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. 2023 Feb 20;2023:gigabyte76. doi: 10.46471/gigabyte.76

Improvements to the Gulf pipefish Syngnathus scovelli genome

Balan Ramesh 1,*, Clay M Small 2,3, Hope Healey 2, Bernadette Johnson 1, Elyse Barker 1, Mark Currey 2, Susan Bassham 2, Megean Myers 1, William A Cresko 2,3,†, Adam Gregory Jones 1,*,†
PMCID: PMC10038202  PMID: 36969711

Abstract

The Gulf pipefish Syngnathus scovelli has emerged as an important species for studying sexual selection, development, and physiology. Comparative evolutionary genomics research involving fishes from Syngnathidae depends on having a high-quality genome assembly and annotation. However, the first S. scovelli genome assembled using short-read sequences and a smaller RNA-sequence dataset has limited contiguity and a relatively poor annotation. Here, using PacBio long-read high-fidelity sequences and a proximity ligation library, we generate an improved assembly to obtain 22 chromosome-level scaffolds. Compared to the first assembly, the gaps in the improved assembly are smaller, the N75 is larger, and our genome is ~95% BUSCO complete. Using a large body of RNA-Seq reads from different tissue types and NCBI's Eukaryotic Annotation Pipeline, we discovered 28,162 genes, of which 8,061 are non-coding genes. Our new genome assembly and annotation are tagged as a RefSeq genome by NCBI and provide enhanced resources for research work involving S. scovelli.

Data description

This article presents a resource (genome assembly) that marks a technological improvement compared to the one previously published in the article, “The genome of the Gulf pipefish enables understanding of evolutionary innovations” [1].

A de novo genome assembly is evaluated based on three primary criteria: accuracy or correctness, completeness, and contiguity [2, 3]. Typically, the correctness of a genome is one of the most challenging features to measure. However, with modern, long-read sequencing technologies, the orientation of the contigs and the gene order of an assembly are highly accurate [4–6]. On the other hand, completeness and contiguity are easier to measure [6–8] yet more challenging to achieve, especially in non-model organisms. The Gulf pipefish (Syngnathus scovelli, NCBI:txid161590, fishbase ID: 3306) genome is an essential resource for the study of comparative genomics, evolutionary developmental biology, and other related topics [1, 9–15]. Given the technological constraints when it was initially sequenced, the first version of the S. scovelli genome is highly accurate and mostly complete, but it leaves considerable room for improvement with respect to contiguity [1]. Here, with the use of third-generation sequencing technology, including PacBio High Fidelity (Hi-Fi) long reads from circular consensus sequences (CCS) and Hi-C proximity ligation from Phase Genomics, we produced a nearly complete chromosome-scale genome assembly that not only improves completeness and accuracy but is also the most contiguous genome yet produced for the genus Syngnathus (Table 1).

Table 1.

Contiguity metrics from QUAST for various Syngnathus species.

Metrics S. acus S. rostellatus S. typhle S. floridae S. scovelli _v1 S. scovelli _v2
Number of contigs 87 8,935 526 6,895 886 526
Largest contig 28,444,102 856,273 9,665,359 61,807,209 23,505,159 30,098,933
Total length 324,331,233 280,208,023 313,958,489 303,298,972 305,995,683 431,750,762
Reference length 324,331,233 324,331,233 324,331,233 324,331,233 324,331,233 324,331,233
GC (%) 43.46 43.08 43.29 43.63 42.95 45.00
Reference GC (%) 43.46 43.46 43.46 43.46 43.46 43.46
N50 14,974,571 88,962 3,046,963 7,845,045 12,400,093 17,337,441
NG50 14,974,571 70,018 3,012,268 7,783,711 11,493,655 20,118,474
N75 11,896,884 34,357 1,098,273 21,150 8,458,319 13,347,818
NG75 11,896,884 15,229 998,421 17,023 7,908,134 15,901,424
L50 8 812 30 5 10 10
LG50 8 1,092 32 6 11 7
L75 14 2,068 72 1,160 17 17
LG75 14 3,492 79 2,003 19 12
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For NGx and LGx calculations, S. acus was used as the reference species. All the Sygnathus genomes (except S. scovelli) were last accessed from NCBI on 2022-July-26.

Context

Evolutionary novelties are widespread across the tree of life. However, the origin of de novo genes and their associated regulatory networks, as well as their effects on the phenotype, remain mysterious in most species. Syngnathidae is a family of teleost fishes that includes pipefishes, seahorses, and seadragons [1, 12–16]. Syngnathid fishes are known for their evolutionary novelty with respect to morphology and physiology. For instance, species in this family have variously evolved elaborate leafy appendages, male brooding structures, prehensile tails, elongated facial bones, and numerous other unusual traits [1, 12–14]. With a variety of mating systems and sex roles [12–16], the syngnathid fishes also provide an excellent study system to investigate the generality of theories on sexual selection and reproductive biology [15, 16]. Advances in comparative genomics and the evolutionary developmental biology of novel traits in syngnathids require the development of additional genomic tools. Among these are well-assembled and annotated genomes [1]. Here, we took a step in this direction by producing an improved reference genome for the Gulf pipefish.

Methods

DNA and RNA extraction

We collected S. scovelli from the Gulf of Mexico in Florida, USA (Tampa Bay), and flash froze them in liquid nitrogen. We pulverized approximately 50 mg of whole-body tissue (posterior to the urogenital opening) from a single male on liquid nitrogen, which we submitted to the University of Oregon Genomics and Cell Characterization Core Facility (UOGC3F) for high-molecular-weight DNA isolation using the PacBio Nanobind tissue kit. We submitted similar (but unpulverized) frozen tissue from the same individual fish to Phase Genomics to generate a Hi-C library using Proximo Animal (v4) technology.

In addition, we used organic extraction with TRIzol Reagent, followed by column-based binding and purification using the Qiagen RNeasy MinElute Cleanup Kit, to extract mRNA from the Brain, Eye, Gills, Muscle/Skin, Testis, Ovary, Broodpouch, and Flap tissues.

Sequencing and assembly

After the size selection of genomic DNA using the Blue Pippin (11 kb cutoff), the UOGC3F constructed a sequencing library using the SMRTbell Express Template Prep Kit 2.0. One SMRT cell was sequenced by the UOGC3F using PacBio Sequel II technology, yielding 33.39 Gb in 2.05M CCS reads (out of 6.298M Hi-Fi reads in total). We sequenced 70.4 Gb of paired-end 150 nucleotide reads (234.6 million in total) from the Hi-C library using an Illumina NovaSeq 6000 at the UOGC3F. The RNA sequencing libraries were prepared using the KAPA mRNA HyperPrep Kit. We sequenced 159 bp paired-end reads using Illumina Novaseq 6000 for each tissue from the RNA sequencing libraries for annotation.

Using the Hi-Fi sequences, we estimated the genome size using genomescope2 (v2.0, RRID:SCR_017014) [17] and meryl (v2.2) [18] with a default k-mer size of 21 (Figure 1). The paired-end Hi-C reads were trimmed using trimmomatic (v0.39, RRID:SCR_011848) [19] with the parameter HEADCROP:1 to remove the first base, which was of low quality. Together with the Hi-Fi sequences, we assembled the first-pass genome assembly in Hi-C integrated mode using hifiasm (v0.16.1, RRID:SCR_021069) [18] with default parameters. The First-Pass assembly refers to the first draft consensus assembly from the Hi-Fi and Hi-C data. We extracted the consensus genome from hifiasm in fasta format and assembled the contigs into scaffolds using juicer (v1.6, RRID:SCR_017226) [20]. We used the 3D-DNA (version date: Dec 7, 2016) [21] pipeline to merely order the scaffolds. The Hi-C contact map of the ordered scaffolds was visualized using juicebox (v1.9.8, RRID:SCR_021172) with no breaking of the original contigs.

Figure 1.

Figure 1.

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Estimated genome size of Syngnathus scovelli based on k-mer analysis using Meryl and Genomescope.

Assessment of completeness and contiguity

To compare the completeness and contiguity of the latest version of the S. scovelli genome against the other Syngnathus genomes (Figure 2), we downloaded the genome assemblies of S. acus (GCA_024217435.2), S. rostellatus (GCA_901007895.1) [22], S. typhle (GCA_901007915.1) [22], and S. floridae (GCA_010014945.1) from NCBI. We used Benchmarking Universal Single-Copy Orthologs (BUSCO v5.2.2, RRID:SCR_015008) [23] in genome mode with the actinopterygii_odb10 database (as of 2021-02-19) to evaluate the completeness of the genome. Also, we used a k-mer-based assessment using Merqury (v2020-01-29, RRID:SCR_004231. [24]) to estimate the completeness and the base error rate. We then used the Quality Assessment Tool (QUAST v5.0.2, RRID:SCR_001228) [25] to estimate Nx and Lx statistics for our assembly.

Figure 2.

Figure 2.

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Cladogram of the five Syngnathus species in this study. This phylogeny is based on the Ultra Conserved Elements among all syngnathids [26].

Annotation using the NCBI Eukaryotic annotation pipeline

The NCBI Eukaryotic Genome Annotation Pipeline (v10.0) is an automated software pipeline identifying coding and non-coding genes, transcripts, and proteins on complete and incomplete genome submissions to NCBI. The core components of this pipeline are the RNA alignment program (STAR and Splign) and Gnomon, a gene prediction program. In this pipeline, the RNA-Seq reads from the various (Brain, Eye, Gills, Muscle/Skin, Testis, Ovary, Broodpouch, and Flap) tissues of multiple samples, including the S. scovelli individual used for Hi-Fi and Hi-C sequence data (SRR20438584–SRR20438604), were aligned to the genome. Gnomon combines the information from alignments of the transcripts and the ab initio models from a Hidden Markov Model-based algorithm to create a RefSeq annotation. This RefSeq annotation produces a non-redundant set of a predicted transcriptome and a proteome that can be used for various analyses. The Eukaryotic annotation pipeline is not publicly available; thus, we requested the staff at NCBI to annotate the S. scovelli genome.

Data validation and quality control

Assembly statistics

With approximately 2 million Hi-Fi reads and 234.6 million Hi-C reads, we generated the first pass consensus assembly with 585 contigs. The N50 and L50 for this assembly were 15.5 Mb and 11, respectively. We scaffolded this assembly to correct misassembles and produced a final assembly containing 526 contigs with N50 and L50 values of 17.3 Mb and 10, respectively (Table 1). This improved version of the S. scovelli genome has around three times fewer contigs compared to the original S. scovelli genome. The NG50 and NG75 are ∼1.75× and ∼2× larger, respectively, than the previous assembly, implying less fragmentation. Our new assembly reduces the number of gaps per 100 kilobase pairs (kb) from 6,837.20 Ns per 100 kb to a mere 0.27 Ns per 100 kbp, owing to the increased contiguity. This new S. scovelli genome is on par with the current best genome in the Syngnathus genus, that of S. acus, which is a complete chromosome-scale assembly. The first 22 scaffolds of the S. scovelli genome are of chromosome-scale in line with the genetic map [1] and the karyotype data [27] with a total length of around 380 Mb (Figure 3), comparable to the estimated genome size of 380 Mb (see GigaDB [28]; Table 2 and Figure 3). In addition, 88.94% of the total assembly length is captured in the 22 chromosome-scale scaffolds. For 15 of the chromosome-scale scaffolds, a single contig makes up the total length; the remaining seven are generally composed of a small number of contigs (Figure 3).

Figure 3.

Figure 3.

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Visualization of contact maps from Hi-C reads for Syngnathus scovelli (v2). The first 22 primary assembly features (blue lines) sum to about 380 Mb in size, which is the estimated genome size for the species. The green lines reflect the individual contigs from the hifiasm assembly that were organized into chromosome-level scaffolds based on Hi-C contact data.

Table 2.

Contiguity metrics from QUAST for the first pass and the scaffolded assembly of S. scovelli _v2.

Metrics Haplotype1 Haplotype2 Primary consensus assembly Scaffolded assembly
Number of contigs 901 544 585 526
Largest contig 21,671,036 23,661,123 30,098,933 30,098,933
Total length 427,545,154 428,155,884 431,749,582 431,750,762
GC (%) 44.99 44.78 45.00 45.00
N50 10,825,652 10,535,849 15,551,623 17,337,441
N75 4,999,310 4,477,557 11,049,644 13,347,818
L50 15 15 11 10
L75 29 30 19 17
Number of N’s per 100 kbp 0.00 0.00 0.00 0.27
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BUSCO and Merqury results

BUSCO results suggest a high degree of completeness as it found 95% of the orthologs in the Actinopterygii dataset (94.7% [S: 93.9%, D: 0.8%], F: 1.5%, M: 3.8%, n: 3,640) when run in genome mode (Figure 4) and the Merqury evaluation suggests that the genome is ∼86% complete with a quality value (QV) of 61.37 and an error rate of 7.3 × 10−5 % (see GigaDB [28] for more details; Tables 3 and 4).

Figure 4.

Figure 4.

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Comparison of BUSCO completeness among all the five Syngnathus species.

Table 3.

k-mer based assembly evaluation for completeness using Merqury.

Assembly k-mer set used solid k-mers in the assembly Total solid k-mers in the read set Completeness (%)
S. scovelli _v2 all 272,969,166 318,487,563 85.708
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Table 4.

k-mer based quality evaluation using Merqury.

Assembly k-mers uniquely found only in the assembly k-mers found in both the assembly and the read set QV Error rate
S. scovelli_v2 6,614 431,737,882 61.3697 7.29504 × 10−7
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Consistent with the BUSCO contiguity metrics, the genome is on par with S. acus for completeness, which is also around 95% complete. Missing genes make up the majority of the remaining 5% of genes. We identified genes likely to be truly missing from the S. scovelli genome and more broadly from members of Syngnathidae (including the seahorses, genus Hippocampus along with Syngnathus) by confirming their absence across the BUSCO results from the present assembly, four additional members of the genus Syngnathus, and six additional Hippocampus publicly available assemblies (see GigaDB [28] for additional details). Of the missing BUSCO genes, 83 are shared among all the species of Syngnathus, and 38 are missing from both genera (see GigaDB [28] for additional details). Future work could profitably explore these missing genes, as some may be related to the interesting novel traits in syngnathid fishes.

Annotation results

After masking about 43% of the genome, the annotations resulted in the prediction of about 28,162 genes, of which 8,061 are non-coding genes (see GigaDB [28]; Tables 5 and 6). The 28,162 genes produce about 59,938 transcripts, of which 47,846 are mRNA, and the rest is made up of other types of RNAs such as tRNA, lncRNA, and others. Out of the 20,101 coding genes, 18,616 had a protein with an alignment covering 50% or more of the query against the UniProtKB curated protein set, and 9,152 had an alignment covering 95% or more of the query.

Table 5.

Gene and Feature Statistics from NCBI Eukaryotic Pipeline.

Feature S. scovelli_v2
Genes and pseudogenes 29,062
protein-coding 20,101
non-coding 8,061
Transcribed pseudogenes 0
Non-transcribed pseudogenes 887
genes with variants 10,398
Immunoglobulin/T-cell receptor gene segments 9
other 4
mRNAs 47,846
    fully-supported 47,491
    with >5% ab initio 89
    partial 39
    with filled gap(s) 0
    known RefSeq 0
    model RefSeq 47,846
non-coding RNAs 12,092
    fully-supported 7,318
    with >5% ab initio 0
     partial 5
     with filled gap(s) 0
    known RefSeq 0
    model RefSeq 10,741
pseudo transcripts 0
    fully-supported 0
    with >5% ab initio 0
    partial 0
    with filled gap(s) 0
    known RefSeq 0
    model RefSeq 0
CDSs 47,855
    fully-supported 47,491
    with >5% ab initio 115
    partial 39
    with major correction(s) 144
    known RefSeq 0
    model RefSeq 47,846
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Table 6.

Detailed Feature Lengths from NCBI Eukaryotic Pipeline.

Feature Count Mean length (bp) Median length (bp) Min length (bp) Max length (bp)
Genes 28,166 11,149 4,361 56 677,970
All transcripts 59,938 3,654 2,773 56 106,526
mRNA 47,846 3,907 3,042 204 98,797
misc_RNA 2,018 3,844 2,824 138 22,974
tRNA 1,351 74 73 71 87
lncRNA 5,304 3,880 1,632 112 106,526
snoRNA 117 123 126 62 319
snRNA 378 142 141 56 196
rRNA 2,920 1,228 154 118 4,380
Single-exon 514 2,381 1,944 358 21,617
    coding 514 2,381 1,944 358 21,617
CDSs 47,846 2,373 1,617 96 97,746
Exons 277,161 325 142 2 38,823
    coding 260,368 299 140 2 38,823
    non-coding 27,774 515 152 9 36,521
Introns 247,597 1,355 160 30 611,280
    coding 235,861 1,207 152 30 611,280
    non-coding 22,579 2,911 304 30 498,241
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Reuse potential

The new version of the S. scovelli genome opens doors to more accurate results by enhancing the comparative genome data analysis and facilitating the creation of robust tools for molecular genetic studies. We generated the original version of the genome to focus on the genetic mechanisms underlying the unique body plan among pipefishes and seahorses. This genome version takes us one step closer to uncovering these evolutionary mysteries and aids in answering other unknown features, such as the effects of sexual selection and mate choice systems on genome evolution.

Acknowledgements

We are truly grateful for the dedicated efforts of Emily Rose and her students at Valdosta State University, who collected the S. scovelli samples crucial for this work (Florida Fish and Wildlife Conservation Commission Permit: SAL-17-0182-E, SAL-18-0182-E). We also thank Jeff Bishop and Tina Arredondo from the University of Oregon (UO) GC3F for library preparation and sequencing assistance. We want to acknowledge the staff at Phase Genomics for their helpful Hi-C technical support. We are grateful to Mike Coleman and Mark Allen for assisting with Talapas Supercomputer Cluster at UO and Benji Oswald with Research Computing and Data Services at UI. We greatly appreciate the support of the NCBI staff for the Eukaryotic Annotation Pipeline. We thank Jacelyn Shu for her pipefish illustrations. We are grateful for the helpful comments and review by Sven Winter and Yue Song on the manuscript.

Funding Statement

This work was funded by National Science Foundation (NSF) Grant 2015419 to WAC and AGJ and Grant 1953170 to AGJ. We also acknowledge the startup funds provided by the University of Idaho to AGJ.

Data Availability

The genome is available on NCBI with the assembly accession number GCA_024217435.2. The genome is annotated via the NCBI eukaryotic genome annotation pipeline, and the annotation report release (100) is available here. Several smaller contigs and contaminant microbes were removed in the annotation pipeline yielding a more robust genome assembly. The sequence identifier for the chromosome-level scaffolds is available in the GigaDB [28]. The NCBI Bioproject accession number is PRJNA851781, the raw Hi-Fi sequence accession is SRR19820733, the Hi-C sequence accession is SRR22219025, and the RNA-Seq sequence files from various tissues are SRR20438584–SRR20438604. Additional data is available in the GigaDB [28].

Declarations

List of abbreviations

BUSCO: Benchmarking Universal Single-Copy Orthologs; CCS: Circular Consensus Sequence; Gb: Giga basepair; Hi-Fi: High-Fidelity; Mb: Mega basepair; NCBI: National Center for Biotechnology Information; not: nucleotide; QUAST: Quality Assessment Tool; QV: Quality Value; SMRT: Single Molecule Real Time; University of Oregon Genomics and Cell Characterization Core Facility (UOGC3F).

Ethical approval

Not applicable.

Consent for publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.

Funding

This work was funded by National Science Foundation (NSF) Grant 2015419 to WAC and AGJ and Grant 1953170 to AGJ. We also acknowledge the startup funds provided by the University of Idaho to AGJ.

Authors’ contributions

Author contributions, described using the CRedIT taxonomy are as follows:

Conceptualization: BR, CMS, SB, WAC, AGJ; Methodology: BR, CMS, SB, BDJ, EB; Software: BR, CMS, HH, MC; Validation: BR, CMS; Formal Analysis: BR, CMS; Investigation: BR, CMS; Resources: MC, BDJ, EB, MM; Data Curation: BR, CMS, MC; Writing – Original Draft Preparation: BR, CMS, AGJ; Writing – Review & Editing: BR, CMS, AGJ; Visualization: BR, CMS; Supervision: WAC, AGJ; Project Administration: CMS, SB, WAC, AGJ; Funding Acquisition: WAC, AGJ.

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  • 28.Ramesh B, Small CM, Healey H et al. Supporting data for “Improvements to the Gulf Pipefish Syngnathus scovelli Genome”. GigaScience Database, 2023; 10.5524/102353. [DOI] [PMC free article] [PubMed] [Google Scholar]
GigaByte. 2023 Feb 20;2023:gigabyte76.

Article Submission

Balan Ramesh
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Assign Handling Editor

Editor: Scott Edmunds
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Editor Assess MS

Editor: Hongfang Zhang
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Curator Assess MS

Editor: Chris Armit
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Review MS

Editor: Sven Winter
Reviewer name and names of any other individual's who aided in reviewer Sven Winter
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
Please add additional comments on language quality to clarify if needed The writing style is quite different to standard scientific English. I would suggest a nearly complete rewrite to make it more concise and more structured.
Are all data available and do they match the descriptions in the paper? Yes
Additional Comments
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> No
Additional Comments I am missing detailed sampling locations and permit information.
Is the data acquisition clear, complete and methodologically sound? No
Additional Comments Permits and sample locations are missing.
Is there sufficient detail in the methods and data-processing steps to allow reproduction? No
Additional Comments I am missing more detailed QC steps and, in general, more details about the methods that were used. How much Hifi data was generated in Gb? Which k-mer size was used for genome size estimation? Was Trimmomatic only used to trim the first base? What is a first-pass genome assembly? What does "with no breaking of original contigs" mean? How can you correct and manually curate an assembly without breaking contigs? Was this a statement or a setting? Why was there no polishing and gap-closing performed? I understand the assembly is based on hifi-reads I would at least mention that no polishing is needed if that is the case, or did hifiasm include a polishing step? There is no explanation of the annotation process or the RNA sequencing. When comparing N50 values it is important to use NG50 instead.
Is there sufficient data validation and statistical analyses of data quality? No
Additional Comments I am missing more QC steps, such as Blobtools, Merqury, etc. to properly validate the accuracy of the assembly
Is the validation suitable for this type of data? Yes
Additional Comments The steps that were done, yes, but they are insufficient, in my opinion.
Is there sufficient information for others to reuse this dataset or integrate it with other data? Yes
Additional Comments I would rather say that there are to many unnecessary tables with no real information in it. List of missing Busco genes? There is no need for that as they are likely just missing due to the assembly quality. List of Accession numbers of Chromosome-level scaffolds without information about what chromosome the accession number belongs to. It is also very strange that the chromosomes are not ordered by length and that they are not listed as chromosomes on NCBI.
Any Additional Overall Comments to the Author 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.
Recommendation Major Revision
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Review MS

Editor: Yue Song
Reviewer name and names of any other individual's who aided in reviewer Yue Song
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
Please add additional comments on language quality to clarify if needed
Are all data available and do they match the descriptions in the paper? Yes
Additional Comments
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
Additional Comments
Is the data acquisition clear, complete and methodologically sound? Yes
Additional Comments
Is there sufficient detail in the methods and data-processing steps to allow reproduction? No
Additional Comments need more detailed paramaters and process about genome assembly. Although using the NCBI pipeline for gene annotation, it is better to give more details too.
Is there sufficient data validation and statistical analyses of data quality? Yes
Additional Comments
Is the validation suitable for this type of data? Yes
Additional Comments
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 (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 paramaters and process about genome assembly and gene annotation. (6) Whether the previous version had any assembly errors and updated in this new one. if exists, please state clearly.
Recommendation Minor Revision
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Editor Decision

Editor: Hongfang Zhang
GigaByte. 2023 Feb 20;2023:gigabyte76.

Major Revision

Balan Ramesh
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Assess Revision

Editor: Hongfang Zhang
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Re-Review MS

Editor: Sven Winter
Comments on revised manuscript 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
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Editor Decision

Editor: Hongfang Zhang
GigaByte. 2023 Feb 20;2023:gigabyte76.

Minor Revision

Balan Ramesh
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Assess Revision

Editor: Hongfang Zhang
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Final Data Preparation

Editor: Mary-Ann Tuli
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Editor Decision

Editor: Hongfang Zhang
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Accept

Editor: Scott Edmunds
Comments to the Author Please work with us to make sure the proofs and DOI are correct.
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Export to Production

Editor: Scott Edmunds

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    The genome is available on NCBI with the assembly accession number GCA_024217435.2. The genome is annotated via the NCBI eukaryotic genome annotation pipeline, and the annotation report release (100) is available here. Several smaller contigs and contaminant microbes were removed in the annotation pipeline yielding a more robust genome assembly. The sequence identifier for the chromosome-level scaffolds is available in the GigaDB [28]. The NCBI Bioproject accession number is PRJNA851781, the raw Hi-Fi sequence accession is SRR19820733, the Hi-C sequence accession is SRR22219025, and the RNA-Seq sequence files from various tissues are SRR20438584–SRR20438604. Additional data is available in the GigaDB [28].

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