The genome sequence of the greater pipefish, Syngnathus acus (Linnaeus, 1758)

We present a genome assembly from an individual Syngnathus acus (the greater pipefish; Chordata; Actinopteri; Syngnathiformes; Syngnathidae). The genome sequence is 359.2 megabases in span. Most of the assembly is scaffolded into 22 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.5 kilobases in length.


Background
The greater pipefish Syngnathus acus is widely distributed in the north-eastern Atlantic around coastlines, to depths of 100 m.Its habitat includes macroalgae beds, intertidal rocky shores as well as sand and mud at estuarine edges (Russel, 2002).However, it is most commonly associated with sea grass (Zostera spp.) beds (Vincent et al., 1995).
Syngnathus acus is a demersal syngnathid, a group characterized by its tubular snout ending in a tiny mouth, and their male parental care strategies.In fish of the Syngnathus genus, eggs and developing embryos are enclosed within specialised brooding structures (a marsupium) located on the ventral side of male fish (Planas, 2022).Pipefish lacking a marsupium seem to produce smaller and less developed plankton larvae, whereas those with a marsupium, like S. acus, give birth to fully formed juveniles which immediately resume a benthic position upon release (Silva et al., 2006).
Like most of the ray-finned fish, (Actinopterygii) fish in the Syngnathidae family feed using suction, using their specialised lengthened and fused snout to catch faster and more mobile prey (de Lussanet & Muller, 2007).
Although ecological research on this species is lacking, the available data suggest high seasonal and spatial variability in distribution and abundances, which may be governed by temperature, habitat and prey availability and potential migratory events (Planas, 2022).

Genome sequence report
The genome was sequenced from one Syngnathus acus (Figure 1) collected from Cawsand Bay, Plymouth, UK (50.34,.A total of 80-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected five missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold count by one, and decreasing the scaffold N50 by 1.84%. The final assembly has a total length of 359.2 Mb in 251 sequence scaffolds with a scaffold N50 of 15.3 Mb (Table 1).Most (92.29%) of the assembly sequence was assigned to 22 chromosomal-level scaffolds.Chromosome-scale scaffolds are named by synteny based on the S. acus assembly GCF_901709675.1 (Figure 2-Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/161584.

Sample acquisition and nucleic acid extraction
A Syngnathus acus (specimen number MBA-211018-001A, individual fSynAcu2) was collected from Cawsand Bay, Plymouth, UK, from the MBA SEPIA vessel (latitude 50.34, longitude -4.15) on 2021-10-18.The specimen was captured using a Naturalist dredge and placed in a suitable container.The specimen was identified by Kesella Scott-Somme (Marine Biological Association) based on gross morphology.The fish was first anaesthetised and then overdosed using Aquased (2-phenoxyethanol).Destruction of the brain was used as a secondary method to ensure the animal was deceased before tissue sampling took place as in accordance with Schedule 1 methodology under the home office licence.The samples taken from the fish were preserved in liquid nitrogen.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The fSynAcu2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Somatic tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on the Pacific Biosciences SEQUEL II (HiFi) instruments.Hi-C data were also generated from tissue of fSynAcu2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with    Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal   and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Maria Nilsson
Senckenberg Biodiversity and Climate Research Centre, Frankfurt am Main, Germany The complete nuclear and mitochondrial genome of the greater pipefish was sequenced.The pipefish are closely related to sea horses and have evolved a highly modified body plan.The genome size is 359Mb.It was not possible to fully phase the assembly.The contig N50 is 4.9Mb, which indicate that the genome is of high contiguity.The complete BUSCO genes are at 94.9% Minor comments: Background: the second sentence "Its habitat includes macroalgae beds, intertidal rocky shores as well as sand and mud at estuarine edges" sounds weird.It sounds like the pipefish live in mud, which I do not think is correct.The mitochondrial genome has not been deposited or separated from the assembly.That makes it difficult for someone that is only interested in the MT genome.It would recommend making it available on its own.Or maybe it is already done, but this is not fully clear from the sentence "The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission".conclusions: The assembly and the assembly report reads well and the high contiguity genome is a valuable addition to understanding the evolution of the highly morphologically modified seahorses/pipefishes.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?

Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: phylogenomics, transposable elements, mitogenomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Jayan D.M Senevirathna
Uva Wellassa University, Badulla, Uva Province, Sri Lanka The manuscript is well-written in a attractive way.The data is clear and less errors.

Suggestions:
The Specimen should be preserved in a museum and record the sample ID of the museum sample.
Table 2 lengths (Mb) should be indicate in three digits.
Corrections: latitudes and longitudes of the sampling location should be written in the correct way.

Figure 2 .
Figure 2. Genome assembly of Syngnathus acus, fSynAcu2.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 359,216,277 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (28,865,858 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (15,280,236 and 8,585,000 bp), respectively.The pale grey spiral shows the cumulative scaffold e count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the actinopterygii_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/fSynAcu2.1/dataset/CANUGO01/snail.

Figure 3 .
Figure 3. Genome assembly of Syngnathus acus, fSynAcu2.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/fSynAcu2.1/dataset/CANUGO01/blob.
Legal and ethical review process for DToL GAL submitted materialsThe materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Figure 4 .
Figure 4. Genome assembly of Syngnathus acus, fSynAcu2.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/fSynAcu2.1/dataset/CANUGO01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Syngnathus acus, fSynAcu2.1:Hi-C contact map of the fSynAcu2.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=TVSC5z-tREy3ilKg1pCxng.

Reviewer Report 10
June 2024 https://doi.org/10.21956/wellcomeopenres.21631.r84108© 2024 Senevirathna J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 3
contains a list of relevant software tool versions and sources.

Peer Review Current Peer Review Status: Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 2
contains 24 chromosome level scaffolds, however in the text it has mentioned 22.If it is only autosomes, please mention.