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: HONEY BEE VIRUSES IN SERBIAN COLONIES OF DIFFERENT STRENGTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Editors,

The manuscript entitled, “Honey bee viruses in Serbian colonies of different
strength (#28925)” by Cirkovic et al. describes virus incidence in Serbian honey bee colonies. This work provides information that is relevant to global honey bee virus epidemiology, as well as provides a baseline for future honey bee monitoring studies in this region. In general, this manuscript is well-written, and the figures are informative.

Points to clarify or address before publication include:

1. Figure 2  - the number of samples analyzed for each virus should be included in the figure (i.e., n= 300) – maybe below each bar or in the figure caption if all samples were tested for all viruses.

2. Figure 3 and Line 81 Defining what the authors mean by “occurrence” and “prevalence” will clarify the results; it seems abundance should be used for qPCR data.
See -  Cavigli et al Apidologie 2015

3. Figure 4 – It is not clear what the main point of this qPCR figure is – maybe it should be a supplemental figure. Detection of a single virus in a sample doesn’t seem like a really important result.

4. Figure 5 – Ct values seem really high. The authors’ could include a supplemental figure or just describe the linear equation that described their qPCR assay and its linear range.
Why are just Ct values shown in this figure when Line 105 indicated that the ddCt method was used?

5. Figure 5 B  This panel would be more informative in the qPCR data was included for both viruses.  It is common for multiple viruses to be detected in any honey bee samples, so likely levels of infection (virus abundance) is more important than presence.

6. Figures 6, 7, 8, 9   -  Accession numbers are required for publication; it looks like there are in the text, but not in the figure captions. The caption should include the length and regions of “genome coding sections”, as described in Lines 110-112 of the text.  What parameters were used to make the tree?  Additional details are required here. I assume the scale bar is nucleotide substitutions, but this should be made clear since phylogenetic inferences can also be based on amino acid alignments.
It seems that all boostrap value less than 85 or some cut-off value are “un-supported” and thus the tree would collapse back at those nodes.  Can the authors emphasize what regions of the trees are supported – maybe with a color change or bold values?

7. Abstract Line 46-47 The last line reads as follows: “For better
understanding of viral diversity in honey bee colonies, additional metagenomic analyses
are needed.”
It doesn’t seem that “metagenomic analyses” are required to obtain virus sequence information – a targeted virus sequence effort or sequencing of virus enhanced honey bee lysates would likely be better and more cost effective.  Since this data is not included in the manuscript, this line should be removed from the abstract.

8. Abstract Line 45 – could be reworded-
“The honey bee virus sequences obtained from bees in Serbia share 93-99% identity with other sequences deposited in GenBank”.   Furthermore, this result doesn’t support the idea that additional metagenomic sequencing is required.

9. Line 63   “accused” should be changes to “cited” or “DWV and mites have correlated with honey bee colony losses in multiple studies . .. .”

10. Line 65 – 67.  There are better general honey bee viruses citations including
Chen and Siede Honey bee viruses 2007 Ad Virus Research
Brutscher et al. Plos Pathogens
The citations can go at the end of the sentence.

11. Line 68 – Martin citation should also be placed at the end of the sentence – this may be a theme throughout.

12. Line 87-88 Authors should include the sampling dates, which may be important for future studies - since virus prevalence and abundance can change in the same colony on a weekly basis.

13. Results section should include the samples sizes (i.e., prevalence is 74% of XXX number of honey bee samples obtained from XX colonies).

14. Line 47 Is the chi-squared test appropriate here or would a student’s t-test be better?

15. Line 150 – 153 – Why can’t multiple infections be considered?  It is quite common that honey bee samples are infected by multiple viruses.

16. Introduction and Discussion could include more citations regarding the number of honey bee colonies lost globally. Perhaps include some recent review articles published in the journal “Current Opinion in Insect Science”.

17. Did all colonies from the same apiary have the same (or more similar) virus profile than those from other apiaries?

18. Could include citations from US (e.g., Cavigli et al Apidologie 2015, and several papers by van Engelsdorp), but the authors may prefer to focus on European countries, which is justifiable.

19. Line 220 – 221 – Not all viruses are transmitted by Varroa destructor (see Glenny et al 2017, LSV2 and mites do not correlate), so it may not be safe to assume that results are due to Varroa management.
This line should be deleted or rephrased.


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: HONEY BEE VIRUSES IN SERBIAN COLONIES OF DIFFERENT STRENGTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Tha manuscript in well written, I have two minor remarks before publications:

1. M&M, Lines 90-92: The complete sentence ''The selected colonies....'' should be checked aggain and need to be expalined in another way, because viral infections in bees can not be excluded according to clinical picture.

2. Figure 4: Not necessary-delete it.
Experimental design: no commnet
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: HONEY BEE VIRUSES IN SERBIAN COLONIES OF DIFFERENT STRENGTH
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work deals with the prvalence of few honey bee viruses in colonies of different strength situated in teritory of Srbia.

The manuscript is well written and presents good connection between introduction and discussion part taking in account the results obtained.

The manuscript fits weel within the scope of the PeerJ.
Experimental design: The aim of research is in accordance with scope of journal and methods are well described.
Validity of the findings: The data seems well analyzed and the conclusions nice stated and supported by obtained results.
Additional comments: Although, list of references fomatting is not a piority of PeerJ
- references are full and clear,
- list must be unified,
- but style is different than jounal recommand; same in manuscript tekst

Line 74. add reference:
Tlak Gajger, I., J. Bičak, R. Belužić (2014): The occurrence of honeybee viruses in apiaries in the Koprivnica-Križevci district in Croatia. Vet. Arhiv 84, 421-428.

All other corrections and recommendations are marked directly in manuscript (attached to this revision report)


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: THE MOST COMPLETE ENANTIORNITHINE FROM NORTH AMERICA AND A PHYLOGENETIC ANALYSIS OF THE AVISAURIDAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is written in a fluent English, and only a few terminological terms (mostly are typos) need to be corrected: see attached pdf with annotated comments.
Both introduction and referenced literature are appropriate.
The structure of the article conforms to PeerJ format.
The figures are appropriate, at a good resolution and add significant information to the text.
Experimental design: This is an original research.
The research questions are well defined, and it is clearly explained how this study fills previous gaps.
Methods are described, but the described results cannot be replicated (see below).
Validity of the findings: The study describes a new specimen and erect a novel taxon with a significant relevance in the field of Mesozoic vertebrate paleontology.

The results of the phylogenetic analysis cannot be replicated with the data matrix provided in the submission.
The authors wrote that they obtained 196 shortest trees of 560 steps each.
Using their data matrix, and performing 1000 TBR rounds, saving ten trees per replication (not one as in the protocol followed by the authors), I obtained 818 shortest trees of 558 steps (two steps shorter than those reported by the authors). I encourage the authors to perform a more exhaustive search than the protocol described in the manuscript, to be sure that their analysis has adequately sampled the tree islands.
Furthermore, there are two problematic elements in the data matrix that need to be solved:
1- The OTU Flexomornis is scored exclusively for five characters, and in all of them, the score is “0”: thus, that OTU is redundant with a subset of the scores of the dromaeosaurid Outgroup, and results a wildcard with no resolution, that is placed in all internodes of the topology: I suggest that the authors remove that OTU a priori from the data set, following the principle of safe taxonomic reduction.
2- The provided data matrix is not properly formatted. In particular, one taxonomic unit (OTU named “shenqi” in the data matrix, which I assume it corresponds to the taxon Shenqiornis) lacks a character score. It is unclear what score is missing, but as a consequence, TNT automatically use the first letter in the name of the successive OTU (“cathyaor” , which I assume it corresponds to Cathayornis) as missing last score for the OTU “shenqi”.
The absence of a character score in one OTU may have significant impacts on the analysis, as it may result in a badly formed string of character states. I encourage the authors to check carefully the scores in “shenqi” and to correct the missing score. Alternatively, that OTU should be removed as potentially spurious.

Another issue, already noted by the authors, is the overall topology resulted for Enantiornithes. The tree obtained markedly challenges the consensus among the majority of the previous studies on enantiornithines. In particular, the placement of forms like Protopteryx and Iberomesornis as more derived than Upper Cretaceous forms like Gobipteryx and avisaurids is quite unexpected, and raises the doubt that character polarity among the enantiornithines was not properly assessed. This suspect is also reported by the authors who stated that “The absence of basal ornithuromorphs is probably somewhat affecting enantiornithine relationships”. I strongly agree with the last sentence, and suspect that the use of just two very derived ornithuromorphs and the absence of basal members of the direct enantiornithine sister taxon has shaped the bizarre internal relationships resulted among Enantiornithes.
To test the hypothesis that the topology among the enantiornithines is biased by taxon sample among ornithuromorphs, I repeated the analysis omitting a priori the two advanced ornithuromorphs from the taxon sample. The resulted shortest trees placed Protopteryx as basalmost enantiornithine (in agreement with the majority of previous studies), and recovered Enantiophoenix and Mystiornis as very closely related to avisaurids (again, in agreement with previous studies). This test demonstrates that taxon sampling among the ornithuromorphs has a relevant impact on enantiornithine topology.
I encourage the authors to repeat the analysis including a couple of basal ornithuromorphs (e.g., Hongshanornis, Archaeorhynchus, or Archaeornithura) and/or to remove the two derived ornithuromophs, because – as the authors themselves noted – the bizarre topology resulted in their analysis is clearly affected by a poor taxonomic sample among the ornithuromorphs. Ichthyornis and Gallus are too derived for being used as appropriate proxies of the ancestral ornithuromorph morphology.

In summary, the published results of the analysis cannot be replicated, due to a series of problematic issues in both the data matrix and the protocol followed. I suggest that the authors:
- Correct the missing score in Shenqiornis,
- Remove Flexomornis, bacause it is a wildcard that lacks any significant score,
- Rename all OTUs in the matrix file with their proper full names, to avoid misinterpretations of the results,
- Perform more exhaustive analyses (for example, 1000 TBR rounds saving 10 trees per replication),
- Add at least two basal ornithuromorphs to the sample and/or remove the two advanced ornithuromorphs.

A last comment is related to the taxonomy introduced in this study.
The authors erect a new genus for a taxon that results phylogenetically bracketed by two species previously referred to the genus Avisaurus. This means that the authors implicitly consider “Avisaurus” as paraphyletic, or eventually, polyphyletic.
It is interesting that in the previous studies including Avisaurus gloriae and Avisaurus archibaldi, their sister taxon relationships was based exclusively on a single feature, the shape of the distal end of metatarsal IV. In the current study, the authors have demonstrated that this single feature cannot diagnose a clade, and, thus, that the diagnosis of the genus Avisaurus is not valid. This must be more strongly remarked in the text. In particular, the authors should explicitly state if they suggest a new genus name for “A.” gloriae, or, alternatively, if they consider “A.” gloriae to be referred to the same genus of its sister taxon, Mirarce.
Note that in Table 2, the new taxon is labelled “Avisaurus sp.”: please, correct that label to avoid further confusion.
An alternative solution is to emend the diagnosis of the genus Avisaurus, in order to include also the species “Mirarce eatoni”, as Avisaurus eatoni: this alternative solution (which, apparently, was an initial option for the authors, as evidenced by the use of “Avisaurus sp.” in Table 2) has the merit of not revolutioning the taxonomy of A. gloriae relative to A. archibaldi, and thus keeps stable the avisaurid taxonomy used in the last two decades.
Whatever the option preferred by the authors, it must be more explicitly discussed and defined, to avoid future misinterpretations of the term “Avisaurus”.
Additional comments: I have no general 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: THE MOST COMPLETE ENANTIORNITHINE FROM NORTH AMERICA AND A PHYLOGENETIC ANALYSIS OF THE AVISAURIDAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comments.
Experimental design: no comments.
Validity of the findings: no comments.
Additional comments: This is well-written paper which describes very nicely preserved late cretaceous north american enantiornithine bird. The paper meets all the criteria of the journal and definitively should be published.
I have a number of rather minor comments.

Line 54. I am not sure that it’s clear what controversy the authors mean. There was a debate on the affinities of the enantiornithes, but apparently not on the identification. Maybe some references (which hare now lacking) would explain this point and clarify this. The discovery of the first articulated skeletons in Spain and China initially had no effect on the acceptance of the Enantiornithes as the clade, because Spanish and Chinese fossils were first described as primitive birds of unclear affinities. It was Larry Martin who united many primitive avians within Enantiornithes in his 1983, 1987 and 1995 (submitted in 1992) papers. His contribution was followed by many others. Luis Chiappe contributed in assigning Avisauridae to the same clade in 1992.
Line 68. The statement about four taxa is not correct. At least, the “Nanantius” valifanovi is known from partial skeleton (Kurochkin, 1996), and more embryos were recently described as Gobipipus (Kurochkin et al 2013).
Lines 152-155. The sequences in which different skeletal elements of the holotype are mentioned is rather unusual. It seems more logical to give them in an anatomical sequence.
Line 179. It will be better to list characters anatomically – first axial skeleton, then forelimb, followed by the hindlimb. The craniocaudal midpoint of the shaft of metatarsal II is not clear. In the pes of bids, usually dorsal and plantar sides are recognized, as well as proximal and distal.
Line 184. “Strongly developed keel” is not a clear statement. Does it refer either to its depth, or craniocaudal extent, or robustness, or all of it in combination? The description on line 245 does not clarify that. It should be specified.
Line 226. Typically enantiornithine pygostyle is not clear, because Pengornithidae have a different one.
Line 268. Given that Pengornithidae have a different proximal humerus, “typical enantiornithine” humerus sounds unclear.
Line 322. Modern birds have differently built carpometacarpi and the morphology of the trochlea carpalis differs in various taxa.
line 510. My former experience shows that 1000 replications may not be enough to find the shortest tree even in a medium-sized dataset. The authors may thus try a larger number of replications to test if more shorter trees may be recovered.
Line 553. Mystiornis was not assigned to Aviasauridae not because of incomplete fusion – as currently stated in the manuscript, bur rater because of complete fusion of metatarsals in Mystiornis – see Kurochkin et al 2011, left column, third line from below. The complete fusion of metatarsals is the character that prevents placing Mystiornis within Enantiornithes but rather unites it with ornithurae. So, the discussion on lines 554-557 should be modified – even the adult Enantiornithes never show a complete fusion of metatarsals throughout its entire length, as in Mystiornis.
Line 628. The morphology of the musculus tibialis cranialis will not affect grasping, as the muscle dorsiflexes the metatarsus. Grasping, in contrast, is produced by plantar flexion by toe flexors.
Lines 670-671. Noguerornis comes from Montsec limestones which are older than 125 MYA (see Selden, Nudds 2012 book), and thus may be the oldest, or at least as old as the oldest Chinese enantiornithines.
Line 727.  Although poorly described, a diversity of the late Cretaceous Enantiornithes from Uzbekistan is notable, though represented mainly by coracoids (described in several papers by Panteleyev).


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: THE MOST COMPLETE ENANTIORNITHINE FROM NORTH AMERICA AND A PHYLOGENETIC ANALYSIS OF THE AVISAURIDAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: I'm thrilled to see this specimen finally described! It is a very important specimen, one of the most complete and best preserved Late Cretaceous enantiornithine, and the authors have done a good job with the description and the superb photographs.
Experimental design: I think the authors need to be careful with the cladistic analysis--it is plagued with missing data. I know they can't do much about it--we just need new and more complete specimens--but the results should be considered as tentative. This said, I found the recovery of North American and South American subclades of avisaurids very interesting.

The description is adequate but it could be more extensive and better for some bones. The description of the vertebrae is very succinct--there's no mention of the parapophyses, the epipophyses, and other important structures that are clearly visible (even labeled) in the figures. This specimen gives the authors the opportunity to provide a more extensive anatomical description of 3-dimensionally preserved vertebrae that are ofter difficult to interpret in the bidimensional material from China. Likewise, in the description of the coracoid there is no mention of the dorsal fossa, which is evident in the figures, and the fact that the coracoidal nerve foramen opens above (i.e., not inside) of such fossa--this is important given the variability of this character among enantiornithines. I strongly encourage expanding the description of these bones.
Validity of the findings: I don't have much to comment on the overall structure of the paper but see my detail comments in the next section.
Additional comments: 1. Between lines 54-56, the authors say that the identification of the El Brete enantiornithines "was initially met with controversy due to the disarticulated and isolated nature of the “El Brete” material, which lasted until the discovery of articulated skeletons from Early Cretaceous deposits in Spain and China." I believe the authors should give credit to the importance of the discovery of the skeleton that would later become the holotype of Neuquenornis. My 1992 paper on avisaurid tarsometatarsi, later cited by the authors, had the goal of documenting the avian and enantiornithine status of Avisauridae. The holotype of Neuquenornis played a key role because it was the first relatively complete specimen interpreted as an enantiornithine (note that this wasn't the case with the initial description of Iberomesornis) that also shared synapomorphies with the isolated bones from El Brete. Therefore, I think the authors should re-write the phrase mentioned above to something like "..until the discovery of articulated specimens from other Cretaceous deposits in Argentina, Spain, and China".

2. Between lines 67-72, the authors say that there are only 4 late Cretaceous taxa represented by articulated specimens: Parvavis, Elsornis, Neuquenornis, and a series of Mongolian embryos. I think this is a bit arbitrary and suggest some editing in order to incorporate other multi-bone discoveries such as Halimornis, Alexornis, and Gobipteryx (originally described as Nanantius by E. Kurochkin).

3. On lines 71-72, the authors refer a series of embryos from Mongolia to Gobipteryx. This has been an unfortunate historical misconception because these embryos, first described by A. Elzanowski, cannot be assigned to Gobipteryx. They can be identified as enantiornithines but assignation to the genus Gobipteryx is not justified. Even Elzanowski recognized that in his original studies. I suggest that the authors don't assign these embryos to Gobipteryx but simply refer to them as 'enantiornithine embryos'.

4. In addition to the Late Cretaceous discoveries mentioned between lines 85-86, the authors should include other remains listed by Chiappe & Walker (2002 - Chapter 11 of the book Mesozoic Birds)--these belong to historical collections by Yale and Princeton.

5. In the vertebral description, the authors use the term post/pre zygOpophysis but the correct anatomical term is zygApophysis (pre/post etc).

6. Lines 250-251, please note that a furrow in the costal surface of the scapula has also been described for the El Brete enantiornithine material (see Chiappe, 1996 - Munchner Geowiss. Abh. 30: 203-244; and Chiappe and Walker, 2002 in Mesozoic Birds).

7. It would be helpful to indicate some of the characters of the omal end of the coracoid in the photographs.

8. Line 263 has two 'the'.

9. Line 268-269, the authors say that the for the humerus, 'the proximal end is typically enantiornithine in profile: concave centrally rising dorsally and ventrally (Fig. 8)". I agree but it would be helpful for the reader to have general references to humeri such as Chiappe (1996 - ) and Chiappe and Walker (2002)--these papers describe the feature mentioned above and illustrate a variety of humeri.

10. Likewise, in line 271, please cite Chiappe (1996 - Memoirs of the Queensland Museum 39: 533-554) for Enantiornis. This paper provides a description and illustrations of the holotype of Enantiornis leali, which were not provided by Walker in his 1981 paper.

11. In line 279-280, the authors say (in parentheses) that the pneumotricipital fossa of the humerus "is perforated only in PVL 4022 [Walker & Dyke 2009]". For the record, Chiappe and Walker (2002) mentioned the presence of a pneumotricipital foramen in the humerus of enantiornithine specimen PVL 4022 several years prior to Walker & Dyke's publication (it was also figured). Here is the exact text used in Chiappe and Walker (2002): "Although the humerus of several euenantiornithines bears a distinct pneumotricipital fossa, a pneumotricipital foramen is known only for an isolated humerus from El Brete (PVL-4022; Fig. 11.8.C)."

12. In regards to the pelvis, the authors may want to make comparisons with the specimens featured in Chiappe & Walker (2002). I can't see it well in the figures but the position of the antitrochanter is important--what do they authors mean by a "dorsal antitrochanter"? Does it mean that it is positioned in the ldorso-dorsocaudal corner of the acetabulum? Labeling the figure of the pelvis would be important as well. The description could be revised, there is at least one type (lease instead of least).

13. Line 365. What do the authors mean by "posterior trochanteric crest"? This is the posterior trochanter that they are referring to. Calling this a posterior trochanteric crest is confusing: it makes it look as if they are establishing a homology with the trochanteric crest of early theropods (e.g., ceratosaurians).

14. Lines 659-664. I'm not sure I would describe the xiphoid process of the new taxon as V-shaped; it looks pretty straight and in this respect not that different from what is visible in some other enantiornithines. Also, I would be careful about comparing this with Neuquenornis because the caudal sternum of the latter is largely missing. Likewise, the hypocleidium of Neuquenornis is incomplete, so I'm not sure you could determine its length.


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: THE MOST COMPLETE ENANTIORNITHINE FROM NORTH AMERICA AND A PHYLOGENETIC ANALYSIS OF THE AVISAURIDAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No particular comments compared to the previous submission: the authors have addressed my comments and suggestions.
I only note that some notes and comments - that clearly belong to the in progress draft and apparently were not meant to be included in the submission file - are still present along the text (e.g., lines 306-7). The authors are invited to carefully check the text and remove any comment or note of the draft present in the text file before uploading the final submission.
Experimental design: The authors have followed all suggestions by the reviewers on both description and phylogenetic analysis.
Validity of the findings: As expected, updating the taxon sample of Ornithuromorpha the relationships among the enantiornithines substantially agree with most of previous studies.
(Although I usually do not evaluate a novel phylogenetic scenario based just on a priori expectations taken from previous literature, the re-analysis has confirmed that the topology in the first submission was clearly biased by taxon sampling, and had to be considered with concerns).
The authors have expanded the comparative and descriptive section, which is welcome given the relative paucity of Late Cretaceous enantiornithines with a comparable preservation as the new taxon.
Additional comments: A few taxonomic remarks. Although they may seem just formal notes, they prevent the manuscript from being emended by future publications on enantiornithines:

The new genus name for "A." gloriae should be registered in ZooBank, otherwise it is not valid under the ICZN: the ZooBank code of Gettyia must be included in the manuscript.

In line 156-7, the proper definition of a node-based clades Avisauridae should be "the LAST common ancestor of Neuquenornis volans and Avisaurus archibaldi plus all its descendants (Chiappe 1993)."
The "last" adjective must be added, otherwise, the clade definition is ambiguous (e.g., even the first vertebrate is a common ancestor of Neuquenornis and Avisaurus: you should explicitly state that Avisauridae refers to the least inclusive among all clades including the two mentioned species).

In line 705, the correct form should be: "GETTYIA gloriae (Varricchio & Chiappe, 1995) new comb.", because the authors have not erected a new species name: they have just erected a new combination for the previously-erected species "gloriae" with a new genus name.


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: THE MOST COMPLETE ENANTIORNITHINE FROM NORTH AMERICA AND A PHYLOGENETIC ANALYSIS OF THE AVISAURIDAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: as stated in my previous review.
Experimental design: as stated in my previous review.
Validity of the findings: as stated in my previous review.
Additional comments: The new version of the manuscript may be published after some minor corrections. The manuscript contains many typos and mispellings, especially in the newly added or edited parts (e.g. lines 64, 89 et cet.), and require careful edition. Some of the added references are not on the reference list. The figure 17 looks unfinished.


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: MICROSATELLITE LOCI DEVELOPMENT AND POPULATION GENETICS IN NEOTROPICAL FISH CURIMATA MIVARTII (CHARACIFORMES: CURIMATIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Yes, the MS is clear and presents sufficient field background. It is also well structured but it presents some issues related with the raw data availability and figures.

The raw data is only partially available. They should be made available for scrutiny since they complement/validate their analyses.

The figures are relevant for the discussion but some issues should be addressed:

- In Figure 1, the small map of Colombia presented on Fig, 1B should be changed to Fig.1A for clarity and better visualization. They should also change the figure caption.

- The manuscript lacks an image of the species. Since it deals with one unique species, it is important for the reader to visualize the species the MS is talking about.

- The manuscript does not present any raw data from the loci analyzed. The authors should present the raw data (electropherogram) since the MS also deals with the development of the microsatellites.
Experimental design: The MS is within the scope of the journal and the research question is clear. The methods are correctly used and the research is relevant since it deals with the Curimatidae, a clade without any kind of paper on this matter.

 I failed to find an ethics statement and cannot precise if the research was conducted following high quality standards. The unique statement provided is related with the agreement between Universidad Nacional de Colombia and Integral S.A., on 19th September 2013)… nothing presented about fishing permissions and sample gathering methods.
Validity of the findings: The main goal here is the development of microsatellites for the Curimatidae, a clade without any kind of previous studies on this field. The authors made it although some issues should be addressed on the discussion and conclusions.

The MS hypothesized about isolation by distance but the authors poorly discussed this hypothesis in the light of their results… they also didn’t presented nothing about this in the final conclusions. If isolation by distance is the hypothesis for the MS, they should be addressed within the MS conclusions.

A second issue is related with the discussion about the ENSO. Imho, the authors cannot use ENSO to explain what they didn’t observed from their results. It seems a bit speculative for me since the genetic differentiation was not significant after the exclusion of both loci putatively under selection for Las Culebras. If they don’t have differentiation, why use ENSO to explain the genetic structure that was not observed?
Additional comments: Some minor comments here:

The authors should check the journal standard for using “et al.” and verify the MS correcting it along the entire MS. Sometimes “et al.” is used sometimes the authors use the entire author list of the manuscript. For instance, check lines 38, 41, and 49 (and also the rest of the MS).

Line 36 - Please add a reference after “… South America.”

Line 68 – Remove the period marker after Barandica

Line 71 – I suggest the use of standard scientific nomenclature when referring the species rather than “vizcaína”

Line 138 - Check number format 5000 / 200,000 / 50,000

Line 213 – I’m not sure about the validity of the comparison with microsatellites from migratory marine fishes. Consider to remove this lines.

Line 224 – a priori, check Italics

Line 272 – Evolutionary potential? Please consider a review here.

Line 273 – “… in the main …”. It seems lost here. Please verify.


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: MICROSATELLITE LOCI DEVELOPMENT AND POPULATION GENETICS IN NEOTROPICAL FISH CURIMATA MIVARTII (CHARACIFORMES: CURIMATIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: I identified lack of attention in writing the manuscript. I made some observations and suggestions below:
1. Did you use next-generation sequencing? Although it is listed as a key word, no results were shown.

2. The language could be improved, particularly in the introduction and in the discussion. Please check carefully the text again.

3. The introduction is quite informative and appropriate, but the references cited in the text are not in the PeerJ norms:
a) For example, lines 38 and 52 (For four or more authors, abbreviate with ‘first author’ et al. and No comma before &)
b) Multiple references to the same item should be separated with a semicolon (;) and ordered chronologically
c) References: are not in accordance with the norms. Check carefully the references.

4. Senseless words in the text “de novo” lines 72-73. De novo is a method of sequencing, but I think it was not used in this study.

Results:
5. How many clones were sequenced? There isn't this information in the ms

6. GenBank accession numbers: There isn't this information in the ms

7. Figure 1: Figure with the delimitation of the basin should be transferred to 1A from 1B. The designation of A and B should appear in the legend.

8. Figure 3: The figures 3b and 3d should be removed (If you are sure that loci outliers are not a technical artifact, you must discuss these results further. The identification of loci under divergent selection is a fundamental step in understanding the evolutionary process because these loci are responsible for the genetic variations that affect fitness in different environments. Understanding how environmental forces give rise to adaptive genetic variation is a major challenge).
Experimental design: 1. The ms fits the standards required for original research articles.
2. The ms is a good example of a well conducted study focused on answering an objective question that is important to conservation of fish fauna in the studied region.
3. The present ms brings the first report of population genetics for a species of the Curimatidae family. These information can be used in the conservation of this species, providing a more complete insight into the evolutionary potential and the short-/long-term persistence in the Magdalena-Cauca basin.

4. Methods was not described in sufficient detail and information to allow replication. Some information was missing, such as:
a) The DNA extraction protocol is according to what author?
b) How many ng of DNA were required for the construction of the genomic library?

5. Was the sampling protocol approved by the Ethics Committee on the Use of Animals? There isn't this information in the ms.
Validity of the findings: No comment
Additional comments: The ms is a good example of a well conducted study focused on answering an objective question that is important for conservation of fish fauna in the studied region. However, I identified lack of attention in writing the manuscript. Thus, I recommend checking carefully some parts of the text and reorganization of figures.


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: MICROSATELLITE LOCI DEVELOPMENT AND POPULATION GENETICS IN NEOTROPICAL FISH CURIMATA MIVARTII (CHARACIFORMES: CURIMATIDAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Landínez-García and Marquez describes a new set of microsatellite loci in Neotropical fish Curimata mivartii. The article is well written, with sufficient references. The approach is well explained and a good example for other authors to follow.
Experimental design: The aim of the work is important. The analyses are performed with a high technical standard and  described in sufficient detail. The methods are well described. A few comments:
The authors should mention how the DNA was extracted.
PCR reactions is redundant
Why the need of using so many (42) PCR cycles in fresh samples?
The authors should have sequenced the amplicons in a few samples to confirm the STR structure.
Validity of the findings: The data is robust and statistically sound. I suggest a PC or MDS analyses to visualize the estimated genetic distances. The conclusion are well stated.
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: MICROSATELLITE LOCI DEVELOPMENT AND POPULATION GENETICS IN NEOTROPICAL FISH CURIMATA MIVARTII (CHARACIFORMES: CURIMATIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Almost all previous issues were addressed/answered by the authors and as far as I am concerned, the manuscript is good to go.
Experimental design: Please check the section 1. Basic Reporting
Validity of the findings: Please check the section 1. Basic Reporting
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: MICROSATELLITE LOCI DEVELOPMENT AND POPULATION GENETICS IN NEOTROPICAL FISH CURIMATA MIVARTII (CHARACIFORMES: CURIMATIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: After the MS review, all questions were answered and recommendations were accepted by the authors. However, microsatellites loci were not deposited in GenBank. That way, I suggest that the authors make the deposit.
The MS fits the standards required for original research articles.
Experimental design: The analyses are performed with a high technical standard and described in sufficient detail.
Validity of the findings: The present MS brings the first report of population genetics for a species of the Curimatidae family. These information can be used in the conservation of this species, providing a more complete insight into the evolutionary potential and the short-/long-term persistence in the Magdalena River basin.
Additional comments: The MS is a good example of a well conducted study focused on answering an objective question that is important to conservation of fish fauna in the studied region.


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: MICROSATELLITE LOCI DEVELOPMENT AND POPULATION GENETICS IN NEOTROPICAL FISH CURIMATA MIVARTII (CHARACIFORMES: CURIMATIDAE)
Review round: 2
Reviewer: 3

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: n/a


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

Input:
Title: A GENERAL PATTERN OF THE SPECIES ABUNDANCE DISTRIBUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall this ms is well written with clear language and well-organized structure. I also commend the author for including the data and the code in the submission. Below are a few minor issues that would require edits or additional clarification:
1. Line 27-30, "this study assumes ... when one investigates any SAD." I have trouble understanding what this sentence means. If Zipf's law for SAD is merely the author's assumption, why would one expect it in nature? Please revise.
2. Line 72, "there is (or is not)" - please change to "is there (or is there not)" for grammatical correctness.
3. Line 119, "Eq. (1) IS based on ...".
4. Line 198, "holds FOR".
5. Line 211, "f_0 is chosen such that the sum of f_r." This sentence is incomplete.
6. Lien 214, "understand" should be replaced with "explain" or a similar word (as the model itself can be understood but cannot understand).
7. Table 1. Please clarify what the range in the "Average" column (e.g., 0.008 in 1.343+-0.008) means. Is it standard deviation or standard error?
8. Reference, 2 and 3 are the same paper.
9. Line 222. What is "the first interpretation of the fractal model"? It is not clear from this paragraph.
Experimental design: The theoretical derivation is clearly laid out, and the data are adequately described. I would suggest the author to include an additional sentence or two at the end of the METHODS section to further explain how the data are analyzed to get the findings in results (i.e., how is p obtained), along the lines of "Ranked species abundances in each sample are fed into Eq. (2) to estimate the value of p."
Validity of the findings: While the analyses are valid, I think the interpretations can be further strengthened. It is perhaps not a surprise that the Zipf distribution fits SAD well (given the universally consistent shape of the SAD), and as the author acknowledged, the purpose of the ms is not to provide another comparison among SAD models. The empirical distribution of p (and the fact that it clusters around 1) is indeed more interesting. However, I do not fully agree with the author's reasoning in the last two paragraphs of the Discussion. The author's conclusion, that p exceeds and approaches 1, is partially based on the mathematical reasoning that the generalized harmonic series converges when p>1 and diverges when p<1 (Line 233-234). However, in reality r can never goes to infinity, since the number of species in a system is always finite. Thus even when p<1, the sum of r^(-p) (Eq 3) is still finite. In other words, there is no mathematical hard bound that forces p>1, which can be seen in Fig.1 where a significant proportion of the communities have p<1.

In addition, I do not fully follow the authors' argument for "a decreasing trend of p" (the second half of the last paragraph in Discussion). The author is correct to state that A_T/T_1 is positively correlated with diversity (Line 239), and that p is negatively correlated with A_T/A_1 (Line 242). However, p approaching 1 (the [incorrect] lower bound) would then imply that the ecological systems are somehow maximizing diversity. Please explain what is the theoretical basis for this implication.

A few additional minor changes that I would suggest the author to make to improve validity of the findings:
1. Line 89-91 provides two interpretations for the fractal model in ecology. I think this part is critically important to set the stage of the current student, and the two sentences included in the current ms are not sufficient. Please consider expanding this section into full paragraph(s) and elaborating on the ecological meaning of the two interpretations, to help readers better understand the context.
2. Line 127. The frequency distributions of SADs for these datasets are actually well known, since they have been extensively analyzed in previous studies, unless the author is referring to the frequency distribution of p when these datasets are fitted with the fractal model. Please rephrase.
3. An interesting finding that the author did not touch upon is the different distribution of p among studies/taxonomic groups (Fig.1). Can you explain/speculate whether the difference in distribution is due to differences in life history, behavior, sampling schemes, or something else?
Additional comments: Overall I think this ms is well written with valid analyses. However, the author's conclusion that the parameter p of the fractal model "exceeds and approaches 1" seems to be in contradiction to the results (Fig.1), where a large number of communities have p < 1. In addition, there are some inconsistencies in logic in the author's argument (the last paragraph in Discussion) to arrive at this conclusion. I would encourage the author to rethink and revise the discussion and strengthen the interpretations of the 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: A GENERAL PATTERN OF THE SPECIES ABUNDANCE DISTRIBUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study by Su, presents a very interesting problem in community ecology and in attempting to understand diversity patterns. As the author suggests, I agree that understanding the shape of the species abundance distribution is a crucial step to understanding the mechanisms regulating biodiversity and community assembly. A lot can be learned from studying SADs, but I also agree that it has been overall problematic. I enjoyed very much reading the manuscript as I found it very well written and easy to follow.

Although the manuscript is interesting and I applaud the intention of providing hypothesis for a general law in SADs, to me they are a bit confusing.I would like a little bit more explanation or maybe change the wording so that it the hypotheses are easily followed. I am not sure how the relationship between total abundance and energy assimilation is laid as a hypothesis for the shape of SADs in every community.
Experimental design: The research question is definitely relevant and the methods are well described and in sufficient detail. The manuscript is extremely clear and very reproducible.
Validity of the findings: I am concerned about the goodness of fit of the de Zipf model on each of the 19000 communities. There is no information in the manuscript regarding the goodness of fit and in this sense it is unclear to me if the distribution of the fractal parameter of the Zipf distribution is just a product of the generalized shape of the log-log relationship between the rank and the relative abundance of species. To clarify this point I performed a Monte Carlo simulation in which I generated 20000 communities, the first 10000 following a Zero-sum multinomial ddistribution with theta drawn at random from a uniform distribution between 10 and 50 and the size of the community drawn from a unifirom distribution between 10000 and 15000. I generated the second 10000 communities using a Poisson log-normal distribution with N=50, mean = 2 and sigma = 1. For each of these 20000 random communities I estimated the value o p. It turns out that the range of p resulted to be between 0.38 and 1.81 with mean 0.95 +- 0.22 and median 0.95. Even though the communities where generated with very different processes (one is product of a zero-sum game and the other one has been hypothesized to be the product of processes ranging from purely mathematical to niche based mechanisms). Does this mean that regardless of the generating process of the community assembly, the diversity and abundance of organisms is regulated by the amount of energy in the community and the transformation of the total energy by each species? Or is it totally and statistical artifact? To answer this, I think the solution would be to fit some alternative models and see if the Zipf with a fractal parameter of 1 is more likely or at least as likely as other generating models that have been previously proposed. If this turns out to be a real pattern then, I think the author has done an immense contribution to community ecology.
Additional comments: Minor comments.

line 211: Something seems to be missing here.
line 230: Should read "fractal and diversity theories"


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

Input:
Title: A GENERAL PATTERN OF THE SPECIES ABUNDANCE DISTRIBUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: I respectfully disagree with the author's rebuttal regarding the last paragraph of the discussion, namely that p is constrained to be close to 1 because of two contrary forces at play.

In the first part of the argument, the author states that "p should be higher than 1", because "if sample size is not fixed in advance and can be allowed to be larger and larger, r might be infinity in theory". Mathematically, r going to infinity implies that species richness S is infinity. However this would never be the case. Richness is always a FINITE (albeit large and/or unknown) number even in theory, as long as our studies are confined on earth. And as long as S is finite, Eqn 3 always converges, regardless of whether p>1 or p<1. In other words, the statement that p should be higher than 1 is derived based on the incorrect assumption that richness can go to infinity.

In the second part of the argument, the author states that "the diversity generally presents a trend of (maximizing diversity) because the energy conversion among species increases the community entropy." I still find this statement quite confusing, and I think would benefit future readers of the paper if the author could elaborate on why this is the case. Does the author mean that the ecological system is maximizing entropy? Or entropy production? Is there a reason why the ecological system is expected to have this kind of behavior?
Additional comments: No additional comments


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

Input:
Title: A GENERAL PATTERN OF THE SPECIES ABUNDANCE DISTRIBUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: As I mentioned in the first review, the paper is in general well written and very clear. It is overall easy to understand and provides enough background of the problem. I would suggest to check a couple of places where there are some grammatical mistakes. For example,
Line 109, is estimated as follows
Line 113, I would delete line 113. I understand that this is in response to reviewer 1 but to me is chela enough in line 108 that equation 2 is used to obtain the MLE of p for each SAD.
Line 261, replace most by strongest or biggest.
Experimental design: No Comment
Validity of the findings: I feel that I would like to see more discussion about the reason why all communities are expected to have a fractal parameter of 1 irrespective of the community assembly process. The author has a very interesting paragraph explaining this that I would like to see added to the discussion. I also encourage the author to add a statement about the R2. I apologized that I missed it the first time but there was no reference in the main text that R2 was calculated for each SAD as a goodness of fit measurement. In my opinion the table that the author added as supplementary material and shows in the rebuttal letter should be also added to the main text.
Additional comments: First of all thank you very much for the honoring offer of working together. I would love to collaborate with the author in future studies attempting to understand how do different mechanisms of community assembly can result in similar SAD predicted by the same fractal parameter. Also I would love to explore what is the relation of the energy transfer and the community assembly process.
Second, I am glad to see that the author does not think that the findings are an statistical mistake. To me it makes now sense that the overall energy in a community determines the diversity irrespective of the community assembly process. However, there seems to be something that I am still not understanding. The author shows in one of his previous papers (Su et al 2016.) that the fractal parameter is related to other diversity indices. In general, you can think about this diversity indices to be a measurement of species richness and the distribution of abundance across species, (i.e. how homogeneous or heterogeneous is the distribution of abundance across species.). This means that in general, the larger the diversity index, the higher the species richness (this is not necessarily true when the abundance is homogeneously distributed across species), which necessarily translates to the fact that the higher the fractal parameter the higher the species richness of a community. How can the author reconcile the huge differences in species richness in communities across the globe with the fact that the fractal parameter in every community should be around 1, or close to 1? Is there something in this explanation that I am missing? I think that the author needs to expand a little the discussion keeping in mind the natural history and the implications that this findings have into understanding the regulation of biodiversity. What happens for example in SAD of communities that have been recently invaded by organisms which have a very high abundance compared to their native places? How can you explain that a community in the tropics with hundreds of species will follow the same rules of a community in the temperate region with just tens of species? Obviously, the energy available for the community is much lower in the temperate region that in tropical regions but then, how does a community trees in a boreal forest can have the same fractal parameter than a tree community in the amazon with at least 1000 species? I am not sure how the increasing trend of community entropy is not related to the generating processes when the generating processes such as speciation, drift and selection necessarily change the entropy of the system by introducing new species, selecting for better adapted ones and randomly monopolizing space? The author states that "the entropy of the community increases with the process of energy transfer" but would this not mean that this results in a larger fractal parameter? If shannon and simpson indices are entropy indices and the fractal parameter is related to them, why would you expect that the fractal parameter is the same for any community in the world? I do think that the findings are very interesting but given the sensitivity of the topic and the importance of the results i would encourage the author to explain this issues in more detail in the discussion so that people more familiar with community assembly mechanisms can understand the interpretation of these results better.


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: FAMILY-BASED WHOLE-EXOME SEQUENCING IDENTIFIES NOVEL LOSS-OF-FUNCTION MUTATIONS OF FBN1 FOR MARFAN SYNDROME
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this manuscript, Pu et al. describe 3 FBN1 gene mutations (c.5027_5028insTGTCCTCC, c.5856delG and c.8034C>A) identified by next generation sequencing in 3 Han Chinese families. These 3 mutations result in premature STOP codons. These mutations are unreported in large public databases gathering variants identified by exome and genome sequencing as gnomAD. These mutations have not been previously reported in Chinese patients (more than 150 reported to date) nor in the FBN1 locus specific databases UMD-FBN1. The manuscript is clear and concise, and results are presented in a manner that is logical.

I do regret, however, the absence of clear clinical description:
- line 126: “…diagnosed with MFS…”
- line 127: “His father (I-1) also had MFS…”
- line 132-133: ”…all had MFS with…”.
I suggest adding a table with a complete and clear description of all symptoms (skeletal, ocular and cardiovascular systems as well as other associated systems) for each of the 23 members of these families.

The second important point is in the way the authors discuss the pathogenicity of these mutations (lines 192 to 205). Even though these affirmations are right, the major point for each of these 3 mutations is the creation of premature STOP codons. The question is then about the activation of nonsense mediated decay (mRNA degradation and resultant haploinsufficiency) or the production of truncated proteins.
Are the mutated mRNAs stable?
Are the mutant proteins detected?

[For more information please refer to:
- Schrijver I, et al. Premature termination mutations in FBN1: distinct effects on differential allelic expression and on protein and clinical phenotypes. Am J Hum Genet. 2002;71:223–237. (PMID: 12068374).
- Tjeldhorn L, Amundsen SS, Barøy T, Rand-Hendriksen S, Geiran O, Frengen E, Paus B.
Qualitative and quantitative analysis of FBN1 mRNA from 16 patients with Marfan Syndrome. BMC Med Genet. 2015 18;16:113. PMID: 26684006
- Faivre L, et al. Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan syndrome or related phenotypes and FBN1 mutations: an international study. Am J Hum Genet. 2007;81:454–466. (PMID: 17701892).]

Publication of such data is important as they often lie within clinical laboratories and are not shared with the medical and research communities. The clinical spectrum associated with FBN1 gene mutations is very large and complex. A lot of data has then to be colligated to identify genotype-phenotype and gene-function correlations.


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: FAMILY-BASED WHOLE-EXOME SEQUENCING IDENTIFIES NOVEL LOSS-OF-FUNCTION MUTATIONS OF FBN1 FOR MARFAN SYNDROME
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study is pretty straight forward and authors followed a standard procedure to identify mutations in FBN1 lead to MFS using whole exome sequencing.
Experimental design: The methods described provide enough detail information for replication. The question of this study not clear. It seems that authors don’t familiar with the rule of mammalian nonsense mediated mRNA decay due to frameshift or stop-gain variants. This study doesn't provide sufficient novelty to fill knowledge gap.
Validity of the findings: The impact and novelty are low.
Additional comments: In this manuscript, authors performed whole-exome sequencing on patients with Marfan syndrome (MFS) from 19 families and identified novel loss-of –function FBN1 variants in three families, two frameshift and one stop-gain variants. These variants were segregated with MFS in families. Authors tried to investigate the role of novel genetic variants in determining MFS clinical phenotype. Authors have discussed the domain location and local conservation of these variants in FBN1 and compared the diversity of clinical status of patients.
Concerns:
1.	All of these variants lead to haplo-insufficiency of FBN1 and individuals carried these mutations should have similar clinical features with MFS based on the clinical criteria MFS diagnosis. The pathogenic variants lead to haplo-insufficiency of FBN1 were frequently seen in patients with MFS.   It seems that authors don’t familiar with the rule of mammalian nonsense mediated mRNA decay due to frameshift or stop-gain variants.
2.	There is insufficient novelty or significance of this manuscript to meet publication criteria in Peer J.


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

Input:
Title: FAMILY-BASED WHOLE-EXOME SEQUENCING IDENTIFIES NOVEL LOSS-OF-FUNCTION MUTATIONS OF FBN1 FOR MARFAN SYNDROME
Review round: 1
Reviewer: 3

Output:
Basic reporting: English.  Unclear/wrong sentences or words are present  throughout the article
                  Row 42:  “major features”. Why major features?
                  Row 51:   “in the genes” , only one gene
                  Row  62: ”relate to the dominant negative”….
                   Rows 63-64:  “Therefore…….MFS”. Marfan syndrome is at the moment associated
                                            with FBN1 gene only. What do you mean with “atypical mutation”?.
                   Rows 104-105   “Then……module”. Unclear sentence
                   Rows 136-137  “Although…..symptoms”  What the Authors mean by “obvious
                                                    symptoms”?
                    Rows 139-140:  “elongation of fingers” = arachnodactily;  what the Authors mean
                                                     by “were attributed to being healthy”?
                    Row 146: “we removed these variants”
                    Rows 152-153  “MFS……….function” english and meaning are both unclear
                     Row 158  “a recurrent insertion”. What the Authors  mean by recurrent? They said
                                          previously that it was a “new muation”
                     Row 176   “several functional mutations”…..All rare mutations” Are the Authors
                                           talking about the three FBN 1 mutations detected in the 3 families or
                                             about other mutations?
                     Row 187-189. English  and meaningunclear
                     Row 198-200 . English and meaning unclear
                     Row 206-207 . English and meaning unclear

Literature references.
References are sufficient. Within the manuscript Reinhardt 2015 is reported buti s omissed in the reference section.

Background.
The background is not very updated. The Author scite many genes associated with marfan syndrome but actually only one, the FBN1 gene, is associated with it.
The Authors mention a “a complex pathogenesis underlying MFS” but do not mention the up-regulation mechanism of TGF-beta signalling.
The Authors use the term “major features”  that has disappeared in the latest guidelines (Loeys et al 2010), substituted by the term “features characterizing the disease”. Moreover they mention only the three clinical  manifrstations by omissing the two genetic features
Finally, it is not clear why they emphasize  the fact thet Marfan patients carry dominant negative or haploinsufficient mutations.

Professional article structure, figs, tables. Raw data shared.
An important table is missing: the one with all the clinical manifestations (such as myopia, mitral valve prolapse, scoliosis, ectopia lentis) regarding the 19 subjects of the three families. Moreover, the Authors should report the cardiovascular manifestations (such as hypertension and dyslipidemias) which can  modulate  some clinical features of the patients.
Moreover, a second Table should report all the mutations detected in the genes associated to TGFbetapathies or thoracic aortic aneurysms/dissections because we know from the literature that milt mutations in these genes may rapresent modifyer genes of Marfan syndrome. The Authors do not mention modifier genes in the background section.

Self-contained with relevant  results to the hypotheses.
The Authors aim to investigate the role of novel genetic variants in determining clinical phenotype.
With this aim I expected a deep presentation of the clinical features detected in all 19 subjects, includin healthy relatives. This is the only way to perform a genotype-phenotype correlation. Therefore this reviewer thinks that important are missing. On the other side they analyzed many genes potentially modifier genes of Marfan syndrome but no data are reported. Also these data would allow improve a genotype-phenotype correlation.
Furtheremore, it is difficult to believe that the Authors found only one mutation in FBN1 gene in each patient. FBN1 not-pathogenic variants  should also be reported in a third Table.
Experimental design: Study subject. The age of the 19 subjects should be reported here or in the pedigree.

Methods. Sufficient.
Validity of the findings: The Authors report 3 new mutations all causin LOF = loss of function. Since they have 3 families they shoud concentrate result and discussion on genotype-phenotype correlation by reporting al the mutations detected in the other genes and all clinical features of the 19 subjectes. Being only 3 mutations I would avoid a comparison of these mutations functions with those reported in literature.

Results.
The resultus are confusing since contain in part information regarding methods.

Discussion.
The discussion is poor and unclear/confused.
The diagnosis of Marfan syndrome is still based primarily on clinical manifestations since  in part of Marfan patients we still do not detect a pathogenic mutation. Of course  the age of diagnosis is around 20 years because earlier most of the times sufficient clinical features necessary to perform the diagnosis are still missing. A correct follow up requires early diagnosis
Additional comments: The Authors performed whole-exome sequencing on 19 individuals from 3 Han Chinese families affected by Marfan syndrome. Their purpose was to investigate the role of novel genetic variants in determining Marfan syndrome as reported in the ‘Background” of the summary.
The article needs deep revision. Clinical and genetic data necessary for genotype phenotype correlation are missing at present.


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: FAMILY-BASED WHOLE-EXOME SEQUENCING IDENTIFIES NOVEL LOSS-OF-FUNCTION MUTATIONS OF FBN1 FOR MARFAN SYNDROME
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article written in English is clear.
The sufficient introduction and background are provided.
The structure of figures and tables are fine.
Experimental design: The experimental design is fine.  It is a standard exome data analysis approach.
Validity of the findings: Impact and novelty are low.
The conclusions are appropriately stated,
Additional comments: In this manuscript, authors performed whole-exome sequencing on patients from 3 families with Marfan syndrome (MFS) and identified novel loss-of –function FBN1 variants, two frameshift and one stop-gain variants. These variants lead to premature termination of FBN1 and therefore, they are pathogenic variants for MFS. Authors have addressed some questions that brought up by reviewers.  However, there are still some concerns about this manuscript.

Concerns:
1.	This manuscript lacks sufficient novelty or significance information to readers. In the abstract, authors purposed to investigate the role of novel genetic variants in determining MFS clinical phenotype. However, a lot of FBN1 pathogenic variants that lead to haplo-insufficiency of FBN1 have been reported and the patients’ clinical phenotypes have been well studied. It may be interested to see whether the clinical phenotypes are difference between Chinese patients with FBN1 PTC mutations and that of patients of European American descent.
In addition, in the discussion, authors propose to conduct protein truncation test to confirm the activation of nonsense mediated decay or the production of truncated proteins. However, such studies cannot generate any novel information for FBN1 PTC mutations because they have been well studied.
2.	Since all of the mutations identified in this study lead to premature termination of FBN1, the discussion of local conservation of the amino acids mutated is not necessary and the figure 2 can be removed.
3.	The FBN1 variants identified in these families are definitely the causative variants for MFS in these families. There are no evidence that mutations in FBN3 lead to MFS (JACC 72(6):605). Therefore, it is not necessary to describe the FBN3 variants.


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: MTBSEQ: A COMPREHENSIVE PIPELINE FOR WHOLE GENOME SEQUENCE ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: Authors use their pipeline to identify SNPs associated with drug resistance and present a table with sensitivities and specificities. While it is absolutely helpful to see how MTBSeq compares to similar tools/pipelines, I note that Sanger sequencing is used as the gold standard, instead of phenotypic DST. If the aim is to provide information on accuracy for detecting drug resistance, DST would be the appropriate gold standard. Please show the calculations with these data as the gold standard, in addition to the current table (as this is helpful to assess the accuracy of SNP calls made using the various pipelines).

Authors recommend using the ‘low-freq’ option to detect minority resistance alleles. Would it be possible to provide some information on the accuracy of this mode for detecting these minority alleles, especially as the authors recommend using it for detection of all resistance-associated variants (lines 205-207)? The numbers might be too small using this dataset; have authors tested on any other datasets with drug resistance?

The authors state they include base quality recalibration in their pipeline, but what positions were used for this? From authors’ previous work (and reviewing the manual on git), these seem to be known resistance-associated mutations, but a supplementary table with these sites listed should be included in the manuscript. This is also important to include, as the reader should know which resistance-associated mutations are being investigated with MTBseq.

Are authors excluding variants within the PE/PPE regions? If so, please indicate this in the manuscript and provide a table listing the positions that are automatically masked.

Line 179 – ‘joint comparison’ – do authors mean they did multi-sample SNP calling was done? Or was this single calling, with VCFs merged afterwards? Please clarify.
Validity of the findings: I note in the Supplementary Table that pncA mutations (it is not indicated which one, so please specify) were reported in low-freq mode for MTBseq, but reported as consensus calls for TBprofiler and PhyResSE. It would be helpful to know what differences in the pipeline were responsible for this, as this suggests the alternative allele was the majority in other pipelines and somehow only minority in MTBseq.

On line 208, authors state their phylogenetic tree is on par with that of TBprofiler and PhyResSE. First, could authors please put these trees in the supplement so this can be assessed by the reader? Also, did both of those tools also use FastTree2, along with the same model of nucleotide substitution? If comparing trees (constructed with the fasta from the author’s pipeline), the same tool and version should be used across all tools.

Figure 2 – As a bootstrap support of 80% is often used to assess confidence, it would be useful to have this indicated instead of 70%.
Additional comments: This pipeline has the potential to be very useful. I really like that it can be run overall and also by module. However, authors state the aim of this pipeline is to assist non-bioinformaticians and those without programming expertise, but this is definitely not ready for such users. One key issue is that the pipeline has only been tested on Linux and no instructions are provided for installation on any other OS, other than to tell the user to set up a virtual machine. This is something that absolutely needs to be addressed if the authors want this pipeline to be used readily. Average clinical microbiologists (and even some bioinformaticians) do not work on a Linux OS. At the least, please set up clear instructions and test this pipeline on a Mac OS.

Regarding the default settings, it would also be helpful to have these stated explicitly for each tool (e.g., the defaults for BWA are not provided). It would also be useful to have a summary table of all the filters that are applied. For example, I didn’t see any filtering on mapping quality or removing reads that map ambiguously, or filtering on allele frequency?

If the pipeline allows users to modify default settings, such that a variety of different thresholds can be used, is it really going to allow for ‘standardized’ analysis across studies? Perhaps useful to elaborate a little on this point.

Minor point - would it be possible to remove the requirement for ‘library’ in the naming scheme or add some flexibility to this? This is not standard naming protocol for many institutions or sequencing centers.


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: MTBSEQ: A COMPREHENSIVE PIPELINE FOR WHOLE GENOME SEQUENCE ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: ### Clear and unambiguous, professional English used throughout.
The article submitted by Kohl et al. for the most part is written well. There are quite a few instances throughout the manuscript where typos were found or sentences were constructed in a confusing manner/ hard to interpret on the first read through. Please address the highlighted sentences/grammar mistakes in the returned review manuscript. Some examples are extra "s" at ends of words, incorrect use of conjunctions or the omission of fullstops.
Furthermore, line 82-88: All one sentence! Rephrase for reading ease.

### Literature references, sufficient field background/context provided.
One of the big flags is the lack of proper referencing, especially for the tools against which MTBseq was benchmarked. Prime example on line 160.
Furthermore, there is no mention of KvarQ, mykrobe, TBprofiler etc. in the introduction. These should be mentioned as they were benchmarked as part of the work presented.

Line 78: "....makes comparison of results virtually impossible". This statement is too strong, please rephrase. Results may not be identical between labs/groups but results are comparable and generally agree for the most part.

Line 69-70: "....no standard analysis pipeline....proposed so far" ....What about TBprofiler?? http://tbdr.lshtm.ac.uk/

Line 149: Suggest phrase change from 'critical issue'.

### Figures relevant, high quality, well labelled and described
Fig1:
Nice and easy to understand.

Fig2:
- Either expand on "outbreak" or provide a reference to the data/publication. All figures should be fully able to standalone and be interpreted without referring to the main body text.
- Check grammar and syntax in FastTree sentence, highlighted.
- References needed for tools used.
- Groups are indicated by coloured labels, but what does "d12 groups" stand for and what is "Support value"? Need to be defined for the less experienced user.

#### Raw data supplied
- Pipeline is fully available on Github but on line 157: 91 strains from Sierra Leone --where is this data? Is it publicly available? Also, data pertaining to what is referred as "Sanger" sequencing/genotyping in Table 1/ Table S1 is missing and should be included.

- Change the supplemental table to .csv/.tsv file format unless it is a requirement of PeerJ to produce supplementary data as an excel sheet.

Fig3:
- "identical spoligotyping patterns" was not stated or mentioned in main body text for these isolates. Lack of continuity with novel information provided in a table legend.

- Either expand on "outbreak" or reference the data source/publication. All figures should be fully able to standalone and be interpreted without referring to the main body text.

- If one isn't familiar with genomic distances and a distance matrix, it is unclear how the 4 isolates are "clearly separated". Suggest adding an explanatory sentence e.g. closely related strains are denoted by smaller pairwise distance values. Need to take audience into account if MTBseq is to be pitched as a diagnostic pipeline and accessible to all.

- "forming a tightly related cluster" --guessing this refers to the phylo tree?? It's out of context here and by what I can see on the tree, wrong?
1024-01, 3929-10, 6331-04 and 6821-03 do not form a tightly related cluster; 6821-03 appears as an outlier and 6631-04 separates from 1024-01 and 39239-10.
Experimental design: Kohl et al. present MTBseq, a pipeline which takes raw MTBC sequencing data, maps it to the reference H3Rv genome, detects variants associated with drug resistance and classifies the phylogenetic relationship between samples. They claim that the pipeline is customizable, expandable and high throughput and can be run on a laptop or desktop computer. They argue that a standardised pipeline is required for data analysis that is
accessible to all, regardless of their bioinformatic skills or specialised working environment.

### Pipeline issues
Line 122-123: "With MTBseq, we aim to provide a high throughput MTBC analysis
123 pipeline that is customizable, expandable, user friendly and, most importantly, standardized."

It is hard to see how MTBseq is high-throughput. The pipeline runs one sample at a time over a maximum of 8 threads and cannot be made high-throughput unless the user goes to a lot of effort to make it so. It goes against the ethos that is suggested throughout the paper regarding ease of use for those less experienced/ with no experience of bioinformatics. Suggest re-word.

With regards to MTBseq being customisable and expandable, I strongly feel that the authors should invest some time in highlighting examples and providing directions for this in their .readme file. Again, ease of use should be a priority here and at the moment there is very little/no direction for how one can change the 'standard' settings for each module in the pipeline or how to go about changing module entirely. For instance, can a user actually swap out the mapper and ensure both its success and compatibility with the rest of the pipeline?

With this in mind, I strongly encourage the authors to provide some toy data for their pipeline. This will not only allow less experienced users to navigate through each module/some of the modules of the pipeline and will provide invaluable information regarding input/output file formats. Refer to "Ten simple rules for making research software more robust", rule 9 (toy data)(http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005412).

I suggest improving checks on system return codes to allow for the pipeline to fail gracefully upon hitting an error. I ran the pipeline and allocated 10G of memory to process 3 toy MTB datasets. The pipeline stalled at the "indelrealigner" stage of the GATK module and I wouldn't have known that the process ran out of memory if the cluster had not notified me so. I could also not see any recommendations in the readme file regarding RAM usage. Strongly suggest fixing this bug. The pipeline ran well up until this error.

# .readme file
Needs to be improved and made more user friendly, especially for those not too familiar with bioinformatics. For example,
"You can ensure that MTBseq is executable from anywhere on your system by adding the MTBseq_source folder to your PATH variable or creating a symlink of MTBseq.pl to a folder that is already in your PATH variable."
would make little to no sense to an inexperienced user. Suggesting for the addition of example code.

- A note: Rule #4 from "Ten simple rules for making research software more robust" : Version your releases. I cannot see a --version flag defined in the readme file for MTBseq?

- usr/bin/perl should be replaced with usr/bin/env/ perl

- Typo (space) in "git clone https://github.com/ngs-fzb/MTBseq _source"

- States in the manuscript and readme for MTBseq that the reference genome can be changed to whatever bacterial reference you like. Some toy data highlighting this would be a useful addition.

- Line 213-215: Placement of this sentence here is odd - suggest switching it to methods.

- Line 124: "Therefore, all parameters are set to default values". What are these?
They should be specified in the manuscript/readme --can only be found in log files once pipeline has run.

- Why were the specific tools chosen over others? No clarity on this provided.

- Line 98: Define InDels

- Line 170: "Low frequency detection mode" has not been defined?
Validity of the findings: #### Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
Kohl et al. present MTBseq, a pipeline which takes raw MTBC sequencing data, maps it to the reference H3Rv genome, detects variants associated with drug resistance and classifies the phylogenetic relationship between samples. They claim that the pipeline is customizable, expandable and high throughput and can be run on a laptop or desktop computer. They argue that a standardised pipeline is required for data analysis that is
accessible to all, regardless of their bioinformatic skills or specialised working environment.

#### Methods described with sufficient detail & information to replicate.
Stating that the MTBseq pipeline is "standardised" and yet fully customisable seems like an oxymoron to me. I strongly suggest re-wording this to reflect what I think the author means i.e. that the pipeline can either be run using default tool parameters or it can be customised by the user if required. "Standardised" suggests assurances that results will be the same regardless of platform used. This is not the case and in fact the authors allude to this themselves in the introduction (although too strongly) in that analysis results tend to vary between labs. Pipeline standardisation can be achieved using pipeline managers or containers (Docker, Snakemake, Nextflow).

- Line 171-172: Which are the 11 strains that could not be resolved by classical genotyping? These cannot be seen easily in Table S1?
- Line 180-181: Again, what are the thresholds for "unambiguous variant detection"? Need to be mentioned.

- Line 204-207: Please expand/speculate as to why this was the case.

- Line 229: "paving the way for efficient application of WGS"...where/how? Sentence is incomplete.
Additional comments: ###Overall
I feel that MTBseq needs improvements in the areas outlined above prior to publication to ensure accessibility and ease of use for those implementing the pipeline in the intended audience.


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: MTBSEQ: A COMPREHENSIVE PIPELINE FOR WHOLE GENOME SEQUENCE ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: In previous review, I requested that authors share the positions used for recalibration as a Supplemental Table in the manuscript. Authors argue it is sufficient to have this file on the GitHub repository and not in the manuscript. I disagree. Authors have use this list of resistance associated mutations when running MTBseq on the 91 strains from Sierra Leone and present sensitivity and specificity of resistance calls (vs. Sanger sequencing) based on these sites - therefore this data should be transparent and readily accessible to the reader as part of the publication. As authors express concern that it would be misleading to include these data, they can add a specific statement that their list of resistance-associated sites may not be comprehensive and that users are encouraged to produce their own preferred variant lists as desired.

I would like to thank the authors for clarifying the filtering process used by MTBseq and adding the relevant citation to Comas et al. 2010 for repetitive regions.

Line 114 - please provide the appropriate website for Picard (I recognize there is no citation for this but a link is needed)
Experimental design: Regarding comparing their pipeline to phenotypic DST - the authors' rationale makes sense to not include this. Thank you for the explanation. I might suggest removing the part about phenotypic data from the following statement, as it is not actually relevant to the aims herein and can potentially cause confusion: "The dataset used consisted of 91 well-characterized strains of a collection from Sierra Leone, for which both WGS data (ENA accession number PRJEB7727) and Sanger sequencing and phenotypic data was available."

Thank you also for adding cautionary comments regarding low frequency variants.

I thank the authors for providing some additional detail about the thresholds for variant calling in Section 3. However, I am concerned by the threshold of only 75% for calling a SNP. This is very low. If we have depth of 100x, this suggests it is acceptable to have up to 25 reads with the reference for this to be called a SNP - when in fact it suggests there may be a mixed population. While I recognize this is the default and can be altered, many users (unfortunately) will simply run this with default settings, taking this as a recommendation of best practice from a group with extensive TB genomics experience. Please increase this to a more confident threshold (e.g., 85-90%, as is found in other published genomics pipelines.

When authors state "variants need to be indicated by four reads mapped in forward and reverse direction" do they mean 2 forward reads and 2 reverse? Or 4 and 4? Please clarify this statement. I assume it is the former, because they then state "at least 4 calls with a P[capitalize]hred score of at least 20."

Please also clarify - are you using the BWA 'MEM' algorithm? This should be explicitly stated in the manuscript.
Validity of the findings: Thank you for the changes made in response to my comments here.
Additional comments: This pipeline is difficult to install and requires a lot of specific software and version numbers. Currently, this is not user-friendly (while I can respect the authors feel otherwise). While I can also appreciate that people who are non-bioinformaticians in the same institution as the authors have anecdotally been able to use this tool, this is not representative of the majority of institutions who would potentially see value in this tool.  Please set up a bioconda or homebrew package to facilitate easier installation for users. This will make the tool much more accessible to others, which authors say is their intent, and this should be a minimum required for publication of this pipeline.

In relation to this, I would note that authors declined the other reviewer's request to present results in tab-delimited format because "the excel format has its advantages especially since both the article and MTBSeq software are not specifically targeted for bioinformaticians and programmers." This seems a bit incongruent with authors' expectation that the user must know basic linux to use their tool; if a user can run linux commands, it's not an unreasonable expectation that they should be able to open a tab-delimited file into a text editor - or Excel - based on their needs.


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: MTBSEQ: A COMPREHENSIVE PIPELINE FOR WHOLE GENOME SEQUENCE ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am happy with the improvements that the authors have made with regards to grammar mistakes, sentence construction and overall readability of the manuscript. The corrections made to figures and supporting information are also overall acceptable. I am happy to see the mix-up with the publicly available datasets is corrected, that the Readme and error logs for the tool have been improved and that version control has been implemented. I still disagree with providing excel sheets rather than .csv/.tsv tables (which Excel will open with ease) as there are numerous formatting issues and errors that can occur (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-1044-7). I recommend this work for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: MTBSEQ: A COMPREHENSIVE PIPELINE FOR WHOLE GENOME SEQUENCE ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have assed only the software implementation and usability.

SUMMARY
=======

Code is not written by a software engineer, but is at a good standard.
No tests or testing scripts are included.
Would benefit from an example run through with data.
Needs to be packaged into Homebrew or Conda before publishing.

COMMENTS
========

Docs should be in Markdown not PDF

These are all core modules and don't need installing
- FindBin (v1.51)
- Cwd (v3.62)
- Getopt::Long (v2.5)
- File::Copy (v2.30)
- List::Util (v1.49)
- Exporter (v5.72)
- vars (v1.03)
- lib (v0.63)
- strict (v1.09)
- warnings (v1.34)

Why is it limited to 8 threads?
"MTBseq is able to run on 1 thread up to a maximum of 8 threads. "

"The re-compiled executables MUST be located within the appropriate folders:"
This is no acceptable, even via symlinks.
If the tools are in my PATH, it should use them.

Please don't distribute .tar.bz2 and untarred versions of the
samtools, bwa, picard and GATK tools

Are you legally allowed to distribute the GATK 3.8 JAR file?
I thought you needed to sign a click through licence?
And that licence is for personal use only?
If people install this on a cluster they may be violating the licence.

Your pipeline produces lots of intermediate files, inc many large BAMs.
Have you considered piping them together?

Much of the pipeline is just iterating over system() and print() commands.
Have you considered using a workflow system like Snakemake, Make, or Nextflow?

MTBseq.pl  --version
Should print ONLY "MTBseq 1.0.1"

Author has written their own FASTA reader/writer, revcom and codon functions.
Did you consider using Bio::Perl or similar?

Lots of 'print $logprint || die' style lines.
This should be wrapped into a separate function.

Could implement your own mean() median() to avoid dependncy on
Statistics::Basic, but that would still leave the non-core MCE module.

The TBtools has masses of hash assignments for SNPs and lineages.
This would be much better configured as a separate config file,
to separate the implementation/code from the meta data.

Needs a --test or --check option to ensure everything is ready and working.
For example, I accidently set my BWA path wrong and got this:
".... 2>> /Users/tmp/tb/Bam/SAMPLE_LIB_FOO.bamlog failed: 32512"
Perhaps check at start of script that 'bwa' exists and works etc first.

Needs a walkthrough tutorial with some public data:
eg.
wget ftp://ena/tb/R1.fq.gz
wget ftp://end/tb/R2.fq.gz
MTB_seq.pl ....

It is not made clear which verion of Java is needed for GATK and Picard?
1.7, 1.8, 1.9 ?

The example command lines say "MTBseq" but I only have "MTBseq.pl" ?

It is not flexible in the filenames it will accept.
Our illumina instrument has _001 etc after the R1:
Sample18231_S81_R1_001.fastq.gz
It would be good to support an "--input_table" of the form
  NAME <tab> READ1 <tab> READ2 <newline>
or similar.

I managed to run MTBfull with 4 threads ok on a single isolate.

It placed everything in the current directory.
This is not ideal behaviour in a command line tool.
Perhaps add an --outdir or similar option.

Most users will not want all the temporary files.
Perhaps have a --clean_up / --sauber_machen option to only keep final files.

END.


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: OPTIMIZING THE TRAINABLE B-COSFIRE FILTER FOR RETINAL BLOOD VESSEL SEGMENTATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1) The language is clear

2) The Literature is the introduction is old. There are many new new references available in the field.
for example:
Line 57:
Recently, a trainable template matching method for vessel segmentation is introduced (Azzopardi and Petkov, 2013b; Azzopardi et al., 2015; Cinsdikici and Aydın, 2009; Fraz et al., 2012a; Hari et al., 2017; Sofka and Stewart, 2006).
This is not recently! 2006, 2009 and even 2012 is too old for this field.

Action to be taken: Add new references to the introduction and related work

3) Many typos here and there.
for example:
Line 88:
complex scenes.4) Detection----> complex scenes, and 4) Detection
Line 103:
shown in 4. ----> what 4? Figure 4 you mean?
Line 104:
difference of Gaussian (DoG) ----> defined before.
Line 111:
(as shown in (1)) --->? Figure 1?
Line 154:
* The optimization---> What *?
Line 233:
point 5 is at separate line where the other points are combined in the previous paragraph.

and many more.

Action to be taken: English editor need to check and validate the language, grammar, spaces and commas.

4) The figures' resolution is low.

Action to be taken: Enhance the resolution to be high

5) Table 3 label is wrong. The authors mean "summary" not "summery"
Experimental design: The research question is clear, and the experiments are well defined as well.
However, the statistics are not very clear. For example, Table 8  should show "that the noise size has no significant effect on the result for all of the given three datasets,"
but where is the statistical numbers to show that the new results has significant differences with the previous results?
Did the authors study the difference in time, space, CPU, or any other factors?

All the results are displayed with no evidence of significant differences
Validity of the findings: The findings are there linked to the original question. I would suggest to have a paragraph about how to choose the value of the suggested parameters?
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: OPTIMIZING THE TRAINABLE B-COSFIRE FILTER FOR RETINAL BLOOD VESSEL SEGMENTATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1. In abstract, “The approach results are better than the other state of the are methods used for vessel segmentation.” is wrong and not precise, your results are not better than lots of supervised methods.
2. The last sentence of abstract is wrong
3. The concept of CORF is wrong, which has been defined in Azzopardi and Petkov, 2012
4. In section 4, the method organized not clear, for example
(1) how would you optimize the three parameters?
(2)”… “threshold(t)” showed in (5)…” may be ““threshold(t)” showed in (6)”
(3) what is the meaning of the sentence “Initially, the three approaches are conducted using the images from the CHASE-DB1 dataset”
(4) in the last paragraph in page 5, the defined symbol “Y” is not used in the paper.
5. in the last paragraph in page 6, for obtain the result Table 3, How do you set each parameter?
6. in table 4 and table 10, are you sure that the result of STARE database is the same as the result of STARE database?
Experimental design: The performance index values in the paper should be less than 1
Validity of the findings: the reference of the expressions in this paper is nonstandard. for example
(1)In the second paragraph from bottom in page 3, some references are “as shown in 3” “as shown in 4”.
(2)In the second paragraph from bottom in page 4, the same references are “as shown in (3)” “as shown in (3)”
Additional comments: No additional comments