparticles compared to the multitude of others being used?  Are they PEGylated or have other modifications or cargo?  More detail on the particles would be appreciated.


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: EMPLOYING TOXIN-ANTITOXIN GENOME MARKERS FOR IDENTIFICATION OF BIFIDOBACTERIUM AND LACTOBACILLUS STRAINS IN HUMAN METAGENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Klimina et al report use of toxin-antitoxin system (TAS) genes in metagenomes as a tool for strain identification in Lactobacillus and Bifidobacterium genera. For this purpose authors created a software program which was compared with existing programs. Using these tools authors report prevalence of different Lactobacillus and Bifidobacterium strains in database available metagenomes from different geographic locations including Russia, USA, Denmark and Japan. They also used this approach to analyze metagenomes from 5 fecal human samples. The results validate the new program and TAS approach as a useful tool for identifying Lactobacillus and Bifidobacterium strains.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: Specific comments
1. The rational of analyzing metagenomes from fecal samples in 5 individuals is not clear. In fact, for the purpose of this study, analysis of database available metagenome data is sufficient. However, whether the authors wanted to validate their in silico analysis with in vitro identification of Lactobacillus and Bifidobacterium strains having the fecal samples would be an advantage. Please clarify the rational of using fecal samples.
2. It is not clear what were the criteria for selection of 4-5 metagenome data from 4 different geographic locations? Were these sex- and age matched? I think that analyzing only 5 individuals are not sufficiently representative to draw the conclusion about the prevalence of different strains of Lactobacillus and Bifidobacterium in different countries. Please state the limitations.
3. Please explain if the TAS method can be used for quantification of Lactobacillus and Bifidobacterium strains in metagenomes and whether the results are depended on the bacterial growth cycle i.e. can be influenced by lack of standardization in fecal sampling procedure.
4. Authors stated that ethical approval and consent to participate were not applicable to this study. What about use of fecal samples from 5 individuals?
5. The paper should be better structured to emphasize the objectives and to clearly present the data.
6. English language should be improved, including rephrasing of several sentences. Ex, line 299-300 sentence can be replaced simply by "country-specific".


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: EMPLOYING TOXIN-ANTITOXIN GENOME MARKERS FOR IDENTIFICATION OF BIFIDOBACTERIUM AND LACTOBACILLUS STRAINS IN HUMAN METAGENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The software tool (TAGMA) was written to allow the users to perform analysis of metagenomic samples with strain resolution. This application might be of interest for a quite broad audience, however a number of issues listed below make the tool difficult and even dangerous to use in its current form:
- Code safety: the current implementation lacks error and exception handling (e.g try-except statements). Unit test should be also included (ideally for every one of the 21 functions implemented), to ensure that the software deals properly with edge cases, non-standard inputs, etc.
- Test data: Please provide a simple example of test data set. Please also provide a test data set when non TA-based markers could be used.
- Dependencies: the TAGMA software tool relies on a number of external dependencies (BLAST, Bowtie. etc), however the repository lacks information on how those dependencies should be installed and configured; how the process should be changed to accommodate Windows and Mac users. Please provide these additional instructions.
- Resources: Please list computational resources required for the tool (RAM, number of CPU). Could it be adopted for use on HPCs? What are the limitations? Which steps of the pipeline can benefit from additional computational resources available? Please also include time estimates for a typical dataset to be analysed.
- Documentation: In the documentation the main program is referred as (‘toximet.py’). However, the repository contains just one python file with a different name (‘bt2_snp_11.py’).
- Documentation: Generally, the documentation is very confusing and should be improved considerably. Currently the GitHub repository contains a README file with some background information on how to use the tool, but poor formatting makes it a struggle to read through: This issue could be solved using appropriate markdown syntax and GitHub Pages.

2. The English language of the paper should be improved to ensure that an international audience can clearly understand the text. The text might benefit from a proof-reading by a native speaker. Some examples, where the language can be changed include Lines 39-41; 59-60; 79-82; 221-222; 225-227; 229-230; 236-237; 242; 250; 267-268; 276-278; 293; 315; 317, etc.

3. The authors mention that they have created a database of toxin and antitoxin genes for Lactobacillus and Bifidobacterium genera (Lines 31-32, 83, 117, etc), however this database has not been made available for the potential readers. It is not completely clear, are the authors referring to a blast database, a list of gene locus tags or is it more than that? In the latter case, the database might be of interest for a broad audience, so some sort of an interface should be provided.
Experimental design: 1. Most importantly, the focus of the manuscript seems to be vague. Currently the manuscript balances between a description of a bioinformatics software tool to perform strain profiling in metagenomic data and a study of Bifidobacteria and Lactobacillus TA II systems occurring in a few human metagenomic data sets. If the software is made the primary focus, the quality of the code and documentation should be improved dramatically, to ensure that the tool receives enough trust to be used by a broader audience. If a study of TA II systems is made a priority, than considerably more data sets, including a wider selection of publicly available human gut metagenomes should be analysed.
2. Provided analysis in the paper considers TAS II systems in two bacterial genera: Bifidobacterium and Lactobacillus. Hence, the title of the manuscript is misleading, in its current form it implies much broader use of the software. If a broader use is indeed plausible, the authors should provide additional evidence that the TAGMA software can be used for other genera of bacteria and archaea. Otherwise, this claim should be reduced and the title of the manuscript - revised.
Validity of the findings: 1. The conclusions the authors are making when comparing TAGMA performance to the existing software tools are not quantitative enough. Some examples include:
Lines 32-34 (‘It was tested on metagenomic samples and shown that it could be used for identification of Lactobacillus and Bifidobacterium species and sometimes for identification of individual strains in the metagenome.)
Lines 324 - 326: (‘TAGMA was compared with existing methods of metagenomic analysis (MetaPhlan, PhymmBL), and at the species level outperformed MetaPhlan2 but proved to be somewhat less sensitive than PhymmBL’)
I think ‘sometimes’ and ‘somewhat’ are not quantitative enough here. Please clearly state limitations of the software along with its sensitivity and specificity.

2. Please also see the comment #1 from the basic reporting section on the code and documentation quality.
Additional comments: In addition, please see more specific comments below:

Abstract:
Background: Please clearly state that the manuscript addresses human/animal type gut microbiomes. Bifidobacteria and Lactobacillus are not the common component of termite gut microbiomes for instance (Lines 16-18).
Results:  I think the ‘results’ part of the abstract lacks sufficient details. It would be great to include types of metagenomic samples, how many of those? Are they publicly available or sequences specifically for this study?
Results: Please add a brief description of what the mentioned programmes are used for (PhymmBL and MetaPhlAn) and their main implementation principles.
Results: Please add a brief description of the principle of marker selection by TAGMA in a nutshell. Currently, it is not clear at all.
Conclusions: Please provide the rationale to focus of RelBE and MazEF superfamilies.
Conclusions: Lines 32-34 (‘It was tested on metagenomic samples and shown that it could be used for identification of Lactobacillus and Bifidobacterium species and sometimes for identification of individual strains in the metagenome.) I think ‘sometimes’ is not quantitative enough here. Please clearly state limitations of the software along with its sensitivity and specificity.

Background:
Lines 47-48: ‘According to their phylum composition gut metagenomes can be classified into three enterotypes [6]. ’ This statement has been challenged recently (Knights et al 2014; Gorvitovskaia et al 2016 ), and the concept of ‘enterotype’ is still not consolidated so this might be worth mentioning.
Lines 51-52: ‘Human microbiota composition has been analyzed mostly at the taxa level or in a lesser degree at the species level’. Species is also a taxon, I think the phrasing is confusing here and should be improved.
Lines 63-65: Is there any evidence that strain variation of Bifidobacterium and Lactobacillus has any impact on human gut microbiome and human health in general?
Lines 82-83: please include the name of the software tool.
Lines 84-85: please include the number of metagenomic data sets analysed.
It would be great to add additional 1-2 sentences to summarise benefits of the TAGMA software tool compared to the existing solutions.

Implementation
Please add a general sentence/paragraph to summarise what the TAGMA software is doing.
Please also list the dependencies and reference installation instructions.
Lines 89-91: not immediately clear how markers are identified and what is a definition of a marker in this specific context. Please rephrase.
Since this section is used to describe the software, there must be a link to the source code.

Methods:
Lines 139-140: please make the referred Python scripts available for the readers.
Lines 187-188: please provide a reference for the Trimming v.0.3 software tool.

Results:
Line 214: please rephrase, ‘slightly less pronounced’ sound very vague.
Lines 229-234: Please clearly state how the TAGMA approach is different from the other mentioned ones.
Lines 240-241: Please include an estimate of gene conservation in the TAS genes. What exactly does it mean (‘not very conservative’);
Lines 252-253: Please include the coverage threshold used to filter out the markers.
Line 264: Please add a reference supporting this statement.

Figures and tables:
Figure 1: Please add the name of the software in the figure legend title.
Figure 1: Please explain what do you mean by ‘valuable positions’.
Figure 3: Please replace ‘Japanese’ with ‘Japan’ in the figure legend.
Table 1: It is not clear what the values in the table mean. Please include additional explanation in the table title or header.

Some additional comments:

Please convert supplementary tables in a csv format to make them easier for readers to reuse and interact with.
GM is used as an abbreviation for gut microbiota too often. Please use ‘gut microbiota’ to improve readability of the text.


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

Input:
Title: EMPLOYING TOXIN-ANTITOXIN GENOME MARKERS FOR IDENTIFICATION OF BIFIDOBACTERIUM AND LACTOBACILLUS STRAINS IN HUMAN METAGENOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: - Please proofread the manuscript and the documentation, English language could still be improved, for some examples please see general comments.

- Lines 206-207: ‘Primary investigation showed that genes of these families are found in all studied Lactobacillus and Bifidobacterium species.’ Please provide evidence or reference for this statement.

- Lines 140-141:
Please add metadata for the files containing toxin and antitoxin genes to the github README. Metadata should contain brief description on how those were generated. Also, in this case, since these are just several small fasta files, the term ‘database’ might be misleading,  I would recommend to change the term to ‘list of genes’, ‘collection of genes’ or something similar.

- Lines 150-151: It is not clear to which exactly python scripts authors are referring here, is it the main ‘toximet.py’ script or something else?
Experimental design: -
Validity of the findings: -
Additional comments: 1). Abstract:
Line 16: ‘Recent research has indicated that each individual may have a unique genotype.’
The statement is too broad, reflects common knowledge which is not so recent. This sentence is also disjoint from the following one. I would recommend starting directly with microbiomes.
Line 21: please spell out ‘TA’, this is the first mention.
Lines 21-23: ‘The objective of this work was to create a software based on polymorphism in TA genes to analyze representation of species and strains of Bifidobacterium and Lactobacillus in human gut microbiota.’
I think emphasis might be wrong here, the main objective should still be solving a defined biological problem, writing a software is just a solution. Please clearly explain the problem and state the rationale of writing the software (are there similar solutions, why they are not suited for certain applications, etc).
Lines 25-26: ‘We created database of toxins and antitoxins genes of these superfamily specific for lactobacilli and bifidobacteria’
There might be a word missing in this sentence.
Lines 29-30:  ‘Based on a limited number of well selected markers TAGMA displays good time performance as compared with results of popular programs PhymmBL and MetaPhlAn.’
Please explain what is ‘well-selected marker’ and add quantitative estimation of ‘good time performance as compared with results of popular programs PhymmBL and MetaPhlAn.’

2) Background:
Line 35: there is a space missing between ‘then’ and ‘1000’	. Also, please replace ‘then’ with ‘than’.
Lines 40-41: please revise grammar.
Line 50: please add references for MetaPhlAn or MG-RAST software.
Line 51: ‘very deep metagenome sequencing’. How deep?
Line 60: Either replace ‘species specific’ with ‘species-specific’ or drop ‘species’.
Line 64: reference is missing.
Lines 82-84: please see comment #3 to the abstract.
Line 89: please provide more background information on ‘in-house’	samples.
Line 90: please briefly describe the results of the mentioned comparison.

3) Methods:
Line 93: please drop ‘basically’;
Line 94: please replace ‘inhouse’ with  ‘in-house’;
Line 98: please replace ‘genes of TAS’ with ‘TAS genes’.
Line 137: please clarify what ‘translates’ mean in this context.
Lines 150-151: It is not clear to which exactly python scripts authors are referring here, is it the main ‘toximet.py’ script or something else?
Line 163: please check the spelling.

4) Results:
Lines 219-220: Please revise grammar.
Lines 222-223: Please use italics for species names.
Line 244: Please explain/ rephrase  ‘good strain marker set’;
Line 249: Please revise grammar.
Line 258: Please revise grammar.
Lies 260-261: ‘The entire pipeline is called TAGMA (Toxin Antitoxin Genes for Metagenomes Analyses) and is available at [https://github.com/LabGenMO/TAGMA].’
Please rename the main python script (‘toximet.py’ to ‘tagma.py’) for consistency or explain the reasons to keep a separate name.
Line 273: Please check the spelling.
Line 279: ‘Lactobacillus genus was represented in a smaller amount’ Please, rephrase.

5) Discussion:
Line 309: please check grammar, typos and misspelled words;
Lines 310-312: please explained what  ‘prolonged maintaining of strains’ means in this context.
Lines 328: Please rephrase ‘pretty large set’


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: EMPLOYING TOXIN-ANTITOXIN GENOME MARKERS FOR IDENTIFICATION OF BIFIDOBACTERIUM AND LACTOBACILLUS STRAINS IN HUMAN METAGENOMES
Review round: 3
Reviewer: 1

Output:
Basic reporting: see general comments
Experimental design: see general comments
Validity of the findings: see general comments
Additional comments: The manuscript has been substantially revised and improved, I have few minor comments.
1. Abstract, 1st sentence is too general, start from “Each individual has a unique microbiota composition emerging from both…
2. l. 20, microbiota is not always…
3. l. 31, strains
4. l. 23, 25, 27, toxin-antitoxin system is abbreviated here as TA while in the following manuscript as TAS, please be consistent.
5. l73-75, delete “individual” at 1st place, what is “individual strain preference” is not clear preference for what?
6. l. 78 what disease?
7.  l. 83, spell out TAGMA at 1st mentioning
8. l. 89, well-selected
9. l. 274, ‘non-shown data’


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: EMPLOYING TOXIN-ANTITOXIN GENOME MARKERS FOR IDENTIFICATION OF BIFIDOBACTERIUM AND LACTOBACILLUS STRAINS IN HUMAN METAGENOMES
Review round: 3
Reviewer: 2

Output:
Basic reporting: I have no further comments.
Experimental design: I have no further comments.
Validity of the findings: I have no further comments.
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: PCR EFFECTS OF MELTING TEMPERATURE ADJUSTMENT OF INDIVIDUAL PRIMERS IN DEGENERATE PRIMER POOLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Raw data are present and available in the SRA as specified.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: The authors hypothesized that variable melting temperatures within a pool of degenerate primers could lead to un-noticed PCR biases. To test this, they adjusted the melting temperature of each possible primer sequence from a degenerate pool by truncating the end of the primer sequence. The authors also suggested that their primer design could be used in real sequencing studies to increase basepair diversity, by introducing offsets into the 16S rRNA gene amplicons.

I appreciated the main premise of the paper, as I’ve thought a lot about 16S rRNA gene primer biases but never really considered the possible effects of melting temperature from the highly degenerate primers currently in use. It’s reassuring, if not terribly exciting, that the authors did not find a substantial effect, and it would be beneficial for the field to know that this isn’t a cause for major concern.

I’m not personally sold on the utility or appropriateness of the truncated primer set for actual sequencing studies – if I were going to go that route, I’d opt for the Lundberg et al. approach of adding nucleotides to the linker region that the authors discuss. I don’t feel that this manuscript sufficiently tests the effects of the truncated primers on environmental samples to put this forth as a viable option (compare with the level of verification needed by Apprill et al. and Parada et al. for context), and I’d encourage the authors to downplay or outright drop this aspect of the manuscript.

There are a number of aspects of this manuscript that would benefit from clarification, specified below.

One major concern: are your samples rarefied or randomly subsampled to an equal number of sequences? If not, the results need to be reanalyzed with that correction applied. Varying sequence numbers between samples are common but confounding with Illumina sequencing. Further, as far as I can tell, the authors might be working with absolute sequence abundance (based on Tables S6 and S7), which is highly problematic if the authors haven’t rarefied their sequences. Essentially every environmental microbiology paper in the last decade has worked with relative abundance of OTUs because we don’t trust the sequencers to be absolutely quantitative.

 Specific comments

Lines 27-28: As noted above, I would discourage this line of discussion in general. But if you want to keep it, rephrase this bit to something like “with minimal addition of exogenous templates.” I read this and was immediately confused about how you’d manage cluster generation, error assessment, etc., without the PhiX spike that the sequencer uses to calculate these – realized in the manuscript proper you’re suggesting less rather than no PhiX, but this reads like the latter. Also Line 70.

Line 75: I don’t feel Caporaso et al. is a useful citation here, as they used the older rather than newer primers, and you didn’t use the EMP barcoding scheme.

Line 120: Please clarify here how the were libraries divided between the two sequencers. Because this seems like a pretty significant confounding factor for a study that’s this focused on the nitty-gritty of sequence analysis. I know it’s in one of the SI tables but you can head off a lot of concern by providing more detail here.

Line 172: Was there a target range of Tm similarity (e.g., within 2 degrees)? Was this the most you could adjust the Tm while meeting the requirements outlined in Lines 164-165 (in which case, help yourselves out and specify that)? Otherwise it doesn’t seem like a very large adjustment in overall range.

Lines 218-220: I don’t feel this is a major caveat – many of the users of the EMP primers purchase individually barcoded primers anyway, as this is the approach the EMP recommends. Your method will still be less expensive than that.

Line 237: To me, the fold-changes reported in Table S6 are at least as interesting as the MDS plots in Figure 3, and I’d like to see them in the manuscript proper. A column chart of the fold-change in relative abundance for select OTUs from standard EMP to shortEMP would be a nice, simple addition.

Line 247: I care less about the within-primer set repeatability and would rather see the Bray-Curtis similarity between primer sets reported.

Lines 241- 261: This section is confusing. It sounds as though you selected an annealing temp and moved on, but then in Line 253 you’re back to talking about the effects of annealing temp. Can you better separate the results of the annealing temp test series and the tests conducted at the optimal annealing temp in this part of the results?

Lines 260 – 261: I don’t buy that, community composition is vastly more important than alpha diversity when it comes to assessing complex microbial communities. If you just want to say that it made sense to continue with your study, fine.

Line 271: What advantage does the ideal score or expect score (be consistent with the terminology) offer over a fold-change or percent difference value that the reader could understand without having to look up a reference to try to figure out the math?

Line 313: And 3’. (Which is potentially more problematic, don’t omit mentioning it.)

Line 318-320: This is a nice hypothesis. This manuscript doesn’t test it. You don’t talk at all about, for example, whether the shortEMP primers pick up novel taxa relative to the EMP primers in the environmental samples.

Line 330: Lake Michigan isn’t a marine system.

Figure 1a: Label 5’ and 3’ ends to make it easier to understand.

Table 1: You should specify somewhere what the ANOVAs are testing (I can guess but can’t verify if I’m guessing correctly).

Figure S1: This is problematic. As I understand it from the legend, the significance test is for within-group variability, but the way it’s presented on the figure looks like it’s testing the median Bray-Curtis Dissimilarity between the two primer sets and finding them to be highly significantly different. Please revise.

Tables S6 and S7: Means of what? I can’t find this in the text or the table.


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

Input:
Title: PCR EFFECTS OF MELTING TEMPERATURE ADJUSTMENT OF INDIVIDUAL PRIMERS IN DEGENERATE PRIMER POOLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see review below.
Experimental design: Please see review below.
Validity of the findings: Please see review below.
Additional comments: The manuscript by Naqib et al. aims to characterize the influence of differential annealing temperatures of common primers of 16S rRNA gene studies.  The research question, while very technical in nature, is an important question since the applicability, if significant, is extremely far reaching since it such a common tool of microbiology and microbial ecology. The paper argues that an overlooked aspect of such studies is the variability of the annealing temperature required for the different variants of the degenerate PCR primers. The researchers experimentally  examine the importance of this variability by trimming the primers of, theoretically, mostly inconsequential bases (in terms of which taxa are targeted). Additionally, they present an idea for how to increase the sequence base calling diversity to improve sequence quality in important positions.

Ultimately the annealing temperature analysis suggests that these modifications did not make a substantial difference in the sequencing output. Therefore, I think that the results will be interesting but not fully adopted, since many labs will not modify their approach a small gain; though it is ultimately ambiguous which approach is “better” , as suggested by the abstract. In fact, the results of a mock community analysis shows that the modifications perhaps made the PCRs less accurate. I find all of the approaches to be of satisfactory technical rigor, but I have some suggestions.

1.)	The authors should consider how the predicted annealing temperatures influences which taxa are over or under represented in the mock communities relative to the input. E.g., are those with lower annealing temperatures amplified more, or vice versa, or no relationship. Such analyses would get at the mechanism and allow the highly technical study to be applied and understood more broadly.

2.)	The authors should provide data that shows that PhiX is actually needed to increase diversity on state-of-the-art MiSeq/HiSeq runs (i.e., 2018 software, hardware, chemistry). Is a full 20% PhiX still commonly used? I thought this problem was not currently as severe as the authors state, and they do not provide a “control” to show that the sequencing quality is poor when the steps they take are not implemented and/or PhiX is not used.

3.)	It appears that all the annealing temperature calculations are for just the 16S primers, but in fact the initial amplifications are done with Illumina sequencing primers attached to the 16S specific primer. How do the common sequences that are added to the 16S primers change their annealing temperatures? It seems that they would influence strongly but I don’t think that this is mentioned. It might be argued that the annealing temperature of the 3’ end of the primers (i.e, the 16S specific portion) is what matters for amplification. With that logic, then the 3’ end of the 16S-specific primer is also probably more relevant that the 5’ end of the 16S-specifc primer. How does that vary between the primers. Can a moving average of the annealing temperature of the primers be calculated? If so, it might help understand the mechanisms for the differences that are observed.

4.)	It would be great if the authors double checked the first time the respective primers were used in the literature. For 515F, this should be cited: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-38. And for historical purposes for the 515F, might also consider citing https://www.pnas.org/content/pnas/82/20/6955.full.pdf and making a historical note (use of an only slightly different 515F in the mid-1980s).


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: PCR EFFECTS OF MELTING TEMPERATURE ADJUSTMENT OF INDIVIDUAL PRIMERS IN DEGENERATE PRIMER POOLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: They have suitably responded to my review.
Experimental design: They have suitably responded to my review.
Validity of the findings: They have suitably responded to my review.
Additional comments: The authors have suitably responded to my original review.