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: DETECTION OF SPREADER NODES IN HUMAN-SARS-COV PROTEIN-PROTEIN INTERACTION NETWORK
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article provides sufficient background information. The theory that was used was clearly stated. But there are several points in the article that need attention.
-	Several outdated references, e.g. in line 81-88. The authors might need to add the updated references.
-	Line 108-111: redundant information with line 91- 102.
-	Line 176: typo error, “hole”.
Experimental design: The authors need to improve in the methods section.
-	The author should add the data collection part. Even though it is noted in the results section, it is necessary to clarify how the data for this study was gathered.
-	How did the synthetic network was constructed? What are the data (protein and interaction) that have been used to construct the synthetic network?
-	Line 248-250: this sentence is confusing. The author might either use PPIN or network, instead of using both PPIN and network. Term of network or PPIN should be consistently used throughout the manuscript.
Validity of the findings: -	The results section need to be improved and organized, can organize the results into subsections.
-	Several terms should be adequately defined, such as level-1, level-2, unique proteins.
-	All figure captions should clearly describe the figures. For example, the authors should add the explanation on the spreader node in Figure 1. The figure caption for figure 1 and figure 4 sound alike.
-	Figure 3 should be rearranged. Figure A and B should be placed on top of Figure C. The caption is confusing. The meaning of the node colors is unclear. Typo error on the caption and the label of the figure.
-	Figure 4 is the SARS-CoV network or SARS-CoV-human network? The difference between SARS-CoV and SARS-CoV-human PPIN should be described in the manuscript.
-	Line 285: redundant terms. PPIN of the SARS-CoV network. It might be either SARS-CoV PPIN or SARS-CoV network.
-	Line 308-309: the complete network has been generated online. Specific tool and parameters used to construct the network should be mentioned in the methods section.
-	Discussion part should be elaborated more. For example, how do the most spreader nodes (at least top 3 or top 5 spreader nodes) relate to COVID19.   How do the network in Figure 4, 5 and 6 relate to COVID19.
-	Can you highlight the significance of the network, proteins and edges identified in this study with COVID19?
-	How does the developed network attribute, spreadability index, and the generated SIS model perform better than existing approaches? How do you know SIS model performs better than other approaches? @as any testing carried out? Please explain.
-	Discussion and conclusion parts can be separated.
Additional comments: The background is well structured and written. However, the authors need to improve on other sections, as followed:
-	Please improve on figures and all figure captions.
-	There are grammatical mistakes and typo errors throughout the manuscript.
-	Several points, such as data collection and the network construction (tools, parameters in constructing the network), need to be addressed in the methods.
-	The organization of results need to be improved.
-	The results need to be critically discussed and concluded.
-	Please be consistent with the term of PPIN and network. Several terms need to be clearly defined.


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: DETECTION OF SPREADER NODES IN HUMAN-SARS-COV PROTEIN-PROTEIN INTERACTION NETWORK
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: All the critiques are adequately addressed and the manuscript is revised accordingly. I am please to see that terminology was fixed and definitions of all the network-related terms were added. I am satisfied with revision and do not have new suggestions.


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: DETECTION OF SPREADER NODES IN HUMAN-SARS-COV PROTEIN-PROTEIN INTERACTION NETWORK
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article provides sufficient background information. The theory that was used was clearly stated.
Experimental design: The experimental design was well defined. The authors have improved a lot in describing the experimental design.
Validity of the findings: The findings and conclusions are well stated.
Additional comments: Authors have made a full effort to revise the manuscript. I suggest accepting this manuscript.


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

Input:
Title: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a Snakemake pipeline for automated assembly tailored to RNA virus genomes.
In the manuscript they describe the individual steps of the analysis pipeline and present a comparison to the
'viral-ngs' pipeline from Broad Institute.

While the manuscript describes the most important steps of the analysis, there are also some important details missing:

- As one example, there is no description on how the final consensus sequence is derived from the mapping. While this is also important to interpret the comparison with the viral-ngs pipeline.
Are missing/uncovered positions masked with 'N' or simply filled with the best matching reference? The latter could lead to shorter assemblies only at the two end-points, as all gaps between contigs were previously filled with the best matching reference by shiver.
- With one central argument for using a workflow system like Snakemake is reproducibility, I also miss the summary of tool parameters that were used to generate the examples for the provided test sequences. Even more severe, it is not documented which sequences were used as references!
- Many tools listed in the dependencies (Fastp, KMC, Mummer, SMALT, BOWTIE, SPAdes) of the pipeline are not mentioned in the manuscript. So it is not clear how/why they are used.
- An overview figure with a flowchart of the complete workflow would help a lot!
- There is no real description how the user would configure and run the workflow, only how a Singularity container could be built.
Experimental design: - For the evaluation I believe that assembly length is not the only important parameter to be considered to evaluate the overall quality like, e.g. the error rate. Recurring to my previous point about the consensus sequence, it is important to be clear how exactly the genome length was defined, whether positions filled from the closest reference are also counted and if this is the same between for both approaches.
I further doubt that a statistical significance of genome length is some valid indicator without considering the qualitative significance of the missing genomic regions.
- The choice of bam files as mandatory entrance to the workflow is a suboptimal choice in my opinion. At least fastq (also gzipped) files should be an additional option. As the authors motivate the bam files by a frequently conducted preceding decontamination step, this should then rather be integrated to the workflow as an optional preprocessing step.

IMPLEMENTATION

- A real manual describing how to setup, configure and run the workflow is missing in the manuscript and also on the github page. There should be examples to explain the user how to configure the workflow and how to start it such that it will use the provided conda environment.

- While the option of using Singularity is documented, the alternative option of how to use the provided conda environment for the workflow is completely missing.
Also, there is no hint to the config file, which is rather hidden in the scripts folder with the confusing file ending ".sh".

- If I see it correctly, the paths to the references, primers and adapters need to be configured within the Snakemake file at the input section of the corresponding rule. The authors should try to implement the Snakemake best practice project structure where the complete configuration can be performed in a single config file.

- The implementation is rather a pseudo Snakemake workflow as most of the steps are contained within quasi sub-pipelines implemented as standard shell scripts.
Thus, the workflow does not make use of many Snakemake features, like, e.g. it does not use its capabilities to distribute the work over multiple cores.

-With the current state, I really doubt unexperienced users will be able to run the workflow.
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: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present a well-written manuscript describing their development of a novel pipeline for the de novo assembly of viral genomes. While the text is well-structured and well written, the current state of the art was not sufficiently well described and relevant references (V-GAP, VirusTAP or VrAP just to name a few potentially competing tools) are missing.

In this vein, I strongly recommend describing the actual problems with viral assembly (such as the minoritary variant problem hinted at in several places) in and existing approaches in more detail. While the introduction does a very good job of describing why NGS and viral bioinformatics are valuable tools, in my opinion it leaves the reader wondering what the actual motivation behind the development of VGEA is (apart from a very broad "it helps assemble viral NGS data").

Minor points:

* Line 61: I would recommend using something like "open view" instead of "unbiased" - while I can see what the authors mean, I feel that this glosses over the (often problematic, also in viral NGS) bias introduced by library preparation in NGS experiments.

* Line 62: I recommend rephrasing this sentence. It is in my opinion unfortunate to directly compare NGS "for virus detection and assembly" with diagnostic methods, that are by design only meant for virus detection. Again, I entirely agree with the point the authors are making, however I find the phrasing unfortunate.

* Line 40: The conclusion could be expanded with an informative, summarizing sentence - I feel like there should be more to the conclusion than the (of course very important) github link.
Experimental design: While opinions on how original combining existing tools into a pipeline is, I think that this is highly valuable work given how many different tools exists. The resulting number of possibilities to combine them into pipelines that solve the same problem is staggering, and it has been well documented that the choice of tools for each step in an analysis can have a massive impact on the quality of the analysis results. I thus feel that the work presented in this manuscript is a valuable, original contribution to the field.

However, in my opinion several points in the description, motivation and evaluation leave significant room for improvement and clarification:

* Since a reference sequence covering gaps in the initial assembly using the closest reference sequence is created using Shiver (lines 144-146) and the final sequence is created by mapping to this sequence (lines 146-148), the potential bias introduced by this use of a reference sequence should be evaluated and the process of generating the consensus sequence from that mapping result should be described in more detail (e.g. what happens with uncovered regions, what happens in edge cases such as the reference being covered by only one or by conflicting reads). This can relatively easily be evaluated by creating simulated datasets, and I feel like this is an important step in ensuring that the final contigs are not biased towards the reference sequence in areas of low coverage or where assembly is hard due to sequence characteristics.

* It is very commendable that the authors have put thought and effort into making their pipeline easy to run and deploy using singularity. However, I would recommend either removing the claim of reproducibility from line 110 or adding an extra sentence describing how this is achieved. Specifically, in order to ensure reproducibility (which is an extremely desirable trait) two points are of major importance: (i) checking for the use of random seeds in the tools within the pipeline and allowing users to set those seeds from outside of the pipeline and (ii) dealing with parallelity: either ensuring that all used tools produce the same result independent of the order of the operations/input files (e.g. when performing mapping with multiple threads, the ordering of sam/bam files might not be reproducible unless special optionss are provided), or offering an option to the user that allows ensuring reproducible ordering (generally at the expense of computational efficiency)

* In the section "Mapping" (lines 138-150) it is not clear to me what the exact process and filtering steps are. Are reference sequences supplied by the user, or is an alignment of potential reference sequences required? Also, aligning contigs to reference sequences using MAFFT is potentially problematic: (i) In case of structural variations (e.g. recombinations, which in some viruses happen within the patient), MAFFT is unable to produce a clean alignment since it performs global alignment and (ii) as far as I know, default mafft is not especially good at dealing with end gaps (such as in contigs only covering part of the genome) - using mafft-linsi might be helpful, but the scripts in the repository use mafft.
Validity of the findings: he performance improvements over viral-ngs presented by the authors are impressive and the datasets used to perform validation of the results have been well documented. However, in my opinion several points require significant further work:

* Most importantly, using only reconstructed genome length as a quality metric does not do justice to the complexity of the task. An especially important metric is the number of errors. This ties into my comment concerning the risk of biases introduced by joining contigs using reference sequences. So, not only the length but also the error rate and types of the assemblies should be compared

* The choice of viral-ngs as the only tool VGEA was compared against is not sufficiently well motivated - there are several other tools that could be seen as competitors: V-GAP, VirusTAP or VrAP just to name a few.

* Again, tying into the bias comment: It is not clear whether the "true" reference sequence was excluded from the reference set provided to VGEA in the comparison. Given that VGEA uses the references to fill in gaps in the contigs, a realistic scenario would be knowledge of all reference sequences available up to the moment when the sequencing run was performed - providing VGEA with reference sequences published afterwards (in the worst case: those resulting from analysis of the NGS data used in the comparison) would provide VGEA with an unfair advantage

* How reads for the comparison were pre-processed should be made clear: Were the same trimmed and background-filtered reads used for viral-ngs as for VGEA?

I feel like only once these points have been adressed the statement "This significantly improves the quality of the genomes obtained from NGS data" in line 188 in the discussion can be made with confidence.
Additional comments: In this manuscript, the authors present a novel pipeline for reference-assisted de novo assembly of viral genomes. This is an important and task in bioinformatics that will profit from further improvements in the available tooling. However, while the manuscript presents a good structure and is well-written, in my opinion significant additional work is required to allow this promising work to achieve its full potential and to make it useful for the community.


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: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a pipeline for viral genome reconstruction from NGS data. The pipeline is implemented in the form of a Snakemake workflow, which preprocesses, aligns and assembles the reads into a consensus genome. The manuscript is well written with perfect use of professional English. Several sequencing data sets were generated for assembly evaluation, these data sets are provided online. However, I do have some comments regarding the background information (introduction) provided:

1. Although viral diversity and quasispecies behavior is mentioned in the abstract, this is not discussed in the introduction. I believe this is an important aspect of viral genome analysis; the authors should expand on the difference between consensus assembly and haplotype assembly (strain-level reconstruction) and clarify the goals of their method in this respect.

2. The authors present a new pipeline for viral genome assembly, but existing assemblers and assembly pipelines are not discussed. There is a whole scala of methods for genome assembly of RNA viruses, as well as review articles evaluating these methods. See for example “Choice of assembly software has a critical impact on virome characterisation” by Sutton et al (2019).
Experimental design: The topic of the research is of high relevance, as viral genome assembly can be very challenging. Some comments regarding the experimental design:

1. The authors should state more explicitly the aims of their research, and explain how their research fills an existing gap. This extends my previous comment regarding the background information: there are many viral genome assembly tools and pipeline available, and it is not clear why these do not suffice. What is the gap that the authors try to fill with VGEA? I do not understand why the authors do not mention or evaluate a recent pipeline called V-Pipe, which I believe serves the same as VGEA.

2. In addition to a theoretical discussion of VGEA in comparison to available tools, presenting a new pipeline also requires benchmarking experiments. The authors compare only to a relatively old approach (viral-ngs) but do not present results for other pipelines. How well does VGEA perform in comparison to other methods?

3. The authors have generated many datasets to evaluate their method on, which is very valuable. However, to truly consider these data sets as benchmarks, there should also be a "ground truth". For example, by generating high quality reads with Sanger sequencing, or PacBio HiFi sequencing. Currently, the authors only evaluate contig length, but this is too limited. Also the assembly accuracy (target reconstructed, error rate, misassemblies) should be considered. Given a ground truth sequence, these statistics can be quickly generated with an assembly evaluation tool like QUAST. It would also be interesting to see how the assemblies compare to the available reference genome.

4. I got very confused in the methods section. The pipeline starts with the splitting of BAM files, but it is not clear how these BAMs are generated. The text suggests that these BAMs are alignments to the human reference genome? Does that mean that VGEA uses all unaligned reads to perform assembly? Later, it is mentioned that reference sequences are used to remove contaminants; how exactly does this work, and which references did the authors use? Finally, the pipeline ends with Shiver creating a reference sequence and reads are mapped to this sequence. But then what is the purpose of these alignments? All of this is not clear from the text, it would really help me if additional details were added, along with a figure that illustrates the entire workflow.

5. The assembly step is performed with IVA. The authors state that "it has been demonstrated to outperform all other virus de novo assemblers". However, IVA was published in 2015 and many other assemblers have been developed since. At this point, there are probably better choices available, and it is important to see how the choice of assembler affects the results of the VGEA pipeline.
Validity of the findings: The software and data sets are available online, but I was unable to find the command lines / config files to enable full reproducibility.

I believe that a more extensive experimental evaluation (see previous comments) would also allow for deeper, more confident conclusions.
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: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Minor:
- Tool / software name typos (capitalization) should be fixed e.g. snakemake (Snakemake), quast (QUAST).
- Some tools are cited multiple times (e.g. shiver three times).
Experimental design: While the authors transformed their workflow into a "real" Snakemake workflow, there are still some features missing and things to be improved:

=== IMPLEMENTATION ===

- While the authors state that "VGEA capitalizes on Snakemake’s multi-threading feature", they are not using the "threads" feature of Snakemake in any of their rules.

- The authors claim that "VGEA allows full customization of the pipeline, so
145 users can modify the parameters used in running their samples". However, the only step to be controlled by the user directly is shiver via its separate config file. For all other rules parameterization via the "params" section of Snakemake together with an augmented config.yaml is NOT possible.

- While the entry point are now the fastq files and the authors mention Trimmomatic and fastp, I think these tools should also be added to the workflow. Shiver (depending on its config file) will apply Trimmomatic but iva runs on untrimmed/filtered reads. While iva could be configured to run Trimmomatic too, the most efficient way would be an explicit trimming step at the very beginning of the workflow together with a quality control. Finally, an aggregation of these results together with the results from QUAST in a single report (multiQC) would be another plus.

- The authors should use the proper mechanism to use Conda with Snakemake, where Snakemake takes care of the environment creation, activation/deactivation instead of letting the user create a global Conda environment first - as proposed on the GitHub readme page.

- Input: Currently the workflow will use all fastq files fulfilling the naming pattern something_R1.fastq something_R2.fastq located in the working directory. The authors could enhance the usability and flexibility and drop this strict naming constraint if they would use the typical samples/units.tsv scheme to be found in all best practices Snakemake workflows.

=== DOCUMENTATION GIT / WEBSITE ===

- The authors should specify which output files will be generated, where they will be found and what information they contain. Probably with a small example run presented on the readme page.

=== MANUSCRIPT ===

- I think the statement "VGEA generates a number of files among which is a CSV file that contains minority-variant information (base frequencies at each position), consensus genomes and several quality metrics to assess the genomes assembled." is somehow misleading. To me this suggests that these files are produced by VGEA in some new, additional downstream analysis step. If I am correct, these files are produced by shiver directly - this should be clarified.
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: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors present a manuscript that has been significantly reworked. It is clear that the comments have been taken seriously and the authors have made a significant effort to address the points that have been raised.

However, there remain a few (minor) points that should to be adressed:

* Lines 115-119: "Most"/"A lot" are redundant with "usually", I recommend dropping one of these from each of the two sentences here.
* Lines 121-124: The authors first describe how daunting a task it is to choose the correct tool from the massive list of available tools, and then go on to describe how they solve "the challenges listed above" by implementing yet another viral assembly pipeline. This is a somewhat unfortunate combination - I recommend just removing the sentence "It can also be overwhelming to pick a suitable software from the huge amount of bioinformatics tools available to carry out various tasks".
* Lines 115-124: There exist both pipelines that are specifically designed to run on personal hardware (e.g. V-Pipe specifically states how it allows choosing tools for each of the steps depending on available computational resources) and to make dependency management easier (e.g. this is a quote from the viral-ngs documentation: "'./easy-deploy-viral-ngs.sh setup' Installs a fresh copy of viral-ngs, installs all dependencies [...]"). It would be helpful if something like a table listing the cited tools and the requirements mentioned in the text was included, with checkmarks/crosses showing which of the tools satisfies which of the requirements. This would allow a quick and clear understanding of the gap that VGEA fills, rather than the general arguments saying that "most" tools "usually" don't fulfill the described requirements.
Experimental design: It is very commendable that the authors have updated their pipeline and the README to better address issues with reproducibility and runnability. However, significant problems remain. Specifically:

In README.md:
* All dependencies could be in one "conda install -c bioconda" line, instead of splitting them across multiple lines
* A description of how to exactly run on a test dataset (e.g. "snakemake --cores n -d data/LASV_L") and what the expected outputs are would be great
* There is no description on how to run the singularity image, a sample command would be very useful
* Having a singularity image as a downloadable artifact for those who are not admins but have permission to run singularity images would be helpful

In the Snakefile itself: After following the instructions in README.md, running the pipeline produces the error:

SyntaxError in line 8 of /home/wojtek/temp/VGEA/Snakefile:
EOF in multi-line statement (Snakefile, line 8)

One reason is that closing quotation marks are missing in the rule in lines 43, 52 and 130, but there are more syntax errors that I have not managed to find (the error persists even after closing these parantheses). These errors need to be fixed before publication. Additionally, in order to generally avoid such problems, I highly recommend creating a minimal test dataset and introducing a github action to make sure that commits do not fundamentally break the pipeline's functionality.

Also, the inclusion of a thorough description of the shiver method has indeed been helpful. However, this raises the issue of the required input files - given the criticality of the quality of the reference alignment for shiver, I feel that this (especially the sentence "producing such an input by automatically aligning a large number of diverse sequences without checking the results would be a bad idea" from the supplementary material) should be explicitly mentioned in the README. This may seem obvious to bioinformaticians but is especially important given the stated goal from the conclusion (lines 210-211): "VGEA was built primarily by biologists and in a manner that is easy to be employed by users without significant computational background".
Validity of the findings: Unfortunately, the authors seem to have taken my comments concerning the  choice of tools (in my opinion too few) and metrics (in my opinion non-optimal) used to compare VGEA against other pipelines as motivation to entirely remove any comparison of VGEA's performance to that of other similar tools. This is highly unfortunate, as this leaves "it's easy to install and run" as the only argument for choosing VGEA - and this is a poor motivation for the choise of a tool to do research with. I thus believe that the comparison needs to be reintroduced and extended (using more tools and better metrics) rather than being removed entirely.

In the current state, the validity of the findings can thus not really be assessed.
Additional comments: The authors have significantly reworked the manuscript and improved it in many places. However, I believe that especially two major points must be adressed before publication:

* The pipeline needs to actually run
* Some form of performance comparison against existing tools needs to be shown in order to allow users to make an evidence-based (rahter than just convenience-based) decition


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: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 2
Reviewer: 3

Output:
Basic reporting: I would like to thank the authors for their detailed responses to my questions. Several things have been clarified, the background has been information has been extended and the goal of the manuscript has become much clearer: to design a pipeline for viral genome analysis that can run on a personal computer.
Experimental design: However, I still have one major issue with this manuscript: where are the results? One of the issues with the previous version of this manuscript was that the assembly results were not evaluated adequately, but the current version has no assembly results at all...

If the research question is "how to design a pipeline to do viral genome analysis on a PC?", the manuscript should *at least* present results on runtime and memory usage for this pipeline, as well as other existing ones. Do other pipelines, e.g. V-Pipe and viral-ngs, indeed require too much resources? And how do other pipelines compare in terms of assembly quality?

The figures and tables shown in the manuscript are literally just the QUAST output on one random sample, with no interpretation. I don't see the added value of this and I would recommend the authors to make their own figures and tables, showing results on resource usage and assembly accuracy.
Validity of the findings: The authors conclude "VGEA was built primarily by biologists and in a manner that is easy to be employed by users without significant computational background". Although the authors have improved the Snakemake workflow substantially, I still don't believe this is true in its current state. The manual provided describes extensively how to install conda and to create an environment with all dependencies, but the only instructions regarding the pipeline itself are:

"Run the snakemake pipeline: snakemake --cores n -d /workingdir/
The location of the human reference genome, the reference, primer and adapter fasta files for shiver and the reference and gene features files for quast can be controlled by adjusting the config.yaml in the VGEA working directory."

If users have to review the manual of the individual tools (bwa, iva, shiver) to set appropriate parameters, I don't see how this is any easier than just running the 3 tools separately. If the authors really want this to be an easy to use pipeline, it would be very helpful if there is more documentation on how to choose parameter and possibly also including a small test case.
Additional comments: line 85: “overlapping region in the genomes”. I think you mean overlapping sequencing reads?
line 120: what is AVL?
line 156-160: I think it is important to indicate here that the superiority of shiver was shown by the authors of shiver, not an independent review.


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

Input:
Title: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thanks to the authors for their responses to my previous concerns and the implementation of the missing features (conda, sample sheet, threads).

However, I still have some issues with the manuscript and the workflow documentation.
Experimental design: In the benchmark section, I feel there is some information missing and also the comparison should be extended:

- As computational efficiency is one major argument, I would like to see the resource footprint for one of the other pipelines mentioned on the same samples to substantiate the central claim that VGEA empowers scientists with very limited hardware.

- For the presented benchmark I miss some crucial information:
  - Hardware specification!
  - Which reference alignment has been used for shiver?
  - How exactly were SPAdes and velvet run? Versions? Parameters? Were they run on raw reads or on the filtered and trimmed reads also used as input for IVA and shiver?
  - Why does the shiver output (which, as I understand is the main result of the pipeline) contain multiple contigs? As I understood shiver would always return a contiguous full length assembly.
  - What is the '-' in Table 2 for SPAdes on sample CV115?
  - The fact that NG50 is always greater than N50 should be addressed. Does that mean, that all assemblies contain a lot of junk/contamination or duplicated segments?
  - Which reference was selected for QUAST and why?

Some minor issues in the Readme.md
  - The listing of the results file tree does not contain shiver for test1 (which should rather be renamed sample1 for clarity), hence it is not described.
  - The given command for a test run gives a wrong path for the test_config.yaml. The folder is .tests (not .test as given in the Readme)

Some problems when running the test example:
  - I received an error for IVA due to some conda/mamba issues (ModuleNotFoundError: No module named 'pkg_resources'). For me it could be fixed by adding "setuptools==52.0.0" and "kmc=3.1.1rc1=h76f5088_0" to iva.yaml.

  - The same problem also leads to an error for shiver (Error running 'fastaq version'. Are you sure that fastaq is installed, and that you chose the right value for the config file variable 'fastaq'?). Could be fixed again by adding "setuptools==52.0.0" to shiver.yaml
Validity of the findings: no comment
Additional comments: It is good to see how the quality of the implementation improved in the two iterations. If the remaining points in the manuscript will be addressed it should be ready for publication.


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

Input:
Title: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors present a manuscript that has again been significantly reworked. It is clear that the authors have gone to great lengths to seriously address all issues that were raised in the previous reviews.

The only remaining point I see is the availability of the data.

Firstly, there is a typo in the data repository: The link in line 131 (https://doi.org/10.6084/m9.figshare.13009997) is correct, however the one mentioned in the data availability statement (https://doi.org/10.6084/m9.figshare.1300999) is wrong (leads to a "DOI Not Found" error page). I suspect that this is supposed to be the same link and that is is just a typo.

Secondly, in lines 192-193, it is mentioned that "We carried out this comparison by making use of five different SARS-CoV-2 test datasets." However, it is not clear which of the 63 datasets available under the figshare link have been used for the evaluation. This should be made clear.
Experimental design: Again, the previous concerns have been very well addressed. I am especially happy to see that automated testing using git actions has been implemented to ensure that the software runs correctly. A quick test confirms that the previously present bugs have been resolved.

One minor point: In the two commands at the end of README.md:

snakemake --use-conda --use-singularity --configfile .test/integration/test_config.yaml -j 1

and

snakemake --use-conda --configfile .test/integration/test_config.yaml -j 1

the test folder has a typo - it should be ".tests" and not ".test".
Validity of the findings: While I am very pleased to see that benchmarking has been re-introduced, I am still not entirely satisfied with how it was performed.

Firstly, resource use and runtime were evaluated for the rules used in VGEA, but there is no comparison of resources use and runtime of VGEA as a whole as compared to the other initially mentioned pipelines. In the introduction, V-Pipe and viral-ngs are specifically mentioned as tools that require too much computational power and/or are too hard to install and thus motivate the development of VGEA. It would be advisable to demonstrate at this point that VGEA actually needs fewer resources or is quicker that these tools (since both provide installation instructions that are similar in simplicity to those of VGEA).

Also, the comparison of assembly results against velvet and spades seems oddly chosen, since velvet and spades are assemblers that are used in pipelines, not end-to-end pipelines in their own right. As such, it would seem more intuitive to compare the results of VGEA against the results of V-Pipe or viral-ngs - unless the performance comparison shows that they indeed need much more resources and VGEA is the only viable option on the used hardware, in which case this should be explicitly mentioned as the rationale for comparing VGEA with these assemblers.

In either case, just contig length/number metrics are in my opinion insufficient. In my opinion, at the very least the misassembly, mismatch and indel metrics from QUAST should be included in this comparison. While it is true that Bradnam et al. recommend using genome coverage as a comparison metric (as mentioned in the manuscript as motivation for using this as a basis for the comparison), it is only one dimension of the COMPASS metrics used and recommended in the cited paper. They even have a section titled "Size isn't everything" in their Discussion and specifically say that " we find that N50 remains highly correlated with our overall rankings. However, it may be misleading to rely solely on this metric when assessing an assembly's quality".

As such, I still find it hard to judge the validity of the findings (or specifically, whether VGEA actually delivers what is described in the introduction as the motivation for its development) based on the data presented here.
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: VGEA: AN RNA VIRAL ASSEMBLY TOOLKIT
Review round: 3
Reviewer: 3

Output:
Basic reporting: The authors have made an impressive effort and addressed my concerns satisfactorily. My only remaining comment is that, in the performance analysis, the assembly statistics evaluated are all related to assembly contiguity and completeness, but not accuracy. For the datasets evaluated no ground truth is known, which explains why accuracy is not evaluated. However, improved contiguity often comes at the expense of reduced accuracy. I therefore believe that it is important to add one sentence to the analysis of the assembly results, pointing out that accuracy was not evaluated, such that readers are aware of this potential limitation.
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: CLINICOPATHOLOGICAL VALUE OF LONG NON-CODING RNA PROFILES IN GASTROINTESTINAL STROMAL TUMOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: Zhao et al describe a data-mining approach using publicly available databases, for the identification of potential long-non coding RNAs (LncRNA) in the pathogenesis of Gastrointestinal Stromal Tumors (GIST). I find the concept quite interesting and I would support its publication if the authors address several major comments on this work.

1. The language throughout the manuscript needs significant improvement and editing. There are several occasions that the language limits the proper comprehension of the actual meaning for the reader. Some of the examples include lines 24, 28, 37, 44, 47, 58, 66, 70, 145-148, 189, 191-192, 232-241, 251.

2. The authors also need to improve and update their reference utilization. In the introduction section, they should provide a more recent and updated source for the clinical aspects of GIST tumors (line 57-60). In addition, in the same section, the authors should expand the scientific background on the role of lncRNA in GIST. More specifically, in line 72 they should include examples of lncRNA that affect prognosis. Furthermore, in the line 73-78, the authors have not included important references for the role of lncRNA in GIST for MALAT1, H19 ( https://doi.org/10.1155/2019/5458717, PLOS ONE 13(12): e0209342.), AOC4P (Onco Targets Ther. 2018; 11: 6259–6269., World J Meta-Anal. Jun 28, 2020; 8(3): 233-244) and extended reviews for the role of lncRNA in imatinib resistant GIST (Oncol Lett. 2019 Feb; 17(2): 2283–2295, https://doi.org/10.1590/1414-431x20198399). Also, the authors should edit their introduction in order to include further information regarding the available treatment approaches and molecular mechanisms that contribute to the pathogenesis of GIST. Another area of significant improvement could be the discussion section. The authors, in line 333-335,  falsely claim the lack of knowledge on the molecular pathways affected by lncRNA LRRC75A-AS1. They cite several papers for its role in osteosarcoma, ignoring its major contribution in the pathogenesis on Triple Negative Breast Cancer (Cell Death Dis 11, 643 (2020), Int J Biol Sci. 2020; 16(2): 251–263. )

3. Another important aspect of improvement is the preparation of the figures. The figures are not consistently labeled, are very small to read and in many cases difficult to follow. I strongly suggest the authors to use high-quality images and prepare them using professional software (GraphPad Prism, Python, Adobe Illustrator)
Experimental design: Although the overall technical approach is correct, I have some concerns regarding the experimental design.

1. Although the original approach is unbiased and the question well defined, with the proper analysis of publicly available datasets for GIST patients, I strongly believe that the later focus only on LRRC75A-AS1 is not justified based on the overall results and the literature. I find the data in figure 5 of little importance and contribution to the field. The authors should revise their approach and generate data showing the pathways that their proposed lncRNAs candidates affect, in a comprehensive unbiased way. I strongly suggest that the authors should show the effect of the expression of these lncRNAs on survival and clinical stage, if this information is available.
Validity of the findings: 1. In figure 2A and 2B, the author should include the Hazard Ratio in their analysis and figures, and calculate the logp-rank for their suggested survival curves.

2. Last but not least, the authors should provide the pairing criteria used for the male and female patients, since this can lead to significant confounding bias. Furthermore, the authors should provide the list of matched patients with statistical proof that their matching is statistically correct. After that, the authors should revise their model and figures.
Additional comments: Zhao et al provide an interesting concept in the already established role of lncRNAs in GIST patients. The authors are strongly advised to re-evaluate several aspects of their manuscript and I would be positive on its publication.


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

Input:
Title: CLINICOPATHOLOGICAL VALUE OF LONG NON-CODING RNA PROFILES IN GASTROINTESTINAL STROMAL TUMOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The description and explanation of this manuscript are clear and nicely readable. All the figures and tables are well organized, except missing a note for * and ** in Table 3. It looks that table 2 is informative, but you need to explain more of any important information we can get from it.
Experimental design: Do you have the gene set enrichment analysis in a subtype of female patients? Any difference between male and female? Can you explain more about why XIST down-regulation may suggest the malignant transformation?

In the ideogram of figure 5, why didn’t you put Chr Y for the whole genome co-expressed protein-coding genes and copy number variation analysis?
Validity of the findings: In the review version, the resolution of figure 1B is not clear to figure out each vertical annotation that is associated with each subtype, but the expression pattern of these 2448 lncRNAs in G1 and G4 subgroups looks similar, and as well as G2 and G3. How can you explain this situation?
Additional comments: This manuscript described a process of how to start by profiling lncRNA expression to detect a specific lncRNA as a prognostic or predictive marker for GIST, which may have higher specificity than using mRNAs. By using different algorithms, LRRC75A-AS1 was detected that showing a higher expression level in the malignant GIST tumor (G3/G4 subtypes). This result could be used for the laboratory evaluation and verified if it can be used as a prognostic marker or progression diagnostic for GIST.


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: CLINICOPATHOLOGICAL VALUE OF LONG NON-CODING RNA PROFILES IN GASTROINTESTINAL STROMAL TUMOR
Review round: 2
Reviewer: 1

Output:
Basic reporting: Zhao et al have provided an extensively revised manuscript that strongly support their model. They have improved their figure quality tremendously making it very easy to follow.
Experimental design: The new data added in the study are well controlled and explained.
Validity of the findings: No comments.
Additional comments: The authors returned a significantly improved version of their work that meets the advanced scientific criteria of the journal. I support the publication of this concept.


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: CLINICOPATHOLOGICAL VALUE OF LONG NON-CODING RNA PROFILES IN GASTROINTESTINAL STROMAL TUMOR
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors addressed the concerns and questions properly and accordingly, the revised version of the manuscript seems to be well written and should provide useful results. However, I still have some concerns and suggestions:
There are 9 images in Fig.1, but in your manuscript, you haven’t explained or mentioned each of the figures you listed. Moreover, in line 186 and line 192, your descriptions are not corresponding to the figures you mentioned. You need to revise those contents and add more explanation corresponding to all the images in figure 1. Similarly, you haven’t described all the figures in figure 3.
Experimental design: No comment
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: EXPRESSION AND PROGNOSIS OF CDC45 IN CERVICAL CANCER BASED ON THE GEO DATABASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Please properly cite any software and databases used in the analysis (including GEO, GEO2R, GO, KEGG, R, ggplot2 and etc) if possible.
2. In Line 87, please be specific on how you downloaded the 257 samples on the TCGA website including inclusion and exclusion criteria. Did you use "CESC" as the keyword? and etc.
3. The non-cancerous tissue samples are too few to compare to (n = 2). Is it possible that any additional samples can be added?
4.

Here are the comments regarding the figures and tables.
1. Please add the value corresponding to the vertical and horizontal cutoff in Fig 1A. The legend in Fig 1B is illegible.
2. Please add the cutoff lines in Fig 5A. The legend in Fig 5B lacks annotation for expression levels and the labels for both axes. Please add the sample size for each group in Fig 5C.
3. Where are there only two variables in the multivariate cox regression shown in Table 1? Please include the comparison and the control group for each categorical clinical variable including stage and grade. Why doesn't the age variable get included in the univariate analysis?

Below lists a list of minor suggestions.
1. In Line 29, there is an additional space between "We" and "utilized".
2. In Line 31 and 95, what is "cox logistic regression analysis"? Do you mean "cox regression"?
3. In Line 93, capitalized "c" in "clinical".
4. Abbreviation is missing for TCGA, FC, FDR.
5. Please include the R version in Line 104.
Experimental design: 1. We highly recommend sharing any codes (for example, R scripts) used in the analysis for scientific reproducibility.
Validity of the findings: 1. Given that the GEPIA consists of TCGA data, the analysis from line 144 wouldn't be considered as an independent verification of Hub genes. Please read more here http://gepia.cancer-pku.cn/about.html.
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: EXPRESSION AND PROGNOSIS OF CDC45 IN CERVICAL CANCER BASED ON THE GEO DATABASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are a lot of grammatical errors and typos in this manuscript. For examples:
- This study aims to identified the hub genes ...
-  ... the module of top 10 genes with the most protein interaction were selected.
- The expression level of hub genes were shown ...
- No space before dot or comma.
- The use of inconsistent capital and small letters.
- ...
Thus the authors are suggested to re-check and revise carefully.

The manuscript lacks detailed information on problem background, and especially literature reviews. No study on cervical cancer-based bioinformatics analysis or CDC45 gene is discussed.
Experimental design: Did the authors concern about batch effect removal when merging three databases?

Source codes should be provided for replicating the methods.

ROC curves and AUCs have been used in previous bioinformatics studies i.e., PMID: 31750297, PMID: 33735760, and PMID: 32613242. Therefore, the authors are suggested to refer to more works in this description to attract a broader readership.
Validity of the findings: Why multivariate analysis did not generate a signature gene according to risk scores?

The authors should have some validation data (maybe from TCGA or other sources).

What are the limitations of this study? It should be discussed.
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: EXPRESSION AND PROGNOSIS OF CDC45 IN CERVICAL CANCER BASED ON THE GEO DATABASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you so much for taking the time to address all my comments and re-analyzing the data. Please make sure that you include a note saying that "The code is available upon request from the authors" given that you won't attach the R code in the supplemental.


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: EXPRESSION AND PROGNOSIS OF CDC45 IN CERVICAL CANCER BASED ON THE GEO DATABASE
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
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: ORGANIC SOIL AMENDMENTS USING VERMICOMPOSTS UNDER INOCULATION OF N2-FIXING BACTERIA FOR SUSTAINABLE RICE PRODUCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clearly presented
Experimental design: A robust design, with strong data
Validity of the findings: These are very clear and useful. The treatment effects were well described and trends effectively defined.
Additional comments: A very good paper and worth publishing. I have some minor comments as follows:

Line 59 - suggest '...and grain yield has a...'
Line 87 - '...spiked wheat...' please clarify the term spiked for readers
Line 133 - '...and 15 m'  Is this altitude?
lines 154-160. This paragraph is rather confusing - please re-write more clearly
Line 164 - suggest '... reactor, which supported a population density of 250 g of earthworms...'
Line 165 - suggest '...reactor was divided...'
line 167 - suggest '...which was processed...'
Lines 201-2 This sentence is confusing - please re-write
line 216 - '...soil was paved...' is 'taken' a better word than 'paved'? '..at the maximum stage...' - what stage?
Line 219 - (Witt et al, 2000).
Line 225 - suggest 'Kelly et al,1999
Line 248 - '...dough stage...' - please clarify
Line 250 - '...the claw...' - please clarify

Results - please clarify that treatment differences stated in the text are statistically valid and not conjectural
Height of panicle and plant section - lines 332-338. Table 4 is referred to - is that meant to be Table 3?
Line 376 - at end of sentence suggest refer to table 4.
Line 390 - Azospicilum - check spelling
Line 440 - suggest 'In agreement with...'
Line 526 - suggest '...for a longer...'
Line 568 - suggest '...treatment and it was...'
Line 620 - suggest '...sources weak and...'

Table 2 - please specify +- values - what error values are they?
Table 3 - Left column treatment abbreviations need a key to clarify
Figures - please check and clarify error bars throughout
Figure 3 - I found this difficult to read and interpret

References - the following references are cited in the text but missing from the Reference list:
Line 100 - Sanati et al, 2011
Line 267 - AOAC 1970


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: ORGANIC SOIL AMENDMENTS USING VERMICOMPOSTS UNDER INOCULATION OF N2-FIXING BACTERIA FOR SUSTAINABLE RICE PRODUCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Organic fertilizers provide essential nutrients to plant. This study reports the effect of vermicomposting of cattle manure mixture with Azolla and rice straw on soil microbial activity, nutrient uptake, and grain yield under inoculation of N2−fixing bacteria using a field experiment in a paddy soil. The manuscript is of interest and generally well written. The data are enough to support their conclusions. But there are some unclear points that should be addressed prior to considering it for publications. Overall, this manuscript is recommended for Peer J after Minor Revision.

Please find some more detailed comments below.

1. Lines 167-170, in general, the optimum moisture content of vermicomposting is about 60%. Why the authors chose 75-80% of moisture content?
2. Lines 171-178, how about the temperature development during vermicomposting? Is the temperature suitable for the survival of earthworms? Also, is the temperature enough to kill pathogens in manures?
3. Figure 3, it is better to use the specific days after transplantation rather than rice growth stages.
4. The total number of tables and figures are 16. It is suggested to move some data into Supporting Information.
Experimental design: no comment
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: ORGANIC SOIL AMENDMENTS USING VERMICOMPOSTS UNDER INOCULATION OF N2-FIXING BACTERIA FOR SUSTAINABLE RICE PRODUCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The amendments to the m/s appear to meet the original comments made. I am happy to recommend publication.


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

Input:
Title: WEAK SPATIAL-GENETIC STRUCTURE IN A NATIVE INVASIVE, THE SOUTHERN PINE BEETLE (DENDROCTONUS FRONTALIS), ACROSS THE EASTERN UNITED STATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript of Garrick et al. provides a very interesting and comprehensive analysis on the populations structure of Dendroctonus frontalis across the eastern United States. The language used describes efficiently the concept, methods and results of this analysis, whereas the authors cite all the necessary lliterature references. I believe however, that the introductory part should be somehow complemented with a few more information regarding D. frontalis itself, before explaining all the previous studies that were done on this species.
Experimental design: The experimental design is really robust. The authors address each approach individually, and explain in details what they will do and why they chose this approach. This works excellently in their case, as they leave nothing vague or questionable, something that facilitates greatly the comprehension of their work. This is further enhanced by providing the settings employed in each software, something that allows the replicate of their analysis. Nevertheless, what I would really like to see included in the Materials and Methods part, are a couple of sentences describing the process used when sampling the D. frontalis individuals. In my opinion, this is important particularly in phylogeographic studies, and should be added, .
Validity of the findings: Following the structure they adopted in the description of the Experimental Design, the authors clearly address each approach, and explain their findings. It is also very important, that their speculation regarding the detection of IBD when only females were included, is learly identified as such, leaving no doubt about that.
Additional comments: The manuscript of Garrick and colleagues provides a very interesting and comprehensive analysis of the population structure of D. frontalis in the eastern United States. The authors adopt a very convenient and reader-friendly format to present the methods used, presenting each approach individually, providing the necessary information therein. This becomes even more important in the next parts (Results and Discussion) as by doing that, they facilitate reader to easily and effectively understand how the authors have reached to these conclusions. The only thing I would like to the authors to add, is some more information regarding D. frontalis in the Introduction, but also some details about the sampling protocol of the individuals analysed.


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: WEAK SPATIAL-GENETIC STRUCTURE IN A NATIVE INVASIVE, THE SOUTHERN PINE BEETLE (DENDROCTONUS FRONTALIS), ACROSS THE EASTERN UNITED STATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article by Garrick et al. assessed several explanations for the absence of spatial genetic structure in a native pest, Dendroctonus frontalis.
The article is well written and includes enough background to demonstrate how the work is relevant for the field and for the management of D. frontalis.
Experimental design: The methods and experimental design are clearly explained. There are many stages of analysis but the structure of the text is very clear. The choice of methods is well justified.
Validity of the findings: The manuscript confirms the lack of discrete population differentiation and isolation by distance in  D. frontalis, based on several, complementary methods, and the authors give several explanations for this result.
As indicated by the authors, these results have strong implications for the management of D. frontalis
Additional comments: Perhaps a paragraph should be added in the introduction on the biology of the species, with a focus on the traits that may affect its genetic structure. The authors already mentionned dispersal distances l59-61, but other traits such as temperature tolerance or reproductive mode could also have an influence.

L404-417 - identifying the best-fit value of K : read Janes et al. (2017) and Tsykun et al. (2019) for an illustration of the method.
--> Janes, J. K. et al. The K = 2 conundrum. Mol Ecol 26, 3594–3602, https://doi.org/10.1111/mec.14187 (2017).
--> Tsykun, T. et al. Fine-scale invasion genetics ofthe quarantine pest, Anoplophora glabripennis, reconstructed in single outbreaks. Scientific reports. 9:19436 (2019)

L462-463 – Add a reference like Lombaert et al. (2010) for bridgehead effect, and like Whitney & Gabler (2008) for rapid evolution in introduced species.
--> Lombaert, E. et al. Bridgehead effect in the worldwide invasion of the biocontrol harlequin ladybird. PLoS One 5:e9743 (2010)
--> Whitney, KD & Gabler, CA Rapid evolution in introduced species, “invasive traits” and recipient communities: Challenges for predicting invasive potential. Divers. Distrib. 14:569-580 (2008)

L508 – see also Roques et al (2012)
--> Roques, L. et al. Allee effect promotes diversity in traveling waves of colonization. PNAS 109 :8828-8833 (2012)

L571-572 – Remove one « Generally »


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: WEAK SPATIAL-GENETIC STRUCTURE IN A NATIVE INVASIVE, THE SOUTHERN PINE BEETLE (DENDROCTONUS FRONTALIS), ACROSS THE EASTERN UNITED STATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment - see below
Experimental design: no comment - see below
Validity of the findings: no comment - see below
Additional comments: The authors provide a detailed analysis of the population genetic structure of the southern pine beetle in the eastern US with the goal of reconciling different results found in the same system. The manuscript is well written and well-organized.

I have two main concerns about this manuscript.

First, I sincerely appreciate the attention to detail and the well-organized approach that was taken in this manuscript. However, I do have concern that these detailed investigations were not undertaken in the previous analysis and associated publications. The onus should be on an author to explore these types of questions when first reporting on the phenomenon. As written, this is a (well-executed) technical piece of work, however, without much of a conceptual contribution. For that reason, while the science undertaken is acceptable, I question the strategy that is being taken here.

Second, related to conceptual contributions, I was a bit surprised that that main line of inquiry was related to techno/analytical solutions to this problem. The assumption seems that if two different studies found different patterns of spatial genetic structure, then something must be wrong with our molecular markers. Ecological context (i.e., timing and outbreak status) was surprisingly not considered. As indicated above, while I do appreciate the due diligence of verifying that the markers and analytical approach were not at fault, I was surprised that more attention wasn’t given to alternate ecological and demographic explanations.

One very logical explanation from my perspective is that the two studies that precipitated this analysis were conducted at different points in time during and outbreak (i.e., 2011 vs. 2019). This idea is mentioned near the end of the MS (line 580 +). Given that you eliminated all other possible explanations, I feel that this explanation should be given greater attention. The spatial genetic consequences of cyclic and irruptive populations are not yet well understood, but I feel that you have likely landed on the most plausible answer in your comment about existing structure being “overwritten” (line 584). Your work may not be “complicated” by this issue of different timings of sampling, but in fact may be driven by them. Please see James et al (2015) and Larroque et al (2019) in this regard. I encourage you to explore this idea in more detail to not only better describe the interesting differences in SGS in D. frontalis at different points of time, but also to add further conceptual content and rigour to this work.

James PMA, Cooke B, Brunet B, Lumley L, Sperling FAH, Fortin M-J, Quinn V, Sturtevant BR. 2015. Life-stage differences in spatial genetic structure in an irruptive forest insect: Implications for dispersal and spatial synchrony. Molecular Ecology. 24(2): 296-309.

Larroque J, Legault S, Johns R, Lumley L, Cusson M, Renaut S, James PMA. 2019. Temporal variation in spatial genetic structure during population outbreaks: distinguishing among different potential drivers of spatial synchrony. Evolutionary Applications 12(10): 1931-1945


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: WEAK SPATIAL-GENETIC STRUCTURE IN A NATIVE INVASIVE, THE SOUTHERN PINE BEETLE (DENDROCTONUS FRONTALIS), ACROSS THE EASTERN UNITED STATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of the manuscript exhibits no further points that need to be improved or enhanced, as the authors have done a very nice work addressing the points raised previsouly. The manuscript is written in a very clear and unambiguous manner, and the literature references are as required. the
Experimental design: The initial concept/question behind this work is something logical that needs to be resolved, and this is very clearly explained in Introduction (Lines 81-93).
The experimental design behind the analysis of this manuscript is very meticulously designed and performed. In particular, the authors pay much attention to the handling of microsatellites in order to gain the most of them, avoiding any possible flaw might have reduced the clarity of the conclusions.
Validity of the findings: As the use of microsatellites might sometimes produce misleading results, the authors have carefully employed numerous methods and protocols that they use in their analysis to avoid such kind of pitfalls (e.g. Lines 255-256/ Lines 267-278). These additional add-ons significantly support the validity of their findings.
Additional comments: Overall, the manuscript by Garrick and colleagues provides a very thorough and interesting investigation on the intra-population structure of D. frontalis across the eastern US, based on the up-to-date approaches in phylogeographic and phylogenetic analyses. For that, I suggest that the manuscript can now be accepted for publication.


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

Input:
Title: WEAK SPATIAL-GENETIC STRUCTURE IN A NATIVE INVASIVE, THE SOUTHERN PINE BEETLE (DENDROCTONUS FRONTALIS), ACROSS THE EASTERN UNITED STATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is very well written, and gives key information for managing a major insect pest.
I particularly appreciated the presentation of the methods, and the discussion of the results, especially the comparison with previous studies.
The authors have responded to all the comments in the previous review.
I therefore recommend this article for publication.
I have just three minor points to note:
l95-109: this paragraph might have been better placed in the discussion, in the "reconciliation with previous work" section for instance. It would make the introduction shorter, and easier to read. This is only a suggestion, however.
l153-156: it is not clear who sampled the specimens and when.
l224: check grammar
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: WEAK SPATIAL-GENETIC STRUCTURE IN A NATIVE INVASIVE, THE SOUTHERN PINE BEETLE (DENDROCTONUS FRONTALIS), ACROSS THE EASTERN UNITED STATES
Review round: 2
Reviewer: 3

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: While some of my previous comments were taken into consideration, my impression is that they were done so only superficially. The focus remains highly technical in nature and still does not adequately address the questions at hand. Greater clarity and an improved framing of the study is needed. Doing the same analysis with just more markers seems like a poor justification to undertake this work, especially when the value of these markers is put into question given the framing regarding previous contradictory results. Similarly, highlighting the contribution of this re-examination as a “roadmap” (line 140) seems strange, as eliminating potential alternate explanations for observed results is just good science.

Specific comments
Line 44 – and outbreak dynamics?
Line 53 – suggest adding your research objective/question here (e.g., statement on line 72) and starting a new paragraph to present life history information.
Line 75 – development “and application” of molecular tools?
Line 100 – what is a “biologically meaningful distance estimate” and how does one distinguish from an un-meaningful one?
Line 107 –context and citation needed for reference to rapid evolution
Line 113 – Here you refer to the unexpected absences as the motivating problem, but is there any a priori reason to consider the absence of structure to be more likely than the presence of structure? One could also frame this study to examine the unexpected structure identified in the first study. Is it not plausible that technical errors could have resulted in spurious detection of SGS? Why is the absence of structure so much more suspect than its presence?
Line 121 – “albeit ephemerally” – do you know this for sure? Suggest remove
Line 122 – reference needed for this idea
Line 126 – why “notwithstanding ... panmixia”? You refer to panmixia above as a potential alternate hypothesis. You could have both panmixia and weak markers, no?
Line 131 – “only those loci …” – Precedent for this approach? Reference needed
Line 140 – seems strange to highlight the value of new molecular markers. Suggest re-emphasize conceptual (vs technical) contributions.
Line 185 – the absence of consideration of outbreak dynamics and demography as an explanation for the differences between the two studies suggests to me that my previous comments were only superficially taken into consideration. Although referred to int eh discussion, this concept should be woven into the manuscript more thoroughly. Population connectivity and effective population size vary through time in irruptive species, and both of these factors affect population genetic inference. The focus on uSat number and quality misses the mark and does little to actually address your question.


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: GENOMIC ANALYSIS OF EARLY TRANSMISSIBILITY ASSESSMENT OF THE D614G MUTANT STRAIN OF SARS-COV-2 IN TRAVELERS RETURNING TO TAIWAN FROM THE UNITED STATES OF AMERICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the professional English language was used, though some corrections are needed
Experimental design: Would require authors to address some concerns. Please see "General comments for the authors"
Validity of the findings: Would require authors to address some concerns. Please see "General comments for the authors"
Additional comments: In the present study, Jian and colleagues sequenced SARS-CoV-2 genomes from five (5) travelers returning to Taiwan from the United States of America. Although this is an important and interesting study, there are a few areas that I would like the authors to address.

Major comments
1.	Line 104-105: The authors should indicate the number of genome sequences retrieved from GISAID and included for the phylogenetic analysis. Based on line 121, it appears that only 37 genome sequences were used for the analysis. Please explain the rationale behind the selection of these 37 sequences. This is crucial because of the 37 sequences, 36 were originated from the USA. Such sequence sampling bias will adversely impact the conclusion. If the total number of sequences included for the analysis was 3,873 (line 139), please include this full phylogenetic tree in Figure 1.
2.	Also, in its current form, Figure 1 is not suitable for publication. Please include the accession number for each sequence, the scale, an outgroup, and remove bootstrap values less than 90.
3.	Line 123-128: --- “Our data showed that the five strains clustered an extended branch-length distance from the ancestral human SARS-CoV-2 virus, hCoV-19/Wuhan/Hu-1/2019 (denoted as WuhanHu1), but were situated among strains (USA) that are mainly from America (Figure 1). This indicated the five imported COVID-19 cases were likely to have originated from contact with infection sources in the USA, not Taiwan”--- Based on the current Figure 1, such claim is rather contentious because (a) the ancestral Wuhan sequence is not situated at the base of the phylogenetic tree, and (b) the current Figure 1 may also suggest that Wuhan case was originated from the USA. Please address the concern raised in Point #1, and see if the authors' observation remains valid.
4.	The authors used genome-wide single-nucleotide variations to establish the haplotype network. This is interesting but would appreciate it if the authors could include a more detailed methodology for this part. The current methodology does not provide sufficient information to enable the readers to reproduce the observation. Also, the sampling bias remains prominent here with 64% of the data originated from the USA.

Other comments
1.	Please check the Reference section; The format does not seem to match the requirement.
2.	Please include citation(s) or source(s).
(a).	Line 55-57: 87% were imported from other countries
(b).	Line 70-72
3.	Unclear sentence structure
(a).	Line 73-76: The SARS-CoV2 genome sequences our five specimens were then compared…”
4.	Add “real-time” to Line 82, since the text is more about real-time PCR, not reverse transcription PCR
5.	Naming should be “SARS-CoV-2”, not
(a).	Line 92:  SARS-COV-2
(b).	Line 111: SARSCoV-2
6.	Table 1: If available, would be nice to include the length of stay of these travelers in the USA.


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: GENOMIC ANALYSIS OF EARLY TRANSMISSIBILITY ASSESSMENT OF THE D614G MUTANT STRAIN OF SARS-COV-2 IN TRAVELERS RETURNING TO TAIWAN FROM THE UNITED STATES OF AMERICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is professional, clear, and informative. I would advise some further English language fluency improvement in order to provide a better reading experience and easier understanding of the content.
Experimental design: 1. The method and materials could add more details regarding the method of sample collection and nucleic acid extraction. Even though the article does state the SARS-CoV-2 testing method reference between line 85 to 87, it would help readers have a better understanding of the whole experimental method with some brief description regarding the sample collection and nucleic acid extraction to ensure good sample processing and quality prior to the subsequent qPCR and sequencing.

2. The guideline for variant case selection could be stated here to help the readers understand how the five cases were selected and under what criteria.
Validity of the findings: 1.	Case 5 (TSGH-29) is significantly closer to Wuhan-Hu-1 strain compared to Case 1, 2, 3, and 4. Please clarify the reasoning of the extended branch-length from the ancestral human SARS-CoV-2 virus.

2.	The discussion section mentioned that Case 1 to Case 4 conform with the findings by Kannan et al., 2020; Vilar and Isom, 2021, but did not mention further findings of Case 5, which is quite unique compared to Case 1 to Case 4 providing the mutated genes were completely different. It would be more comprehensive if Case 5 could be further investigated and described.

3.	The five cases all had moderate to high viral load. Together with the infection of the mutated SARS-CoV-2, if possible, please describe if the patients have more severe symptoms or special treatments needed compared to the wild type. And if there are any suggestions regarding if the border control should be strengthened or if mandatory variant testing for COVID-19 positive patients should be implemented in order to prevent more deadly variants from spreading to countries based on the clinical significance.
Additional comments: In general, the article provided me extensive information and knowledge regarding the mutated COVID-19 virus. I would appreciate it if the article could also mention deeper insights regarding the importance of preventing the variants from spreading and how the variants could adversely affect public health to provide more supportive facts to the conclusions.


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: GENOMIC ANALYSIS OF EARLY TRANSMISSIBILITY ASSESSMENT OF THE D614G MUTANT STRAIN OF SARS-COV-2 IN TRAVELERS RETURNING TO TAIWAN FROM THE UNITED STATES OF AMERICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Jian et al. present the analysis of five SARS-CoV-2 samples from patients with a travel history from USA to Taiwan in March and June 2020.  The manuscript focuses in the analysis of the D614G change that has been related with a higher transmissibility.
Experimental design: The samples are described as “isolated” but there is no description of culture of the virus. The genomes are obtained using a previous PCR enrichment procedure (ARTIC?) but this is not described in the manuscript. In the current scenario, where there are hundreds of thousand available genome, five genomes seem to be a small sample for analysis. The authors must remark the importance of analyzing this number of samples to understand the evolution of SARS-CoV-2 specific markers.
The analysis of the samples is correct but too basic, a neighbor-joining tree with relatively few numbers of samples, considering the available genomes in the databases, provide scarce results. The D614G change is found in most strains currently circulating around the world and there are several studies that deal with its origin and spreading.
Validity of the findings: no comment
Additional comments: I suggest deepening the classification of the samples using pangolin software (https://pangolin.cog-uk.io/) and to establish the genetic changes using for example the glue application (http://cov-glue.cvr.gla.ac.uk/) that will increase your genome information.


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: GENOMIC ANALYSIS OF EARLY TRANSMISSIBILITY ASSESSMENT OF THE D614G MUTANT STRAIN OF SARS-COV-2 IN TRAVELERS RETURNING TO TAIWAN FROM THE UNITED STATES OF AMERICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I applaud the authors' effort and initiative in addressing all the comments. However, the topology of the phylogenetic tree in Figure 1 does not support the authors' claim. A few matters to consider

1) Phylogenetically speaking, the Wuhan strain does not appear to be an ancestral strain (though a better way to illustrate this may be through genealogical analysis by estimating the tMRCA using BEAST). As of now, it appears that CTYale046/2020 is the ancestral strain, which is contradicting.

2) Lineages clustering in Figure 1: For example, based on Supplementary Table 1, isolate CACZB1066/2020|EPI_ISL_437066|20200416 is categorized as lineage B.46. However, in Fig 1, this isolate was intermingled between TSGH29 and WuhanHu1, both of which were categorized as Lineage B. Please check the clustering and topology of every single isolate in the tree (not just the TSGH isolates), to safeguard the accuracy and reliability of the claim.

3) I am not certain if Hepatitis B virus strain will be a suitable outgroup. I would suggest the authors include the endemic human coronaviruses - OC43, HKU1, NL63, and 229E - as an outgroup.
Experimental design: As mentioned above
Validity of the findings: As mentioned
Additional comments: In general, the work is interesting and important, though some confirmatory analysis is required.


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: GENOMIC ANALYSIS OF EARLY TRANSMISSIBILITY ASSESSMENT OF THE D614G MUTANT STRAIN OF SARS-COV-2 IN TRAVELERS RETURNING TO TAIWAN FROM THE UNITED STATES OF AMERICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revision of the article addresses all the questions and suggestions with professionalism. No other comments are made.


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 SPLEEN BACTERIOME OF WILD RODENTS AND SHREWS FROM MARIGAT, BARINGO COUNTY, KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text is written fairly clearly, however, there are a number of grammatical issues that are distracting and detract from the paper. Additionally, there are several improvements that could be made to the basic reporting of this paper.
Firstly, there is very little literature review or additional context provided. The first line of the background statement discusses EIDs, but the authors do not discuss EIDs in depth, nor how microbiome metabarcoding is useful for monitoring EIDs. More information on prior microbiome studies on small mammals and the usefulness of the spleen as a sampling tissue is needed. The current introduction discusses only surface-level topics about small mammal pathogens.
There are also no stated hypotheses, although the authors do calculate Shannon diversity and assess differences among different species in the discussion. There is clearly an opportunity to test for differences in diversity among the different species, but the authors make no attempt to do this. In addition to conducting this analysis, the authors should also discuss the meaning of high/low Shannon diversity in the context of EIDs, as well as more discussion about the specific bacterial groups detected.
There are a few issues with some of the figures:
Figure 1: The font is entirely unreadable
Figure 2: Please provide sample sizes for each small mammal group
Figure 3: This figure is easier to read than figure 1, but is still challenging to see the bacterial groups.
Figure 4: This would be better as an ordination. At the very least, the samples should be grouped and labeled by small mammal species. It looks like Bartonella was very common in about half of the samples. Are these individuals from the same group? Additionally, the diverging color scheme is inappropriate for these data.
Figure 5: Providing statistical information on this plot would improve interpretation (e.g. which means are significantly different?)
Experimental design: There was no specific research question aside from a goal to sequence and identify the spleen microbiome. This study could be improved by examining differences in bacterial composition among the different rodent species. Similarly, more detail about the samples is needed (despite the citation to the more in-depth previous study, further information about sampling dates, storage method, and species breakdown is needed).
It is not entirely clear what knowledge gap is being filled, as the introduction does not review metabarcoding studies in small mammals, nor does it discuss whether the species examined in this study have been investigated in this manner before.
The metabarcoding and bioinformatics methods are generally clear and well-described; however there is very little discussion of rarefaction methods and diversity calculations/analyses.
Validity of the findings: There are no statistical tests of diversity differences among species, yet the authors claim in the discussion that diversity in Acomys is higher than in other genera. A test for diversity differences should be conducted if they want to support their claims with the data. Additionally, some kind of ordination of bacterial communities among genera (plus rays for specific bacterial groups) would also be more informative than the results as they are currently presented. Again, there was no clear research question posed in the introduction, so it is impossible to connect the conclusions in the discussion with the original study goals. A better review of the literature, justification for the study, and meaningful research question would improve this paper. Finally, the conclusion section did not seem to reflect the actual data from the study. It could have been written without any of the information from the study (i.e. the finding that the spleen acts as a microbial filter and that the 16S rRNA region can be used to characterize microbiota are findings from Ge 2018). While this study replicates the Ge 2018 study, the authors do not provide a clear rationale for why their study furthers our understanding (aside from saying that the specific species from the study location were examined). However, if the authors are able to provide a more in-depth analysis, especially of bacterial diversity, in additional to details about the ecology of the small mammal species and why this matters for disease transmission, then this study would provide a meaningful extension to the Ge 2018 study.
Additional comments: The data and sequences from this study are important, as they document bacterial communities in a range of small mammal genera of disease concern (e.g. Rattus) in Kenya. However, the study needs far more context and rationale, including a clear research question.


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 SPLEEN BACTERIOME OF WILD RODENTS AND SHREWS FROM MARIGAT, BARINGO COUNTY, KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are many errors in the paper. I forged ahead and read it anyway. It seemed to have promise. Introduction to the methods of barcoding was extremely superficial. The authors mix up classification with identification of mammals. Several references are incomplete.
Experimental design: no comment
Validity of the findings: Any student who had a basic course in mammalogy can identify a shrew versus a rodent. There was a lot of wasted effort sequencing these mammals when someone could just look at  them. Also since there were no specimens deposited in museums the findings cannot be replicated or ever checked. Did the authors just throw away the mammals after the materials were taken from them?
Additional comments: Line 56: Some species are spread around like Mus musculus and M. domesticus and Rattus norvegicus and R. rattus and a few Sciurus spp. plus some others. Nutria (Myocastor coypus) have been transplanted from Bolivia to various continents. Beavers (Castor canadensis) are running rampant in Argentina, but for the most part those are just a few species of rodents that are sufficiently wide-spectrum in their ecological niches that they can be moved around by humans and then thrive where they are introduced.
Line 66. Rats and mice may harbor organisms like viruses and bacteria and helminths that can cause disease in animals, they do not transmit the disease. They may act as reserviors of the various pathogens that cause disease in other hosts.
The reference for these statements is incomplete:
368 Lindahl, J. F., & Grace, D. 2015. The consequences of human actions on risks for infectious 369 diseases: a review. 1, 1–11.
Line 76 paragraph. The authors mean identification instead of classification. Classification means the process of delimitation, ordering, and ranking of taxa. Identification means to find out what the species of mouse is in hand.
Line 78. Morphological classification means nothing. What the authors mean is identification of rodents using morphological characters.
LIne 85 - Accurate reconstructions of what?
I think that the literature on the barcoding initiative should be consulted to write this section.
https://ibol.org/about/dna-barcoding/

Line 237: I see no reason to go to the trouble to create a phylogeny of species that are clearly very distantly related. It does not make logical sense to spend the time and resources to do this and there is nothing that can be derived from it.f
Line 240: Then why do you say that morphological methods for identification is poor in the introduction? Here you show that this is not the case.

Unfortunately this paper was ill conceived: Trying to id the mammals using molecules when a look at the skull by any competent mammalogist would give the ID instantly. It is NOT POSSIBLE to mix up the identification of a shrew with a rodent unless the person doing the ID had never seen one before.
I cannot say much about the species of bacteria that were found in the spleen. The work seems good in that regard.