Reviewer name and names of any other individual's who aided in reviewer	Yunyun Lv
Do you understand and agree to our policy of having open and named reviews, and having your review included with the published papers. (If no, please inform the editor that you cannot review this manuscript.)	Yes
Is the language of sufficient quality?	Yes
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Are all data available and do they match the descriptions in the paper?	Yes
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Are the data and metadata consistent with relevant minimum information or reporting standards? See GigaDB checklists for examples <a href="http://gigadb.org/site/guide" target="_blank">http://gigadb.org/site/guide</a>	Yes
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Is the data acquisition clear, complete and methodologically sound?	Yes
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Is there sufficient detail in the methods and data-processing steps to allow reproduction?	Yes
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Is there sufficient data validation and statistical analyses of data quality?	No
Additional Comments	Appropriate modifications
Is the validation suitable for this type of data?	Yes
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Is there sufficient information for others to reuse this dataset or integrate it with other data?	Yes
Additional Comments
Any Additional Overall Comments to the Author	The main contribution of this article is a new chromosome-level genomic assembly of humpback puffer with NGS, TGS and Hi-C reads. The high-quality of the assembly is reflected in the results and figures. However, I feel some results should be checked and some sentence should be rephrased until it could be officially received. I list the lines with errors or that need to be rewritten. In summary, I feel this article could be acceptable after major revisions. Abstract: “ Despite interesting biological features, such as its very inactive nature, tetrodotoxin production and body expansion mechanisms, molecular research on the humpback puffer is still rare because of the lack of a high-quality reference genome.” I feel this sentence should be rephrased. I understand a high-quality genome resource should benefit molecular research that focused on this species. However, I don’t think the reason of rare molecular studies is due to a lack of genomic assembly. In addition, “inactive nature” should be replaced by other words as my opinion. “scaffold N50s”? “Based on the genome, ~61.5Mb (18.11%) repeat sequences were also identified, and totally 19,925 genes were annotated, 99.20% of which could be predicted with function using protein-coding function databases.” I feel this sentence should be rewritten. In addition, the inconsistence appears in the abstract and results. There are 90.1% genes could be annotated with biological function as author’s announcement in section of genome annotation. This mistake should be fixed in the revision. ‘Finally, a phylogenetic tree was constructed with single-copy gene families from ten teleost fishes.’ This sentence just tells me you have done the phylogenetic analysis, but where the result (phylogenetic status of humpback puffer according to your analysis). The humpback puffer genome will be a valuable genomic resource to illustrate possible mechanisms of tetrodotoxin synthesis and tolerance, providing clues for future detailed studies of biological toxins. I understand this genome would be valuable for further studies, but I cannot see any clues for studying biological toxins in this article. Data Description The author mentioned slight sex dimorphism occurred between male and female, however, the sampling sex used to genomic assembly is not described. ‘Different from other species of predatory pufferfish, the humpback puffer is so inactive that it only moves when the food is right in front of it’ I feel confusion in this sentence. The humpback pufferfish is not predatory, or just owes lazy behavior in food seeking process? ‘Previous studies have proved that the content of the toxicity in the humpback puffer varies greatly in different seasons, so it can be edible when its skin and internal organs are removed’ I feel the former has no logical relationship with the latter. ‘In addition to these biological characteristics, the compact genome size of humpback puffer is roughly about 385 Mb, which alongside other pufferfish species which have been used to study intron evolution, makes it an ideal model species for genetic study’ This sentence is too long that bring difficulties to read. In this study, we provided a chromosome-scale genome of an adult humpback puffer that will allow us to study features such as mechanisms of tetrodotoxin synthesis, expansion defense, body differences between males and females, and genome size. Comparative genomics analysis can help to better understand the phenotypic evolution and special gene families of the Tetraodontidae. This sentence may mislead the readers that this article should contain those analyses related to the mentioned features, but actually not. Thus, I feel it should be rephrased. Methods To be honest, I feel the section title should be “Materials, Methods and Result” as the content actually contains the three aspects. In the assembling process, the authors assembled draft assembly with only using of stLER reads but without PB data, and the PB data was only used for closing gaps? I have not used TGSgapfiller, but I think the function of this program may be limited in gap close but not expand the assembled contigs. Why do not use the PB reads in initial draft contig assembly? How to define chromosomes number (18) in 3dDNA pipeline should be described. “1,945 bps coding region” should be “1,945 bp ” As mentioned above, the inconsistence appears in this section and Abstract ‘We firstly used MUSCLE…..’ Type error. There is a redundant space between ‘to’ and ‘align’ in this sentence. The author should describe how many fossils used to correct the phylogenetic topology and which nodes be corrected. In addition, I am curious that why parameter of alpha is 0.431879.
Recommendation	Major Revision