The genome sequence of the nine-spined stickleback, Pungitius pungitius (Linnaeus, 1758)

We present a genome assembly from an individual male Pungitius pungitius (the nine-spined stickleback; Chordata; Actinopteri; Gasterosteiformes; Gasterosteidae). The genome sequence is 480.4 megabases in span. Most of the assembly is scaffolded into 21 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.57 kilobases in length.


Background
The nine-spined stickleback (Pungitius pungitius) is a small freshwater fish that belongs to the family Gasterosteidae and is widely distributed throughout the Northern Hemisphere with populations in North America, Europe, and Asia (Kottelat & Freyhof, 2007;Page & Burr, 1991).The species' conservation status was last assessed by the IUCN in 2012 and is classed as "least concern" due to its broad geographic distribution, large population sizes, large number of sub-populations and lack of major threats (IUCN, 2022).The species is known for its tolerance to a wide range of temperatures, salinities, and water qualities and inhabits both coastal marine and freshwater environments including rivers, lakes, ponds, and estuaries (Wootton, 1976;Wootton, 1984).P. pungitius primarily feeds on benthic and planktonic invertebrates, sharing a similar diet with three-spined stickleback, Gasterosteus aculeatus (Hart, 2003;Hynes, 1950;Wootton, 1984).Where these two species occur sympatrically, P. pungitius displays a heightened preference for weeded areas, which potentially serves as a mechanism to avoid competition (Copp et al., 1998;Hart, 2003).The reproductive behaviour is characterised by paternal care where males construct and guard nests containing small clutches of eggs (Maitland, 2004;Wootton, 1984).Due to its wide distribution, ecological versatility, and genetic diversity, the nine-spined stickleback has emerged as an ideal model organism for studying various aspects of evolution, behaviour and genetics (Merilä, 2013; Peichel & Marques, 2017).Its suitability as a model system is further bolstered by its ease of captive breeding and the existence of nine congeneric species, compared to the three for G. aculeatus, which provides the opportunity for extensive comparative studies (Merilä, 2013).
We present a chromosomally complete genome sequence for P. pungitius, based on one male specimen from Cottingham, UK, as part of the Darwin Tree of Life Project.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Together with a recently published high-quality genome assembly by (Varadharajan et al., 2019), the P. pungitius genome presented in this study will provide an important resource for comparative genomic and evolutionary analyses of fish and other vertebrates.

Genome sequence report
The genome was sequenced from one male Pungitius pungitius (Figure 1) collected from Thwaite Lake, Cottingham, UK (53.78, -0.40).A total of 61-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 44 missing joins or mis-joins and removed 3 haplotypic duplications, reducing the assembly length by 0.13% and the scaffold number by 6.42%, and increasing the scaffold N50 by 3.9%.
The final assembly has a total length of 480.4 Mb in 174 sequence scaffolds with a scaffold N50 of 21.0 Mb (Table 1).Most (97%) of the assembly sequence was assigned to 21 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (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/134920.
The specimen was taken from the pond by Bernd Hänfling using a hand net, and identified by Alan Smith.The specimen was transported alive to the University of Hull and left to recover fully in an aquarium for a week before any sampling commenced.The specimen was euthanised in a lethal dose of MS-222 and tissue dissection was carried out by Bernd Hänfling within 30 minutes of euthanasia, and the tissues were immediately shock-frozen in liquid nitrogen.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The fPunPun2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.
Muscle tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.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.
RNA was extracted from muscle tissue of fPunPun2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from muscle tissue of fPunPun2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator.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  4 -that scaffolds are assigned by busco genes tax rules, but it isn't explicitly mentioned in the main text.
Genome annotation: I was hoping for annotation data alongside the genome data.The RNA-seq data is mentioned several times but only at the end of the article do we find out that it will be used in the future for genome annotations -I recommend mentioning this earlier in the data note.

Minor:
There is a Pungitus pungitus genome from 2020 (GCF_902500615.1).The introduction mentions such prior work, but some comparisons should be done in the manuscript to show potential advantages of the new assembly.
Some of the assembly statistics (contig N50 = 1.4Mbp) seem to be much lower than expected for HIFI reads, which can yield T2T assemblies of some chromosomes in fish species with much larger and more repetitive genomes.It should be discussed, what was limiting the assembly continuity (contig-level).In this regard HIFI readlength distribution, estimates of heterozygosity and a repeat analysis should be presented.
The authors state: "While not fully phased, the assembly deposited is of one haplotype."It seems to be a standard phrase in this type of articles and as mentioned before I am not sure if this is correct.If I read the methods correctly, the chromosomal scaffolds will represent a mosaic of the two haplotypes.Or has Hi-C data been used to disentangle chromosomal haplotypes during hifiasm?This needs to be clarified.
The authors state "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", this also seems to be a standard phrase in articles of this type.But looking at the PacBio protocols it seems the ratio needs to be much higher (at least 3.1X) to not loose your DNA (Procedure & Checklist -Using AMPure PB Beads for Size-Selection (pacb.com)).

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

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: Genomics, Bioinformatics, DNA/RNA Sequencing technologies 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, however I have significant reservations, as outlined above.
The ability to colonise new habitats and adapt to changing environmental conditions has led to the establishment of several evolutionary lineages and ecotypes of nine-spined stickleback.The genetic structure of the species has been shaped by geographic isolation among several glacial refugia resulting in two allopatric phylogeographic lineages in Europe (Teacher et al., 2011) and by multiple independent colonisations of freshwater from marine habitats (Bruneaux et al., 2013; Mobley et al., 2011; Shikano et al., 2010).Adaptive evolution associated with these demographic processes has in many cases resulted in the repeated evolution of similar morphological and behavioural phenotypes across different populations (Mobley et al., 2011).Recent research indicates that this phenotypic convergence is largely underpinned by non-parallel genetic changes (Wang et al., 2020).

Figure 2 .
Figure 2. Genome assembly of Pungitius pungitius, fPunPun2.1: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 480,450,674 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 (35,745,030 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,005,440 and 16,732,017 bp), respectively.The pale grey spiral shows the cumulative scaffold 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/fPunPun2.1/dataset/CASGFK01/snail.

Figure 5 .
Figure 5. Genome assembly of Pungitius pungitius, fPunPun2.1:Hi-C contact map of the fPunPun2.1 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=MCjOpVtaT4mHi8fRg1_3Dw.

INSDC accession Chromosome Length (Mb) GC%
Pungitius pungitius, is an important ecological and evolutionary study organism alongside other stickleback species.Its genome provides a resource for understanding the genetic basis of a wide-range of repeatedly evolved phenotypes across populations and species.The protocols, methods, and datasets are overall clearly presented.I wonder to what extent this assembly differs from others.The study by Varadharajan et al. is mentioned in the Background, but it isn't clear how different the assemblies are (or the origin of the individual sample used for sequencing).There have also been more recent improvements to this other reference Kivikoski M et al. (2021 1 ) and Wang D et al. (2024 2 ), which include attempts to identify and separate the X and Y chromosomes.The most recent assembly appears to have superior assembly metrics and BUSCO completeness.A comprehensive comparative analysis is not necessary but it seems relevant to at least reference these articles.Please provide definitions and separate numbers for missing joins and mis-joins for clarity.What does 'scaffolds are colored by phylum' mean and what does 'no-hit' represent?This is explained a bit in Figure Frederic Chain Biological Sciences, University of Massachusetts Lowell, Lowell, Massachusetts, USA Michael Olufemi Biological Sciences, University of Massachusetts, Lowell, Massachusetts, USA The nine-spined stickleback, ○ Manual Curation: ○ Figure 3: More information would be helpful here for the naive reader on how these data ○ are generated.