Improvements to the Gulf Pipefish Syngnathus scovelli Genome

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The Gulf pipefish Syngnathus scovelli has emerged as an important species in the study of sexual selection, development, and physiology, among other topics. The fish family Syngnathidae, which includes pipefishes, seahorses, and seadragons, has become an increasingly attractive target for comparative research in ecological and evolutionary genomics. These endeavors depend on having a high-quality genome assembly and annotation. However, the first version of the S. scovelli genome assembly was generated by short-read sequencing and annotated using a small set of RNA-sequence data, resulting in limited contiguity and a relatively poor annotation. Here, we present an improved genome assembly and an enhanced annotation, resulting in a new official gene set for S. scovelli . By using PacBio long-read high-fidelity (Hi-Fi) sequences and a proximity ligation (Hi-C) library, we fill small gaps and join the contigs to obtain 22 chromosome-level scaffolds. Compared to the previously published genome, the gaps in our novel genome assembly are smaller, the N75 is much larger (13.3 Mb), and this new genome is around 95% BUSCO complete. The precision of the gene models in the NCBI’s eukaryotic annotation pipeline was enhanced by using a large body of RNA-Seq reads from different tissue types, leading to the discovery of 28,162 genes, of which 8,061 were non-coding genes. This new genome assembly and the annotation are tagged as a RefSeq genome by NCBI and thus provide substantially enhanced genomic resources for future research involving S. scovelli .

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  1. Abstract

    This work has been published in GigaByte Journal under a CC-BY 4.0 license ( and has published the reviews under the same license.

    **Reviewer 1. Sven Winter **

    I am really sorry, and I do not want to sound mean, but this manuscript needs major improvements in structure, writing, and data validation. It violates so many standard practices of scientific writing. I have never seen anybody cite a full title of a previous manuscript. There is absolutely no need for that. The annotation is labeled as an improved annotation, but its results are only listed in the abstract, and it is not mentioned how it is generated anywhere other than the data availability section. That the genome is tagged under RefSeq by NCBI is absolutely unnecessary information in the abstract, this is just a label, and it tells not much about the quality. I would urge the authors to restructure the manuscript. Start with a short description of the species and why the species and its genome is important as an introduction, then focus on a detailed data description with methods and basic results such as assembly statistics (importantly not just scaffold N50 but also on the contig-level!), Busco, Merqury completeness and error rate, genome size estimate, annotation (repeat and gene), etc. There is really no need for 30 pages of useless supplementary tables (please also make sure that next time you sort the files during the submission so that the pdf does not start with 30 pages of tables). The data cannot support any information about gene loss, as there is so much of the assemblies not properly anchored into chromosomes. I would also try to improve the Hi-C contact map figure. There is really no need for the blue and green boxes and the assembly label at the x-axis. I may have overlooked it due to the writing style, but I would like to see mentioned how much of the assembly is in the chromosome-scale scaffolds and how much is unplaced. I like the improved assembly, it just needs a much better presentation in form of a well-structured manuscript, and unfortunately, in its current form, it clearly is not well-structured. There are plenty of other data notes available as templates. I personally would always opt for a more traditional manuscript structure (Introduction, Methods, combined Results and Discussion), but that is my personal preference. I hope my comments are helpful, and I am looking forward to seeing a revised version in the future.


    Thank you for the improvement of the manuscript. It is now easier to follow and includes more information as before. It was a bit difficult to see the changes as they were not highlighted and the lines are not numbered. Despite that, I have only a few minor comments that should be addressed easily so that the manuscript will be ready for publication soon. Line numbers in the comments refer to lines of the specific paragraph/section.

    DNA and RNA extraction: L7:such as? If you listed all tissues, please remove such as, if you sequenced RNA for nor tissues please add them.

    Sequencing and Assembly: L5: 159 bp is an uncommon read length. Was this just a typo, or how did that come to be? L10: remove "the" before juicer; otherwise, it sounds like an actual fruit juicer instead of a bioinformatics tool ;-). Same for 3D-DNA in the line below. Please make it more clear in the text if you sequenced the RNA for each tissue separately or in one library. L11-12: I am not convinced that not allowing for correction was the right approach. Did you test how the results would look with corrections enabled?

    Assembly Statistics and Quast Results: Quast calculates assembly statistics so I am not sure why the header needs to include both. L5: Please avoid using "better" but instead rephrase so that is is clear that the NG50 is 1.75x larger than the previous assembly. "Better" is not clear.

    Busco and Merqury results: I would not claim that Busco says the genome is 95% complete, as busco only tries to find genes that are supposedly orthologous in Actinopterygii. So I would rather say Busco suggests a high completeness as it finds 95% of the orthologs. Also, all genes in the Busco dataset are supposed to be single-copy orthologs; therefore, I would not say that 93% are conserved single-copy orthologs, as the remaining duplicated or fragmented genes could just be assembly errors. Please also state the Merqury QV value, and I would suggest stating the error rate in %. I still find the discussion about missing Busco genes strange, as since Busco 4 or 5 the datasets all got much larger and the Busco completeness values went down in most assemblies, even in well studies taxa as mammals. With recent datasets, it is very unlikely to get much more than 95-97%. In my opinion, it is rather a sign of too large and incorrect Busco datasets than evidence for missing orthologs. I would at least add that point to the discussion.

    Table 1: Please follow standard practice in scientific writing and add separators to the numbers in all tables (main text and supplementary), e.g., 28444102  28,444,102. Otherwise, they are difficult to read.

    Annotation Results: L3: 20,101 coding genes, 18,616 genes … Please check throughout the whole manuscript for consistent style.

    Data Availability: L2: Annotation report release 100. What does "100" stand for? Also, "at here" sounds not correct; please remove "at". L4: Table S2 does not show the scaffold identifiers. L5: please state the complete BioProject accession not just the numerical part.

    Supplementary data: Please change numbers in all tables to standard format e.g., 21,671,036

    **Reviewer 2. Yue Song **

    (1) Please state clearly how much CCS Hi-Fi data has been produced by sequencing and hic-data finally used for chromosome assembly after filtration, not just the number of reads. (2) Please state clearly the estimated genome size using Hi-Fi data. 

    (3) What is the process for “correct primary assembly misassembles”? Please described in detail. (4) In Table 1, I noticed that the difference between the new and previous genome of S.scovelli is more than 100M (about 25% of the size of the newly assembly). Otherwise, most of genome size of Syngnathus species ranged from 280-340 Mb, I think take some explanation of these extra sequences is necessary. (5) Need more detailed parameters and process about genome assembly and gene annotation. (6) Whether the previous version had any assembly errors and updated in this new one. if this exists, please state so.