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

Input:
Title: GENOME-WIDE IDENTIFICATION OF NEW REFERENCE GENES FOR RT-QPCR NORMALIZATION IN CGMMV-INFECTED LAGENARIA SICERARIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors use clear and professional language throughout the paper.
The structure is suitable as well as the figures and other information available.
Experimental design: Relevant work to ensure the robustness and accuracy of future gene expression studies

Methods described with sufficient detail and information.
Anyway, some consideration should be taken into account:

Line 121 to 127 -once RNA extraction is described these paragraphs should be reformulated and included in the point: “RNA and first strand cDNA preparation”

Line 129- “RNA sequencing”: although the authors have used the RNA-seq data for the selection of candidate genes, this point is beyond the scope and objectives of this article. Should be withdrawn.

Line 177 – 10mM/ml should be replaced by 10mM
Validity of the findings: Line 284 to 286- “…stability of the 18 and 18 candidate…” should be replaced by:”…stability of the 18 candidate….”

Line 429- to 431 (legend Fig 2)- “As shown in the figure [….] infection” this is a result/conclusion, therefore should be included in the text and not in the legend.
Additional comments: No comment


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF NEW REFERENCE GENES FOR RT-QPCR NORMALIZATION IN CGMMV-INFECTED LAGENARIA SICERARIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written in an unambiguous and technically correct style. The Authours used a professional English language.
Introduction is well-structured. The background of your study provide context to the information discussed throughout this paper. Although the background information include both important and relevant studies, I suggest to read and add in the introduction this recent reference: Wamg et al. GourdBase: a genome-centered multi-omics database for the bottle gourd (Lagenaria siceraria), an economically important cucurbit crop. Scientific Reportsvolume 8, Article number: 3604 (2018).
The structure of the paper is conform to format of ‘standard sections’.
Figure and tables should be improved. The Authors should decide if reported the same value in graphs or table, in this way are redundant.

In the paper all results are relevant to the hypothesis and are not sufficiently argumented by discussions
Experimental design: This paper matches within the Aims & Scope of the journal. This research is relevant and meaningful to improve the knowledge in the plant-pathogen intercation (Lagenaria-CGMMV).
This is the first study that evaluated the candidate reference genes in this pathosystem.
M&M are well-described and structured, with high technical standard. All methods are reproducible by another investigator. Some aspects should be clarify (see comments of Authors)
Validity of the findings: The paper contains some interesting and useful data, and, thus, yields new information in this research area. Data are robust but there are some problems in the stastistical analysis.
Discussions and conclusions should be improved.
Additional comments: The manuscript “Genome-wide identification of new reference genes for qRT-PCR normalization in CGMMV-infected Lagenaria siceraria” by Zhang et al. represents a comparison and choosing of reference genes. This is the first study in which a set of candidate RGs was analyzed in terms of their expression stability in bottle gourd infected by cucumber green mottle mosaic virus.
The author used three different statistical algorithms to compare these reference genes and obtained the most stable genes. In my opinion, all data are reliable and sufficient. Furthermore, the Authors evaluated the expression analysis of two target genes, chosen for RGs validation, involved in adenylate isopentenyltransferase (IPT) and DNA-direct RNA polymerase subunit (DdRP).
The data obtained and discussed are useful and provide novel insights into this area of applied research.
However, some parts of the manuscript need some improvements before the publication.
In particular:
Title
The authors should change “qRT-PCR” into “RT-qPCR” and check in whole paper.

Abstract
L30: RGs are not preferred, but they are the most stable identified in this study.

Introduction
L40: The Authors should improve this sentence. Lagenaria siceraria is a specie belongs to Cucurbitaceae family.
L43 Change “utilitarian” into “horticultural”
L97: Correct the typing error
L100: RefFinder is an online available tool that integrates some algorithms. On the basis, of your data (table 2 and table 2) we cannot assume 100% efficiency for all genes and RefFinder outputs may be biased because PCR efficiencies are not taken into account. I suggest to delete this analysis.

M&M
The Authors should improve the description of the number of RGs obtained by literature and RNA-Seq and explain this in the text and in the captions of figures and tables.

L121-124: The authors should report these informations in the “RNA and first strand cDNA preparation” section
L124: Add this method “microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer”. Check the brand of Bioanalyzer.
L127: Add the reference.
L125: The authors should add some information on the extraction of CGMMV by infected plant.
L140-141: The Authors should improve this sentence and delete “could”.
L162: In this section the Authors should add the method used to obtain PCR efficiency (E) for each primer pair and regression coefficient (R) as suggested by the guidelines “Minimum Information for Publicationof Quantitative Real-Time PCR Experiments (MIQE)” (Bustin et al. 2009)
L182: The authors should indicate quantification cycle as “Cq”, according to the RDML (Real-Time PCR Data Markup Language) data standard (http://www.rdml.org), and reported by the guidelines “Minimum Information for Publicationof Quantitative Real-Time PCR Experiments (MIQE)” (Bustin et al. 2009).
L187: Delete RefFinder
L193 and L202: Correct the typing error
L209: The Authors should improve this section, because some RGs are reported in Table 1 and other in table 2.
L217: The name reported for some genes are not in agreement to Table 1
L220: Check the number of RGs, in Table 2 there are 20 RGs.

L227 (Fig.S2): Negative control should be add in each experiment, as recommended by MIQE guidelines. In Fig. S2 the negative control are not indicated in the melting curve.
L229 (Fig. S3): The Authors should improve the text at the bottom of the panel A.
L233: Delete R2, the values of single RG are reported in the tables.
L285: The Authors should improve this sentence
L300-305: Delete these sentences.
L312-314. Combined use of geNorm, NormFinder and BestKeeper to select and validate the best RGs generated substantial discrepancies in the final ranking due to different mathematical models associated with each algorithm, such as demonstrated in previous studies. To overcome differences in the ranking of RGs, how did the Authors obtain a final ranking? Explain the used method in Materials and Methods.
L313-314: The authors should choose the number of RGs on the basis of the results obtained by GeNorm (L271-272; L290)

Discussion
L328-338: Add some references.
L348-374: The discussion should completely rewritten on the basis of correct interpretation of their interesting results.

Conclusion
The conclusion should completely rewritten on the basis of correct interpretation of their interesting results.

Table 1: Check the name of gene and add the reference for each RGs.
Table 2: Check the name of RGs
Figure 3: The Authors should add the means of “M”
Table 3: The Authors reported in this table the values indicated in Fig. 3A and 4A, respectively. In Fig. 4A some values are negative, check these.
Table 4: The Authors reported in this table the values indicated in Fig. 3C,3E and 4B, 4C, respectively. In Fig. 4B and 4C some values are negative, check these.
The Authors should decide if reported the same value in graphs or table, in this way are redundant.

Reference: Check the reference in the manuscript


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF NEW REFERENCE GENES FOR RT-QPCR NORMALIZATION IN CGMMV-INFECTED LAGENARIA SICERARIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Zhang et al. brings an important contribution for the genetics and breeding of cucurbitaceae. The species has not only economical potential per se, but also as a rootstock source for watermelon and other cucurbit crops. The use of appropriate reference genes is of paramount importance for the normalization of gene expression and the proper identification of differentially expressed genes in transcriptomic analyses.
Experimental design: The experimental design used by the authors is correct and the tools (software) used to evaluate/validate the reference genes are the current used by the scientific community. The authors were successful in identifying reference genes for leaves ( LsWD, LsGAPDH and Ls H3) and fruits (LsH3, LsGAPDH, Ls P4H and Ls CYP) of Lagenaria siceraria. The use of GeNorm and NormFinder, as well as Bestkeeper and Reffinder was appropriate, allowing the authors to validade and demonstrate the quality of their results. Vn/Vn+1 checks were used to indicate that the group of RG candidates was adequate.
Validity of the findings: The findings are relevant, since they can provide tools for geneticists/breeders working on this crop for a better assessment of genes involved in the response to CGMMV. The correct identification of genes responding to infections can expedite the process of identifying resistance genes and transfer these to commercial genotypes.
Additional comments: Clear, well written manuscript and elegantly presented. The objectives were fulfilled.


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF NEW REFERENCE GENES FOR RT-QPCR NORMALIZATION IN CGMMV-INFECTED LAGENARIA SICERARIA
Review round: 1
Reviewer: 4

Output:
Basic reporting: I think that this manuscript was informative, systematic, logical and comparatively well written although several typos to be corrected stood out in the manuscript.
Experimental design: Each experiment was generally well organized to achieve the objectives. However, I can’t hardly point out my concerns as below.
1.	Do the authors believe that the newly developed RGs are applicable to other bottle gourd genotypes. I would like to hear the authors’ logic explanation on this issue in addition to the genotypic situation (an inbred line, F1 hybrid, local variety with heterozygosity/heterogeneity, and so on) of the accession ‘Hangzhou Gourd.’
2.	Why didn’t the authors clarify the race or isolate name of CGMMV used as the inoculum. Similarly with the upper question, why did the authors exclude an unstable and/or altered expression pattern of the new RGs depending on races?
Validity of the findings: Collectively, the authors newly suggested the stably expressed reference genes for qRT-PCR normalization in the bottle gourd plants infected by CGMMV based on the results obtained from various approaches. They might be worthy of notice for the genomic studies of the bottle gourd with a long history. However, it is a flaw that the novel RGs have limited availability, that is, restrictively in CGMMV-infected condition.
Additional comments: 3.	The authors should show the pictures of leaves and fruits with typical symptoms caused by CGMMV, merged with Fig. S1.
4.	What are the methods for cultivation and management of bottle gourd performed according to Li et al. (2016). Li et al. (2016) described those very briefly in their article. Please provide enough details, which set readers at ease from apprehension regarding any biotic/abiotic stresses.
5.	Please mention when the fruits sampled, and the total RNAs were extracted from which part of the fruits.
6.	I recommend that the authors put forward evidence to support that the bottle gourd plants were not contaminated by other viruses such as WMV and ZYMV, which can easily infect bottle gourd plants as well.


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

Input:
Title: GENOME-WIDE IDENTIFICATION OF NEW REFERENCE GENES FOR RT-QPCR NORMALIZATION IN CGMMV-INFECTED LAGENARIA SICERARIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written in an unambiguous and technically correct style. The Authours used a professional English language. Introduction is well-structured and a new reference has been added as suggested my previous revision. The structure of the paper is conform to format of ‘standard sections’. Figures and tables have been improved. In the paper all results are relevant to the hypothesis and are sufficiently argumented by discussions
Experimental design: New information have been added
Validity of the findings: The paper contains some interesting and useful data, and, thus, yields new information in this research area. Data are robust and some information in stastistical analysis has been provided.
Discussions and conclusions have been improved.
Additional comments: The Authors reported in the revised manuscript, all suggestions recommended by reviewers. Revised paper has been improved and discussion and conclusion have been rewritten on the basis of their obtained results.


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

Input:
Title: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The reporting would benefit from some changes.  The terminology could use some work.  For example, ‘mortality’ is often used where ‘fatality’ would be more appropriate, as fatality refers to a death event whereas mortality refers to a rate, e.g., deaths per 100,000.  The term ‘mortality rate’ is redundant.

‘The number of bears killed by people for all other reasons [than hunting]’ might be more concisely represented by ‘the number of non-hunter-killed bears.’  Basically the study is about bears killed either by hunters or non-hunters.

Specific editing comments

Line 3		I suggest breaking the first sentence into two sentences, the first ending with conservation.  I suggest merging the next sentence by replacing the period with a comma and the ‘however’ with a ‘but’.

Line 8		Replace ‘for’ with ‘including’.

Line 9		Delete the first ‘for’.

Line 15	Add comma after ‘office’.

Line 20	Delete hanging parenthesis.

Line 39	The reasons for non-hunter fatalities ought to be summarized in the Introduction.

Line 114	Table 1 identifies the number of female bears as 37.  However, I suggest skipping all of the analysis of gender differences in reporting of bear fatalities.

Line 118	Here is a good example of why it would help to include a table summarizing the circumstances of fatalities assigned to the non-hunter category.  This bear’s collar was attached to bottles and tossed into the river, probably bringing some laughs to whoever did it.  But how does this act of research vandalism, by itself, support the determination that the bear was killed by the non-hunting public?  What if the vandal(s) found the bear dead already, either killed by a hunter or by natural causes?  Left as is, the assignment of the bear to the non-hunter fatality category seems like a leap to guilt.  There must be an additional reason for the category assignment.

Line 122	This entire paragraph and much of the next paragraph can be deleted without loss to the main result of the study.

Line 141	This paragraph is discussion material, so belongs in the Discussion section.  I also suggest deleting all discussion about sex differences in reporting rates.

Line 164	Yes, the sample size is small. For this reason, I suggest revising the following clause by adding a statement of uncertainty such as ‘might indicate.’

Line 169	 Replace included with include.

Line 174	The second reason seems the same as the first.

Line 175	The sentence beginning with ‘In addition’ lost me.  I suggest rewriting it.

Line 179	Is there any basis for this speculation about more male bears being killed in hunter camps?

Line 192	Why would a hunter report not shooting a bear because it was collared?

Line 194	This paragraph includes multiple conclusions that are unfounded or over-confident.  Some of the statements could use citations, and some could use more caution.

Line 204	The citation does not appear in the list of references.

Line 207	I suggest deleting the sentence on the time period beginning in 1995.

Line 208	I could not understand the sentence about managers and researchers knowing something by far…

Line 211	‘…nature of human-caused mortalities…’ seems vague.  It would help to be more specific.

Check the referencing.  McLellan 1998 is cited on line 21, but is not listed in references.  The same for Servheen. Check all of them.
Experimental design: Grizzly bears participating in a telemetry study that was begun in 1979 are used in this paper as a training data set to obtain what are assumed to be the true proportions of bears killed by hunters versus non-hunters.  The ratio of hunter to non-hunter fatalities in the training data is then used to adjust an agency data base of bear fatalities for the proportion of non-hunter deaths that were unreported.  This is an approach often used to adjust fatality estimates for the proportion of fatalities not found during fatality monitoring where human activities caused wildlife deaths.  The number of detected fatalities F is divided by a fatality detection rate D informed by trials imposed on the searchers who, ideally, are blind to the trials.  The essential form of the adjustment, F ̂=F/D  , has its origin in Horvitz and Thompson (1952), but multiple revisions or additions have since been made to their estimator to suit specific challenges (Korner-Nievergelt et al. 2011).  Applied to the bear study, F is the number of bears reported killed by hunters, D is the ratio of hunter to non-hunter killed bears in the training data, i.e., the telemetered bears, and F ̂ is the estimated number of bears killed by humans who were not hunting.

Before continuing, I should interject three points.  One is that the paper needs to simplify the analysis by eliminating comparisons of outcomes by gender.  The case for gender differences in reported fatality rates is weak, where percentage differences really represent small numerical differences in bears of one gender or the other.  Second, whether to include suspected causes of death should be decided, and either the results should include or exclude fatalities of suspected cause.  I suggest using only bears of known cause of death, but I doubt that it makes much difference either way.  Third, the study results should include only one time period, which is the period over which bears were captured for the telemetry study.  Breaking out results over a more recent time period is justified by an assumed greater reporting accuracy in recent years, but no evidence is provided in support of this assumption.  Why would reporting be less accurate during 1980-1995 than during 1995-2015?  Using the more recent time period yields a greater adjustment to the number of bears killed by non-hunting humans, but at the cost of relying on an even smaller sample size.  I suggest using only the study period 1980-2015.  In summary, the paper would be stronger by restricting its focus to those bears reported as fatalities caused by hunters and non-hunters and with and without telemetry units, i.e., only four numbers.  These four numbers would be 10 collared bears killed by hunters, 9 collared bears killed by non-hunters, 70 uncollared bears killed by hunters, and 10 uncollared bears killed by non-hunters.

The Horvitz-Thompson estimator, or any of its analytical descendants, can be highly sensitive to the effects of bias and error.  A relatively small change in D can greatly affect the adjusted number of fatalities.  If only one fatality was found or reported, whereas 50% of the training data were found or reported, then 1 ÷ 0.5 yields 2.  But if D = 0.1, then the adjusted fatalities increases to 10 (1 ÷ 0.1), and if D = 0.01, then the adjusted fatalities increases to 100 (1 ÷ 0.01).  The outcome of the fatality reporting of telemetered bears makes a huge difference to the estimated number of bears killed by non-hunters.  Therefore, the training data set used to derive D must be reliable.

The reliability of the training data in the bear study bears scrutiny for several reasons.  D is a ratio unaccompanied by an error term.  The ratio that was reported for the training data was 1.11 (10 ÷ 9), which would yield an adjusted number of non-hunter fatalities of 63.  However, D was based on a small sample size, meaning that a shift of one fatality from the hunter to non-hunter category, or vice versa, would change D to either 0.90 or 1.375 depending on the direction of the shift.  Applying these outcomes to 70 reported hunter deaths, the adjusted non-hunter fatalities could be either 51 or 78 bears killed by non-hunting humans, or 19% lower or 25% higher than the reported number of 63.  A shift of only 2 fatalities to the other category would change D to 0.73 or 1.71 depending on the direction of the shift.  Applying these outcomes to 70 reported hunter deaths, the adjusted non-hunter fatalities could be either 41 or 96 bears killed by non-hunting humans, or 35% lower or 52% higher than the reported estimate of 63.  The paper should include an assessment of uncertainty of the study result due to small sample size.

References Cited

Horvitz, D. G. and D. J. Thompson. 1952. A generalization of sampling without replacement from a finite universe. Journal of American Statistical Association 47:663–685.

Hurlbert S. H.  1984.  Pseudoreplication and the design of ecological field experiments. Ecological Monographs 54:187-211.

Korner-Nievergelt, F., P. Korner-Nievergelt, O. Behr, I. Niermann, R. Brinkmann, and B. Hellriegel. 2011. A new method to determine bird and bat fatality at wind energy turbines from carcass searches. Wildlife Biology 17:350–363.
Validity of the findings: Another reason to scrutinize the reliability of the training data goes to the assumption that the ratio of hunter to non-hunter fatalities of telemetered bears represents the same ratio applied to non-telemetered bears.  The paper implies that this assumption is valid because the telemetry units were small and difficult to see from a distance (line 107).  However, this assumption could be flawed for two reasons that were not discussed in the paper.  First, hunters using scoped rifles were likely to notice the telemetry units.  If only one bear was spared by a hunter who noticed the telemetry, and had that bear later died as a result of an encounter with a non-hunter (train, car, or self-defense), then the adjusted fatalities due to non-hunting human causes would have shifted from 51 to 63.  Had two hunters made this decision and both bears later died due to non-hunting human causes, the shift would have been from 41 to 63.  Secondly, the assumption might be flawed if telemetered bears shift their behaviors as a result of capture and handling.  Alternatively, telemetered bears might have been captured because their behaviors differed from non-captured bears, making them easier to capture.  For fatality rates of telemetered bears to represent those of non-telemetered bears, the telemetered bears would have had to have been a random sample from the bear population and their behavior unaltered as a result of capture and handling.

Lack of experimental design adds another reason for interpreting the result cautiously.  Even a mensurative study can benefit from the basic tenets of experimental design such as replication and interspersion of treatments, use of a control treatment, and appropriate spatial and temporal scales (Hurlbert 1984).  As a wildlife ecologist I understand that studies of species such as grizzly bear rarely allow for the implementation of experimental design tenets.  In the case of this study, the treatments of hunters and non-hunter killers of bears were mixed but not interspersed in the same study area, and there was no replication and no control treatment (sizable areas with no hunting allowed).  Although experimental design tenets are understandably absent or at best weak due to the nature of the animal, the study result should be interpreted cautiously.  I suggest that the paper present the under-reporting of non-hunter-killed bears as a possibility – a possibility that warrants focused research on the question.

Even in raising the possibility of under-reporting of non-hunter killed bears, I suggest that it would help to add more details about the bears reported killed this way.  The paper lists the types of causes of death other than hunting, but because the under-reporting of non-hunter deaths is central to the paper, I suggest adding a table that summarizes the circumstances associated with each bear assigned to this category.  How many were killed in self-defense?  How many were killed by cars?  It would help to report whether any of the bears assigned to the non-hunter fatality category carried bullets or showed other evidence of wounding by hunters.  Confounding factors affecting only one or two bears in this study can greatly change the study’s outcome.
Additional comments: I appreciate the opportunity to review this paper, which presents an interesting and important possibility that grizzly bear fatalities caused by non-hunting humans is under-reported.  I believe that the essential conclusion of the paper is likely correct, but I also believe that the evidence leading to the conclusion is not yet entirely convincing.  The approach of using a training data set of grizzly bear fatalities to inform the estimate of non-hunter killed bears in the larger population has real potential, and raises interesting questions and challenges related to ensuring the estimate is accurate.


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: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract "Thus about 12% of the human-caused but non-hunter kills were reported. Between 1995 and 2015, the rate was 21%, suggesting an improvement in reporting rates" These 2 statements are both unclear.
"The study area may have low reporting rates because it is >40 km of gravel road from a Conservation Officer office so reporting is difficult and there are no residences so there is little concern of a neighbor contacting an officer." Minor issue but begs the question of whether reports can be called in or mailed in later or something.
"Across British Columbia (BC), McLellan et al (2017) found the death of almost twice as many males as females of each cohort ended up reported."
This statement is not clear enough. As with the abstract, the authors should clarify . Also this sounds a bit like they are publishing the same data again?
The literature review in the Introduction is inadequate (also see below for assumption made without considering the counter-evidence). Given the authors' goal is to understand unreported mortalities and the disappearances of female brown bears, I would have expected a thorough review of recent literature addressing cryptic poaching (Liberg), undocumented mortalities (Treves), differential risk (Borg, Adams, Schmidt) in wolves, but none of this work was cited. Indeed the introduction reads as if the authors are almost the only ones studying this issue. This is a weakness that rises up again in their assumption ()see below) and surfaces in the abstract (see above), and may undermine the methods.
Experimental design: "Assuming the rates and causes of death of collared  bears are the same as uncollared bears, as has been done to estimate survival rates and population  trends,…" Despite providing 7 citations, I am troubled by this assumption and the authors should be also. The assumption was shown to be unreliable for Alaskan wolves and for wolves in the lower 48 (citations above). The reasons for unreliability of the assumption is that collaring did not happen randomly but some individuals were more likely to be collared. Furthermore it appeared that in some cases collard and uncollated animals faced differential mortality risks and rates. I am concerned the authors are ignoring a rather substantial literature beyond grizzlies that has shed light on the central problem they are investigating. Also see above.
The descriptions of hunters and recreationalists in the study area deserve a citation to evidence.
I think it would be clearer throughout if the authors use "permitted" or "licensed" for the legal killing of bears by a permitted hunter rather than "hunted" which was ambiguous in several places as it could be interpreted as a hunter with a permit for ungulates shot a bear illegally. Line 124 is a perfect example of the potential confusion.
"When collared bears were killed for other reasons, they too were often reported. If they were not, then we reported the deaths to the Conservation Officers." This needs clarification. Who is the subject doing the reporting in the first sentence? "often" is a value judgment unless data are presented. How did the authors know if a collared bear's death had not been reported? I can guess but slightly too much left to the imagination of the reader here. Then this sentence either answers my concern with 'often' or muddies the water: "Most collared bears (95%) that were known to have been killed by people for any reason are in the CI database." Does this 95% include those legally hunted and the legal government control? If so it is misleading coming on the heels of sentences pertaining to other causes. I would separate such reporting (truly mandatory) from the other types of mortality that are not so clearly mandatory, i.e., would a driver know to report a collision with a collared bear? And by the way these look like results so I suggest reorganizing.
Lines 103-110: Several problems here. First the authors gloss over the possibility (probability) of bears with non-functional collars out there. What did they do with those? Recent work cited above addresses this issue as a bias to precisely the type of analysis the authors are conducting here, so it cannot be ignored. The second issue is the assumption. While it is excellent practice that the authors are very clear and twice identify their starting assumptions, it is not so great that they do not propose sensitivity analyses for both components (risk and rate) of those assumptions. By the end it felt like they did not care if the assumptions were valid.
Lines 114-121 are almost clear.  Because the supplementary data contain 102 dead bears if I am understanding the dataset (better labeling would help), then you might want to end by clearly stating what was included or excluded.. Please clarify. Also the one case of a collar strapped to a bottle and thrown in water being classified as human-caused makes sense but can't it be classified a bit more precisely? After all it wasn't a vehicle collision right?
Validity of the findings: I am deeply concerned that the entire validity of the findings hinges on the 2 starting assumptions that collared and uncollated bears die at the same risk and rate. I cannot encourage publication without quantitative evaluation of these assumptions. And please note both assumptions are important and may be partially correlated only not perfectly correlated. For example, if collared bears face a higher rate of mortality than uncollared bears for whatever reason but the risk patterns are the same, then perhaps the author's estimates are somewhat biased upwards or downwards (a question of accuracy). But if the risks faced by uncollared and collared bears differ then either the precision of their estimates would be off or their inferences might be erroneous.
Line 123-128: note recent literature from 2017 addresses deaths of unknown causes. One of the clearest conclusions coming out of that work is that unknown causes are NOT legal hunting or legal government killing, therefore the calculations in these lines MUST include the unknown causes because the authors are explicitly calculating a ratio of legal to other causes. There is no scientific rationale for excluding them as was once done under the erroneous assumption that unknown deaths are estimable from known deaths.
Line 136: "Assuming the collared sample is the true ratio,…" The authors missed the conclusions of the wolf research cited above. Neither sample is "true", both are biased but in complementary ways in some cases.

Although I find their basic conclusion is probably correct that the vast majority (79-88%) of uncollared bear deaths were not in the governmental database, I worry about the accuracy of their calculations for the reasons stated above. It's very difficult to evaluate if the sex difference they claim to find is real or an artefact of their assumption and handling of collared bear data.
Additional comments: "Furthermore, the death of female bears may be more commonly unreported than males (Ciarniello et al. 2009, McLellan 2015), which could have important implications for population trajectories."
While everyone of your readers understands that females give birth so directly affect population dynamics, this statement is misleading. It implies males are unimportant to population trajectories which the authors know is inaccurate. Expose implicit assumptions as such or rephrase to accord male brown bears their role in population dynamics (e.g. infanticide., etc.).


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: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The writing, structure, and information provided in this manuscript is appropriate for a scientific article, and the raw data are supplied.
Experimental design: The subject matter is of interest to a narrow field of scientists.  The results are quite specific to the study area in southern BC, and to BC in general.  The analysis/math involved is quite rudimentary, so therefore I would expect a more rigorous review of previous literature and a greater attempt to put this work into a larger context and show how it adds to previous work.  I am not convinced it fills an identified knowledge gap as currently presented.
Validity of the findings: The challenge of estimating unreported mortality of grizzly bears is daunting.  Although information from the deaths of radio-marked bears is the most appropriate data for helping to estimate unreported mortality, sample sizes are generally quite small given the relatively high survival of adult bears and the resulting low number of observed deaths.  Therefore, even with the most representative data, inference may be subject to sampling variance.  Still, Cherry et al. (2002) proposed a method for estimating unreported mortality based on a Bayesian analysis using a prior distribution informed by the reporting rate (ratio of reported to unreported) for grizzly bear deaths documented among sample of radio-marked bears. This manuscript uses a similar approach (although non-Bayesian), but there are significant differences in the assumptions, and for that reason I have some concerns.

The analysis presented is logical and clearly serves to demonstrate that human-caused mortality (other than hunting) is under-reported.  If the intent of the manuscript is primarily to make this point, then it should be titled like “Reporting rate low for non-hunting human-caused mortality of grizzly bear in the Flathead Valley, BC Canada”.  However, the current title suggests that the intent of the manuscript is not just to show that reporting rate is low, but to estimate (or perhaps predict) it.  I would presume this estimate might then be used in other population analyses.  This is where I have some concerns.

First, unlike Cherry et al., the current authors do not actually utilize information about an observed reporting rate to estimate the true reporting rate.  On line 99, they state “when collared bears were killed for other reasons [than hunting], they too were often reported.”  How often?  Table 1 indicates that there were 12 non-hunting mortalities and 5 natural mortalities that were observed among radio-marked bears.  Given the sample size, the observed reporting rate may have been as low as 6% (1 of 17), as high as 94% (16 of 17), or in between.  By sex, it could have ranged from 10% to 90% (n = 10) for females and 14% to 86% (n = 7) for males.  These ranges certainly include the estimated 12% reporting rate obtained in their analysis, so at the very least, I think this observed reporting rate should be provided so that a reader can compare.  If the two estimated reporting rates are similar, it would add support to the validity of the estimate.  If not, it might bring the estimate into question.

Cherry et al. attempted to explicitly estimate the reporting rate for causes of death which were known or assumed to have <100% reporting.  As such, they specifically excluded agency removals from analysis, because they were assumed to be an accurate count.  Again, they were utilizing the ratio of reported to unreported within a small sample to extrapolate to the population.  It is a subtle difference, but this is not what the current authors did.  They argue that “like all bears killed legally by hunters, all collared bears that hunters killed were reported.”  In other words, hunter kills have a 100% reporting rate.  So, in essence, this current analysis assumes some quantifiable ratio between the number of bears that died from a cause of death with 100% reporting to the number of bears that died from causes of death with <100% reporting.  I would argue this is a more tenuous assumption.  On the one hand, I can see the rationale.  One could argue that all causes of death are most correlated with population size, so that if hunting mortality goes up, non-hunting mortality also goes up.  On the other hand, though, there is the argument that hunting mortality may be inversely correlated with some sources of mortality, like poaching.

Examining the difference in reporting rate between sexes is also a major them in the manuscript.  This appears motivated by the McLellan (2017) paper, which states: “We also found the death of almost twice as many males as females were reported. There are 3 reasons why fewer female deaths were reported than males: an unequal sex ratio at birth, reporting errors (Schliebe et al. 1999), or females have higher natural and unreported human-caused mortality rate…Although sample sizes were small, 2 of 2 deaths of female bears monitored in the mountains of central British Columbia by Ciarniello et al. (2009) and 3 of 3 female bears in the mountains of southwestern British Columbia (McLellan and McLellan 2015) died of natural causes. It is also possible that, because of their protective behavior when with cubs, more females than males are killed by ungulate hunters and not reported (Mace et al. 2012, McLellan 2015).”  So, after arguing that natural mortalities (which we can certainly assume have <100% reporting) may partially account for the “missing” female mortalities in the CI database, the authors select to exclude natural mortalities from this current analysis and focus entirely on human-caused mortalities.  Why?  This seems like an odd choice, especially given that female natural mortalities were more numerous than male natural mortalities within the radio-marked sample (4 versus 1 in Table 1).  Of course, if natural mortalities are included in the analysis, then the estimated reporting rates would be even lower.

So what is the proposed application of this analysis?  Is it relevant to anyone else studying grizzly bears? For example, are the authors proposing that the estimated 12% reporting rate be used in conjunction with the CI database to estimate total human-caused mortality?  It would appear so, given lines 141-151, where they do just that.  Again, I am not claiming that this is wrong, but perhaps the authors could provide an argument for why they chose not to directly estimate reporting rate to obtain the estimate of total mortality.  Are they, perhaps, suggesting that the estimated reporting rates obtained from a radio-marked sample are biased high?  If so, then I think this point might be made more strongly.  In fact, perhaps this is where this manuscript needs to go, to make it more useful for biologists working in other areas.

I think the math used to derive the 12%, 18%, and 5% reporting rates (total, male, female, respectively) should be more clearly described in the results and/or shown in the table.  I don’t think it should be left to the reader to do the math.  For example, “While the ratio between the number of legal hunter kills to non-hunting kills was 0.833 for the collared sample when the suspected kills were included, it was 7.0 (70:10) for the uncollared bears in the CI database. Assuming the collared sample is the true ratio, by applying the ratio to the number of uncollared legal hunter kills (70/0.833), we derive an estimate of 84 non-hunter kills.  Since only 10 uncollared non-hunter kills were reported, this would indicate that only 12% of the non-hunting human-caused mortalities were in the CI database.”  I also think the authors should clearly state that they are applying the observed ratio (from collared bears) to just the uncollared bears, and explain why.  Again, if this estimate is meant to be applied to real world data for management purposes, it would be instructive to explain how data obtained from collars should be treated.

Reporting rate varies by cause of death.  Although the authors touch on this, they are not particularly direct about it.  It would be nice if the causes of death among radio-collared deaths were reported, along with whether or not they were reported.  Without that, I can only surmise from lines 167-181 that most of the mortality was either illegal or natural.  It is completely expected that reporting rate would be low for those causes of death and low reporting rates have been previously reported (Costello et al. 2016 - grizzly bear demographics in the Northern Continental Divide Ecosystem).

In summary, I think this manuscript could be greatly improved by comparing the radio-marked reporting rate to the estimated reporting rate obtained using this current method, followed by discussion of the factors that might account for any differences.  Another option is evaluate the estimated reporting rate with other population analyses.  In its current form, the manuscript provides little that is useful for biologists working in other areas, perhaps even within BC.
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: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper has been cleaned up and written more clearly.  More could be done to improve the writing, however.  Many superfluous phrases could be cut or simplified.
Experimental design: There is no experimental design, which is typical of studies such as this one.  However, without experimental design elements, there is good reason to interpret the result cautiously.  See below for more on study design issues.
Validity of the findings: The study’s approach is really very simple, using algebra to solve for the expected number of non-hunter killed bears in a government data base to point out that the expected number is much larger than the observed number.  The fundamental weakness of the paper, which I tried to point out in my first review, is its zero degrees of freedom when comparing the observed value to the expected value.

As I suggested in my comments on the first draft, the telemetered bears should be seen as the training data set on uncollared bears, although it is questionable whether the training data set is truly representative of the uncollared bear population.  The new draft of the paper is an improvement over the earlier draft by pointing out that telemetered bears might be unrepresentative of uncaptured bears, but then some of this improvement is lost when the paper dismisses the likelihood of the captured-bear bias on the unconvincing grounds that three capture methods were used.  In my earlier review I also pointed out that had hunters seen the collars and decided not to shoot only two collared bears, and had those bears died by human agents other than hunters, the shift in the expected number of uncollared non-hunter killed bears would have been from 64 to 41, a 36% reduction. The new draft introduced yet another source of potential bias by revealing that collars were designed to disengage from bears within <1 year to 6 years (this potential bias should have occurred to me during my reading of the earlier draft).  Whereas bears in the training data set could have ended up in the mortality data set during the time they were collared, uncollared bears could have ended up in the data set during their entire lifespans until the study period was curtailed.  I am not sure how significant this potential bear-year bias might have been.  Another relatively small bias might have influenced the study finding had any of the collared bears dispersing out of the study area might have returned without their collars.  By pointing out all these potential biases, I don’t mean to argue that the study result is wrong; my point is that ample reason exists to interpret the result cautiously.

I noticed that my notation in my first review did not transfer from my Word document to the PeerJ reviewer form, so I’ll change it slightly to facilitate transfer.  Consider the following equation, which I prefer over the equation in the manuscript because my version isolates the term the authors wish to estimate:

F’ = F/T,

where F’ is the estimated number of unreported fatalities of uncollared bears caused by non-hunting humans, F is the reported number of fatalities of uncollared bears caused by non-hunting human agents, and T is the training data expressed as the ratio of reported collared hunter-killed bears to collared non-hunter-killed bears, or in this case, and combining genders, it is 71/(10/9) = 64.  This estimate of 64 is the expected number of uncollared non-hunter killed bears in the data base, and is larger than the observed number of 10.  This is really the message of the paper – that 54 (84%) bear fatalities caused by humans other than hunters are missing from the data base.  However, this outcome is weakened by the comparison of one observed value to one expected value, involving 0 degrees of freedom.  The outcome is a comparison of two numbers that cannot be compared in a statistical hypothesis test.

I suggest the study’s finding warrants a letter-length paper pointing out the magnitude of difference between the observed and expected numbers of non-hunter killed bears as an intriguing indication that non-hunter killed bears might be under-reported.  Even between drafts of this paper, 1 bear got added to the uncollared, non-hunter killed bear category, thus indicating the vulnerability of bear fates to investigator assumptions and interpretation.  Decisions over which bear goes into which category can substantially change observed and expected values because the numbers in the training data are small.  And then there is the issue I raised in my review of the earlier draft that experimental design elements are lacking from this study, which is another reason to interpret the finding with caution.

Breaking down the analysis by gender further weakens the analysis by diminishing the numbers serving as training data.  The numbers of bears in the training data set are too small by gender to support calculations of expected values from ratio metrics.

Finally, I think the paper is improved with the inclusion of more detail about the fates of telemetered bears.  I would even encourage more detail. I suggest that the strength of the paper is in the story it tells about human-caused bear mortality, of which the estimated expected number of unreported human-caused bear mortality is a part.

Manuscript Line

119	“Based on these factors…” is too vague an explanation for how it was determined whether bears were killed via cryptic poaching.

148	I could not understand how PopTools was used to generate confidence intervals.  Was PopTools applied to the training data?  Or also to the counts of hunter-killed and non-hunter killed bears in the data base?  It seems to me the only appropriate application of randomized subsampling would be to the training data.

199	How was a confidence interval derived from a count within one sampling unit?  Counting my family of 4 within my single household sampling unit, I get 4 and not 2 to 6.  A count is not an estimate.
Additional comments: Replies to responses by cited comment number

1	Regarding the definition of mortality, I see that the first definition of a Google search on ‘mortality’ was used.  I know this because I got the same definition from Google based on this query.  I suggest scrolling down the Google output to a reliable dictionary such as Merriam-Webster, where you would see that mortality has different meanings than fatality, and that ‘mortality rate’ is redundant.
9	I see that my suggestion to not report gender differences was followed, but that doing so was deemed unwise.  If unwise, then perhaps it would have been best to decline to follow my suggestion.  This said, I explained why the division of results by gender differences was distracting and unhelpful to the paper.  The numbers are too small, and so are the differences. But that is my opinion.  Another approach would be to demonstrate that the gender differences are of sufficient magnitude to justify reporting fatalities by gender.
The paper continues to report results by gender, but without suggesting differences in fatality rates between genders.  This work-around to my suggested revision solved part of the problem, but still burdens the paper with too many findings.  The results are difficult to follow.  And I continue to wonder why it is so important to break out the results by gender, except that by doing so the paper implies gender differences.  Doesn’t work for me.
18	Hunters love to talk?  I’ve known many hunters.  Some love to talk.  Some do not.  Some say what they want the regulators to believe.  The stated assumption about hunter behavior appears speculative, rather than based on any evidence.


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: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I can relate to being defensive in responding to a critical review.  Nonetheless, I have to chuckle at the irony of the authors response to my review.  They assert that I totally misunderstood their manuscript, and I respond by asserting that they totally misunderstood my review.  Looking back at my original review, I can sort of see why the authors concluded that I did not understand their analysis, but I assure you I did.  Let’s start fresh.

(1) The revision includes a formula, but, may I suggest that they change it to the one that Reviewer #1 suggested and include the citation?  I recognize the authors arrived at it independently, but the earlier use of the method is still worth acknowledging.  Reviewer #1 said, “The essential form of the adjustment, F ̂=F/D , has its origin in Horvitz and Thompson (1952), but multiple revisions or additions have since been made to their estimator to suit specific challenges (Korner-Nievergelt et al. 2011).”  I think this form of the equation is simpler and more intuitive.  It also works very well with the “ratio” language used in the text, such as “The ratio between the number of permitted hunter kills to the number of human-caused mortalities but for other reasons is much different for the uncollared bears in the CI database than it was for the collared sample.”  The value D in the formula above is exactly that ratio.

(2) Line 63-65 says “The difference in the ratio of bears killed by permitted hunters to bears killed by people for other reasons between the uncollared bears in the government database and the radiocollared sample provides an estimate of the number of unrecorded human-caused mortalities of uncollared bears.”  I get this and agree, certainly when comparing these ratios within the same set of years in the same area.  Please take a minute and just consider these questions: (a) For management purposes, are there any cautions for extrapolating this estimate to other areas or to other years in the same area?  (b) Is there an effect from having a limited number of hunting permits, which may fix hunting mortality to a specific number (and potentially affect that ratio of hunting to non-hunting mortality)?

(3) I accept the argument for not including any natural mortality.  Given that natural mortalities are of no interest, I suggest you remove them from Table 1. I also suggest the following format for the table (picky, I know):
		                  Hunting	      Non-hunting
Collared	      Male   	6	              4 (2)
	              Female	4	              5(1)
	              Total   	10	              9

Uncollared     Male	         45	               8
	              Female	26	               2
	              Total	        71	             10

(4)  The discussion begins with a brief review of previous estimates of the proportion of non-reported deaths among non-hunting human-caused deaths in BC (53%, 62%), which were based on samples partly obtained within the same study area and the time frames overlap significantly.  This is followed with the estimate from the current study and a similar study further north in BC (84–88%, 90%).  The differences are notable, but the authors do not support or advocate for either estimate as better, more realistic, or less biased.  A reader might conclude that surely the authors believe this current estimate is better, hence the publication of a new estimate, but are we correct?  Which should a wildlife manager use for management purposes?  It may not be obvious, but much of my original review was centered in this very question.  I was encouraging the authors to provide more context for the need for another estimate, and some discussion of the pros and cons of the old versus new approach.  Interestingly, it was obvious from the author responses to my review that they are skeptical of the previous estimate and made several references to unrealistic assumptions.  Is this not important to discuss?  For example:

          (a) THEY [Cherry et al.] HAD TO MAKE TWO ASSUMPTIONS THAT WERE CLEARLY INCORRECT.
          (b) I THINK THIS REVIEWER MISSED THE METHOD WE USED AND ASSUMES THAT WE ARE ESTIMATING THE REPORTING RATE FOR COLLARED BEARS AS I DID 20 YEARS AGO (MCLELLAN ET AL. 1999) AND AS DID CHERRY ET AL. (2002).  WE DO NOT USE THIS METHOD AS IT MAKES ASSUMPTIONS THAT WE KNOW ARE INCORRECT.
         (c) My original review and response comments:  Are they, perhaps, suggesting that the estimated reporting rates obtained from a radio-marked sample are biased high?” NO, THIS IS NOT ASSUMED. If so, then I think this point might be made more strongly. In fact, perhaps this is where this manuscript needs to go, to make it more useful for biologists working in other areas.

So, all told the authors seem to be saying…Estimating reporting rate from a collared sample has 2 ASSUMPTIONS THAT ARE CLEARLY INCORRECT, it results in non-reporting rates which are lower than the non-reporting rate estimated in this study, but IT IS NOT ASSUMED that it is biased?  Please explain.

(5) Despite the views of the authors, I was completely aware that the current analysis was not utilizing information about how many of non-hunting, human-caused, radio-collared deaths were reported in their analysis.  I was simply asking for them to report this number or proportion.  Does it support your estimate or not?  If not, please provide some explanation.

(6) I agree with the decision to combine sexes and eliminate the discussion about any differences.
Experimental design: no other comments
Validity of the findings: no other comments
Additional comments: no other comments


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

Input:
Title: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 3
Reviewer: 1

Output:
Basic reporting: I have nothing more to add from my first two reviews.
Experimental design: I have nothing more to add from my first two reviews.
Validity of the findings: The findings could be true, but the high uncertainty and potential biases warrant cautious interpretation.  In my opinion, the paper presents an intriguing result with too much confidence that the result is true.
Additional comments: Probably not a great idea to insult the reviewer.  I was only trying to help.


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: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 3
Reviewer: 2

Output:
Basic reporting: Fine
Experimental design: Fine
Validity of the findings: Fine
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: ESTIMATING UNRECORDED HUMAN-CAUSED MORTALITIES OF GRIZZLY BEARS IN THE FLATHEAD VALLEY, BRITISH COLUMBIA, CANADA
Review round: 3
Reviewer: 3

Output:
Basic reporting: Line 246 terminology should be kept consistent, so in this case I would suggest “cryptic poaching” instead of “cryptic killing”
Experimental design: Past studies, briefly mentioned in the introduction, have attempted to estimate unreported mortality of large carnivores. How does this study differ from those? Why does the method being used by these authors better apply to grizzlies, or at the very least build on the work already conducted? The introduction currently reads as though the work previously done has very little application to unreported grizzly mortalities. I struggle to belief that is true, but if it is, I would love to know why. There is a mention of this in the discussion but I think it would improve the rigor of the manuscript and the introduction to include it more in the introduction.
Validity of the findings: Line 245, why do the authors assume that bears had been shot and their collars destroyed in this case?

I think the authors have done a good job of addressing the concerns of previous reviewers regarding the assumptions made.
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: VIRUS DISCOVERY IN ALL THREE MAJOR LINEAGES OF TERRESTRIAL ARTHROPODS HIGHLIGHTS THE DIVERSITY OF SINGLE-STRANDED DNA VIRUSES ASSOCIATED WITH INVERTEBRATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is a thorough and well written analysis of 44 DNA CRESS viruses identified in different arthropods.
Experimental design: Complete genomes of 44 novel viruses were obtained by rolling circle amplification, restriction digest and cloning.  The methods are appropriate and rigorous.
Phylogenetic analysis is also appropriate.
Validity of the findings: The data is robust.
Additional comments: It appears the sampling of arthropods was simply opportunistic. The authors could comment on what they believe would be an optimal sampling strategy to capture viral diversity.

Small circular DNA virus genomes have been identified as contaminants of various reagents (e.g. silica columns). What steps did the authors take to rule out the possibility that the detected viruses were actually from the putative host species and not perhaps an adventitious contaminant of a reagent?


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: VIRUS DISCOVERY IN ALL THREE MAJOR LINEAGES OF TERRESTRIAL ARTHROPODS HIGHLIGHTS THE DIVERSITY OF SINGLE-STRANDED DNA VIRUSES ASSOCIATED WITH INVERTEBRATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes the isolation of putative viral circular ssDNA sequences from a number of members of arthropod lineages, many of them not known previously to harbor ssDNA viruses. The novelty of these sequences is clearly shown, as is their diversity. As such I find that it is appropriate for Peer J.  I am particularly impressed and gratified to be informed that CRESS DNA virus genomes have been found in arachnids, apparently for the first time.

I have a couple of suggestions for improvement. The main one is to emphasize the apparent chimerism in Rep protein sequences as described in Kazlauskas, Varsani and Krupovic, 2018. This chimerism is apparently highly prevalent in “unclassified” CRESS viruses which makes the construction of phylogenetic trees of whole Rep protein sequences problematic in terms of virus phylogeny.  The authors might consider reducing the number of sequences presented, particularly in Figure 1 to reflect this.  Figure 1 is overwhelming and it is not clear what information is to be conveyed in this figure.  Having hundreds of separated lines for each sequence with nearly illegible text is excessive, in my opinion.  I understand that the authors wish to present the diversity of their newly discovered sequences, but that could be done with considerably fewer sequences.  I suggest simplifying this figure (and possibly Figures 2 and 3 as well), for the main text but providing the entire figure as a supplemental figure.  I find the icons to be very useful and aesthetically pleasing, consider adding an icon label for the endogenous viruses in Figure 3.

It would be extremely useful to readers if a table with species demarcation percentages for different CRESS-DNA virus familes were presented.  As currently in the manuscript it is very confusing to have different cutoffs for different families.
Experimental design: No comment in addition to those stated above.
Validity of the findings: No comment.
Additional comments: Specific comments:

I find the use of “PI” throughout a little confusing, consider defining “pairwise identity” in each section of the manuscript.

Lines 123-125.  Consider expanding this statement to include chimerism.

Line 204.  Consider either adding “with default parameters” or state whether alternative start codons or genetic codes were used to define ORFs.

Line 225: on (date) or in April 2018

Line 363 “of” species

Line 397 following: Please describe how is was determined that this 99% PI is not a contamination.

Line 437. Please describe how “non-significant” is defined.

Line 439. Consider reminding readers of the CEV abbreviation here.

Discussion. Please describe how virus families are defined.

539-540 – I find the “ancestral sentence” to be excessively speculative, particularly given recombination in Rep sequences.

Figure 2, Consider adding star to figure legend/on figure.

Footnote 3 of Table 1 Please clarify what parentheses mean.
Table 2.  Please clarify why there are multiple accession numbers for some viruses.


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: VIRUS DISCOVERY IN ALL THREE MAJOR LINEAGES OF TERRESTRIAL ARTHROPODS HIGHLIGHTS THE DIVERSITY OF SINGLE-STRANDED DNA VIRUSES ASSOCIATED WITH INVERTEBRATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clear, well written and logically structured. The introduction and background information are appropriate and demonstrate the context and relevance of the study. The structure is good and the figures are relevant, clear and nicely presented. The raw data can be accessed via genbank and the alignment can be opened and visualised with MEGA.
Experimental design: The manuscript and design of the study are scientifically and methodologically sound and fit well within the scope of the journal. The question of to what extent is CRESS DNA virus diversity represented in a sample of diverse invertebrate hosts is clear and the results are to provide useful information to expand knowledge about the diverse and still largely unexplored ssDNA virome of invertebrate hosts. The methods for sample processing and analysis are appropriate and clearly explained.

I just have a few minor comments to add where extra clarity/information may be added:

L157: What was the buffer for homogention? Further SM buffer after the three washes?
L187-192: How was it ascertained from the agarose gel whether the ends were sticky or blunt?
L196: what did you class as significant similarity?

L373: You previously described 78% similarity as a cutoff, but here you describe a novel virus  FlyACV-2 as being 92% similar to one previously described. In a couple of cases including this one, and in the paragraph starting L388 it is not clear to me how exactly the boundary is decided between what you are classing as an isolate/specific virus species. A sentence could be added for clarity.

L402,408: could you provide examples of the shared genomic features.
Validity of the findings: The results of this exploratory study are sound. The sample size was covers a broad range of hosts/locations/time points, but the low level of replication is fine in this study as no assumptions were made about prevalence within hosts or the full extent of the diversity present in invertebrates. There are limitations to this study, as with any which explores viral diversity in a broad range of hosts ie, the sample size per host species is small, whether the viruses detected originate from true/active infections, possibility of contamination from infected food sources, etc, but these are discussed in the discussion, and although there is an amount of speculation in the discussion it is clearly presented as such.
The evolutionary history of the viruses discovered including using CEVs appears robust, yet is not overstated. The finding of distinct cycloviridae clades for example is informative and supported but the authors highlight the need for further sampling to resolve other relationships further.
The conclusions are appropriate. The authors have discovered a significant amount of previously unknown CRESS DNA virus diversity and the data indicates that the diversity in invertebrate hosts is far larger than that of CRESS DNA viruses infecting other taxonomic groups.
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: SEQUENCE VARIANT ANALYSIS OF RNA SEQUENCES IN SEVERE EQUINE ASTHMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, however this manuscript could benefit from following HGVS nomenclature (http://varnomen.hgvs.org/). In the manuscript the word 'variant' is used both to represent a variable position in the genome (a SNP) as well as to represent a specific base (A,T,C,G) at a SNP. This should be clarified.
Experimental design: The methods regarding alignment, variant calling, assessment of protein function, and validation with Sanger sequencing are well done. Disclaimer: I am not an expert with the programs used for assessing protein function.

The experimental design to associate genetic variants and gene expression to the asthma phenotype in this paper is very poor. The authors do not perform any statistical tests to associate the identified genetic variants or gene expression to asthma.
Validity of the findings: no comment
Additional comments: General Comments:
The manuscript titled "Variant analysis of RNA sequences in severe equine asthma" by Tessier et al. aligned RNAseq data with STAR, called and filtered DNA sequence variants with GATK and VEP, identified novel variants with VEP, assessed each variants' impact on protein function with PolyPhen2 and SNAP2, and argue that two of these variants impact protein function which are also relevant in asthmatic horses. The objective was to assess the reliability of calling variants from RNAseq data. Reliability was determined by comparing genotypes called by GATK from the RNAseq data to Sanger sequenced data. The methods regarding alignment, variant calling, assessment of protein function, and validation with Sanger sequencing are well done. Therefore the main objective of the paper is well done. Notably, the version of GATK used (v3.2.2) is years out of date (a more recent v3.3 was released in 2014), however re-analysis with v3.7 or v4 is not required.
However, the paper is severely hampered by attempting to find a connection of DNA variants and RNA gene expression to asthma disease status. No statistical tests were performed in this manuscript to quantify if gene expression or DNA variants are relevant to asthma. The authors never state that they have a significant association nor blatantly misrepresent their findings. However, by focusing on two specific variants (identified after a procedure to identify "variants of interest") that have altered genotype frequencies between 10 asthmatic and 14 non-asthmatic individuals. They consistently imply that these variants are relevant to the asthmatic phenotype throughout the manuscript when no statistical tests have been performed. For example: in the conclusions the authors write "The heterozygous mutant genotype of PACRG was more prevalent among asthmatics while the homozygous-WT genotype of RTTN was more prevalent in non-asthmatics," and in Results they write "Expression of PACRG overall was slightly higher in asthmatic than non-asthmatic horses." Presenting these data in such a manner could be very misleading to non-experts that read this manuscript who might not realize that no statistical association to asthma was found or even performed.
Regarding DNA variants and asthma:
Asthma is a complex trait which likely has many small effect loci that effect disease susceptibility or progression. This is why in horses GWAS for SNPs associated with severe equine asthma with ~370 individuals (~350k marker density) or ~650 individuals (~50k marker density) are still underpowered to find a single variant that is significantly associated to asthma after genome wide multiple testing correction (Shakhsi-Niaei et al. 2012, Schnider et al. 2017). For reference, meta-analyses in humans are reaching sample sizes in the tens of thousands (and even hundreds of thousands) to find significant associations (Ferreira et al. 2014 doi:10.1016/j.jaci.2013.10.030, Zhu et al. 2017 doi: http://dx.doi.org/10.1101/133322). With 24 samples the likelihood that the authors found a significant association after multiple testing correction is very low.
1.	Specifically – Results:
a.	Although important to present in this paper, the estimated genotype and allele frequencies from 10 asthmatics and 14 controls are likely unstable within each group because the sample sizes (10 and 14) are too small to accurately represent population frequencies.
2.	Specifically – Methods Lines 176-195:
a.	They describe a procedure to identify variants of interest (i.e. variants that impact protein function and are relevant to asthma).
b.	This section is supposed to be a GWAS to test for how well any particular SNP predicts asthmatic disease status (to identify variants of interest and relevant to asthma). They should test all potentially impactful high confidence protein modifying variants for association to asthma in a linear model (logistic regression, DiseaseStatus ~ Genotype) and correct for multiple testing by FDR or Bonferroni correction (example α/NumberOfComparisons).
i.	Notably to help reduce multiple testing correction burden they could remove variants violating HWE in control individuals, use a MAF > 0.05, etc.
c.	The authors 'by-pass' the multiple correction burden and identify 'variants of interest' essentially by filtering for variants "present in all asthmatic horses but not all non-asthmatics".
d.	This procedure is statistically unsound and does not statistically link any variant to asthma.
In regards to RNA sequencing and asthma:
There are some very strange sentences in this manuscript that attempt to support differential expression (DE) results from one of their other manuscripts [56] that found the majority of genes (81 of 111) to be upregulated. Example, in the Results lines 219-222 and specifically in the Discussion lines 309-313: the authors attempt to summarize the expression of all ~20,000 protein coding genes as 'slightly higher' in 'some' asthmatic horses after challenge by visual inspection of aligned sequences in IGV.
1.	This is unacceptably subjective and is not a result that can support their previous paper.
a.	The authors recognize that this procedure had 'limited precision', however this is not enough to justify its presence in the paper.
2.	At best, viewing the number of aligned RNA sequences would represent the raw counts. However, raw counts are never analyzed for differential expression; all counts must be normalized (usually by the library size) before one can make a conclusion on the biological implications. With raw counts the observed differences in sequence depth could simply be due to more sequences being sequenced in some individuals.
3.	The authors need to perform a DE analysis (GeneExpression ~ DiseaseStatus) with normalized counts before attempting to support their other paper.
The authors present unacceptable connections between gene expression in the two 'interesting' genes PACRG and RTTN in the Results section lines 249-254.
1.	Here they present a range (min-max) of counts-per-million (CPM, a normalized RNAseq count) counts in asthmatics and non–asthmatics.
2.	Minimally, the authors must provide mean and/or median CPM counts per gene in each group so the reader can imagine the distribution.
3.	However, again the final sentence states that "Expression of PACRG overall was slightly higher in asthmatic than non-asthmatic horses."
a.	Again, although not explicitly stated this sentence is clearly present to make the reader think that there is a difference (and specifically upregulation of genes genome wide) between asthmatic and non-asthmatic horses when no statistical analysis (DE, or a t-test) was performed, and the range of CPM counts for asthmatic and non-asthmatic horses are almost perfectly overlapping anyways (asthma 30.7-66.3 and non-asthmatics 25.1-65.6). Are the means different? Is there differential expression?
Specific comments:
1.	Abstract - Results:
a.	Line 61-63: What are the frequencies for the PACRG heterozygous genotype, and RTTN homozygous genotype in asthmatics compared to controls?
b.	For RTTN the authors write "homozygous mutant phenotype". I believe they meant genotype. A phenotype cannot be homozygous. If the genotype determines the phenotype, please refer to the increased frequency of the genotype.
c.	Minot point, but important.
i.	Use of "mutant" should be avoided when referring to DNA variants. Nomenclature (at least for humans) regarding DNA sequence variants has been standardized and I recommend that the authors follow guidelines at: http://varnomen.hgvs.org/ . More specifically: http://varnomen.hgvs.org/bg-material/basics/ under the Mutation and polymorphism section.
1.	"In some disciplines the term “mutation” is used to indicate “a change” while in other disciplines it is used to indicate “a disease-causing change”."
2.	It is generally preferred to avoid terms like "mutation" and use more neutral terms like “sequence variant”, “alteration” and “allelic variant”.
ii.	for examples see http://varnomen.hgvs.org/bg-material/simple/ .
iii.	The authors are describing a deleterious effect on function at the protein not DNA level.
2.	Results – RNA-Seq coverage:
a.	Lines 219-221: Remove from results section "With the IGV tool large-scale genomic data sets are visualized in real-time over a wide range of resolutions [72]."
i.	Not a result
b.	Line 221-222: After visual inspection the number of aligned RNAseq reads were determined to be "slightly higher" in "some" asthmatic horses following challenge.
i.	What was the value of the "similar" coverage across horses and challenges?
ii.	The change in depth of coverage needs to be quantified.
iii.	How many asthmatics?
iv.	Is this result relevant to the conclusions of this paper?
3.	Results – Variant calling and filtering:
a.	Lines 227-228: which variants and what is 'lowest prevalence'. Please clarify reference/non-reference and which base, and quantify frequency.
b.	This section would benefit by following HGVS nomenclature.
4.	Results – Protein alignment:
a.	Specify "Amino acid sequence alignment" in tittle of this section.
5.	Results – Sanger sequencing:
a.	Should this section be titled "Confirmation of RNAseq with Sanger sequencing"?
b.	I bring this up because the total number of individuals is different if the reader thinks the authors are referring only to the Sanger sequencing.
c.	Line 267: please specify the number of asthmatics and non-asthmatic horses.
6.	Results – Expression of PACRG and RTTN:
a.	Why is it relevant to differentiate cpm values in asthmatic and non-asthamtics when no differential expression analysis is performed? Or should we trust the genotype calls more in asthmatic vs. non-asthmatic?
b.	What is the mean/median expression in cpm for asthmatics and non-asthmatics?
c.	Lines 253-254: This sentence needs to be removed or clarified with some statistical analysis (see below).
d.	Although not explicitly stated, this sentence implies that there is differential expression and upregulation of PACRG in asthmatic horses. A differential expression analysis or t-test must be applied for this sentence to remain in the paper.
e.	Asthmatics have expression of 30.7-66.3 and non-asthmatics have expression values 25.1-65.6. With the information provided these distributions seem to be nearly perfectly overlapping. I see no support for the last sentence (lines 235-254).
f.	Was this gene upregulated in their other study [56]?
7.	Results – Sequence alignment:
a.	This section presents the evidence (genotype frequency imbalances between case and control individuals) for the connection of these two DNA variants to the asthma phenotype.
b.	Unfortunately, no statistical test is performed to quantify how well these variants predict disease status.
i.	Specifically, this section would greatly benefit from logistic regressions DiseaseStatus ~ Genotype. One for each variant. Importantly, these regressions should not only be performed on the 2 variants discussed but ALL potentially impactful high confidence protein modifying variants. The exact number of variants that should be tested is not listed in the methods section however, from EVP the authors list 26619, 24527, 28909, and 28451
ii.	Additionally, to do this in a more appropriate manner I would require genotype to be a significant predictor of DiseaseStatus at α < 0.005. As this represents a bonferroni correction for 10 independent tests.
c.
8.	Discussion:
a.	Line 310-312: Again, summarizing the depth of coverage for aligned reads for 20,000 protein across multiple individuals like this is inappropriate. These very subjective sentences should be quantified or removed.
i.	Quantifying the depth of coverage would be beneficial if attempting to support variant or genotype calls.
ii.	However, given the next sentence (312-313) it seems like the authors are attempting to state that genes are upregulated in asthmatics (after visual inspection of aligned reads). Which would support their other paper [56]. However, differential expression analyses are only done after normalization of counts. Therefore, observing the raw counts might just mean that more sequences were sequenced in 'some' asthmatics and have no biological meaning. This sentence does not support a conclusion on differential expression.
b.	Line 312-313: When considering the previous sentence and this sentence together. It seems as though the authors are attempting to state that the majority of differentially expressed genes (81 of 111) were previously found to be upregulated as stated in reference [56].
i.	"prominent changes" doesn't mean upregulated.


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: SEQUENCE VARIANT ANALYSIS OF RNA SEQUENCES IN SEVERE EQUINE ASTHMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Major comments:

1. What was the degree of relatedness of the horses in the institutional research herd. if there was a degree of relatedness then this may reduce the likelihood of identifying variants related to disease.

2. The authors state that "10 were missense mutations, coded for proteins and had SIFT scores <0.01" It would be better if all 10 variants were identified in a supplemental table as they only go on to discuss the  PARCG and RTTN variants only.

Minor comments:
1. Sequence coverage. the authors state that there was  "slightly higher  coverage in some asthmatic horses following challenge”  - were the genetics regions only adequately covered for variant calling in the asthmatic challenge samples? If so were these trimmed from the data set before comparison with non-asthmatic samples? Otherwise this would give flase positive as  SNPs would be more likely to be found in asthmatics due to better coverage.

2. Was the Sanger sequencing carried out on cDNA or gDNA? specify
Validity of the findings: While the variants identified in PARCG and RTTN are of interest and affect plausible biological pathways in relation to airways disease, I feel the the authors should explicitly note in their discussion that there was no significant difference in genotype frequency between the asthmatic and non-asthmatic horse groups.
Additional comments: This is a well written paper that utilises established bioinformatic methods to identify coding variants from RNA seq data. The potential significance of variants in 2 genes, PARCG and RTTN, are analysed using in silico methods and genotypes were verified using Sanger sequencing.  The significance of the observation is limited by the small group sizes and I feel that fact that there is no difference in variant alley frequency between groups should be explicitly acknowledged in the discussion and conclusions (e.g. lines 403 - 404) as noted 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: SEQUENCE VARIANT ANALYSIS OF RNA SEQUENCES IN SEVERE EQUINE ASTHMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article is clearly written and well-referenced.

Some of the figures are not needed (see specific comments below).

No hypothesis was stated. it seems that the authors have incompletely analyzed their data relative to their objective.
Experimental design: I have several questions and comments relative to the experimental design, and I believe that this is a potentially powerful data set that should be re-analyzed and in some places additional experimentation done and  additional data [resented (see specific comments below).
Validity of the findings: I believe that some of the authors findings are not valid and the study suffers from inadequate data quality controls and lack of statistical analysis (more detailed comments below).
Additional comments: ABSTRACT

Lines 36-37: The author’s statement--- “The disease in horses has complex inheritance
37 including both dominant and recessive patterns that are ill defined.” ---is contradictory and incorrect based on the current literature, one can conclude that the inheritance is complex, but there is not good evidence for either dominant or recessive patterns of inheritance for RAO.

INTRODUCTION

Lines 93-120 seemed to be more appropriate for the discussion than the introduction; in particular since there is not equine data that I am aware of that directly implicates cilliary dysfunction in RAO, and because the author’s objective was not to evaluate cilliary function or these two genes at the start of this research.

METHODS

Line 132-133 It is clear that the control horses were exposed and biopsies taken after 3 days of exposure, but it is unclear if the asthmatic cases were biopsied when clinical signs appear (i.e. 1-3 days) or at 3 days. Please clarify.

There are several places in the methods where results are reported. These data should be moved to the results section. For example:

Line 181: Suppl. Figure 1

Lines 182-188: “In asthmatic horses, a total of 26,619 pre- and 24,527 post-challenge variants were identified, respectively, while the corresponding numbers were 28,909 and 28,451 for non-asthmatic horses. Approximately 30% of variants were novel and not previously described. The types of variants and their coding region effects are summarized in Suppl. Figures 2 and 3. For further variant selection using VEP the inclusion criteria were 1) missense mutation in protein-coding sequence; and 2) predicted to cause loss of protein function.”

Lines 191-194: “Ten variants present in all asthmatic horses (before or/and after challenge) but not all non-asthmatic horses were identified. Of these, only two variants were expressed in all samples and were more prevalent in asthmatic compared to non-asthmatics.”

Line 194: Why would the authors not evaluate the putative functional effect of all discovered variants---this seems like a much better approach, in particular since their sample size is too small to clearly delineated differences in variant frequencies especially with the multiple testing burden with thousands of variants identified.

Line 203: The authors use Sanger sequencing of bronchial cDNA to “confirm” variants identified by RNAseq and to genotype the horses. However, this is not the appropriate approach. The SNVs that are detected should be present in genomic DNA and genomic DNA would be the appropriate reference material to confirm genotype. Unlike experiments that are interested in identifying novel somatic mutations in RNAseq (which is where variants calling in RNAseq originated); these authors are using RNAseq to identify functional genomic variation (instead of doing whole genome sequencing that is the most appropriate, but is more expensive option). To conform what was found in the cDNA genomic DNA is most appropriate for this application--unless you were looking to confirm novel splice variants---which unfortunately the authors failed to do.

No statistical analysis appears to have been performed.

COMMENTS ON THE RESULTS PRESENTED IN THE METHODS

Lines 182-188: Because, the authors were not looking, nor should they expect, novel somatic mutations in RAO (as stated above), it is very disconcerting to find huge differences in the variants detected in the cases (or controls) before and after challenge. To the reviewer this suggests one of three issues with the dataset:

1.	The data quality was insufficient to accurately call variants resulting in widely different variant calls across the two time points. Unfortunately the reviewer cannot determine if this is the case as the authors fail to report sufficient information regarding the RNAseq data and its quality, or the variant quality scores of their cut-offs used (missing form methods) this cannot be assessed (see below).
2.	The differences are due to the presence or absence of particular genes in the dataset at the different time points. Again without information about the read depth, number of expressed genes or the overlap in the expressed genes between each time point, this cannot be assessed.
3.	The read depth was insufficient to accurately capture low abundance reads resulting in different genes being captured in each library.

RESULTS

Lines 219-222: The authors do not report sufficient information regarding the RNAseq data, for example:
1.	The total number of reads collected per sample or minimally mean/median and range.
2.	The average mapped read depth and coverage. This should be calculated; figure 1 is not useful or adequate to determine the utility of the data.
3.	The total number of genes identified in samples from each time point and how those genes overlap across case/control and time point which would be important to report to address #2 above.
4.	An indication of read quality.

Lines 255-226: the authors need to report variant quality scores and the cut-off used. The reviewer suspects that the large differences in variants called are do to poor quality reads and/or insufficient quality control. In its current format supplemental figure 1 is not that useful. A Venn diagram of the overlap in genes detected across the samples would be more useful.

 Lines 227-228: These data should not be reported a “prevalence.” Allele frequencies should be reported and allele frequency differences should be calculated across all the variants and the authors should use a statistical analysis to come to the conclusion that allele frequencies are different. Authors should consult a statistician to perform statistical analyses of their data.  The authors also need to be aware of correction for multiple testing as they consider allele frequency differences in the data.

Lines 224-233: Why did the authors not evaluate other changes in the data besides SNPs? RNAseq give a great opportunity to look a splice variants and it’s disappointing that this was not done.

Lines 249-254: The methods do not adequately describe the gene expression differences and the analysis that led to these results. Where the data normalized? What was done to correct for multiple testing, what where the p-values or q-values, what was the FDR? It would be more useful to have the mean and SD (and/or fold-change) for the expression in the groups and have some sort of statistical reliability associated with the results.

Lines 266-280: See comments above authors need to do comparisons to genomic DNA.

Lines 284-287: As stated before the authors should present allele frequencies (as this is a complex trait. PACRG allele frequencies are not statistically different between cases and controls: Cases G=0.4, A=0.6, and controls G=0.43 and A=0.57.

Lines 289-292: Allele frequencies should be reported and a statistical test performed. This test should also account for multiple testing given the 100s of variants identified (and ideally actually tested for differences).

DISCUSSION

Lines 297-300: This statement is true, so why did the authors not report depth, quality, read mapping quality, variant quality and make appropriate cut-offs in their data?

Line 309-312: IGV viewer not adequate and the authors cannot make conclusions about similarity of differences in expression based on this method.

Lines 332-334: The authors need to go back and correct for these omissions in their data analysis.

Lines 349-354: These comments about the PACRG alleles are not appropriate as the frequencies are not statistically different.
Lines 360-361: Adding a statistical test would allow the authors to make a true statement here.

Lines 402: The authors have not demonstrated this statement with their data.