The genome sequence of the European plaice, Pleuronectes platessa (Linnaeus, 1758)

We present a genome assembly from an individual Pleuronectes platessa(the European plaice; Chordata; Actinopteri; Pleuronectiformes; Pleuronectidae). The genome sequence is 687.4 megabases in span. Most of the assembly is scaffolded into 24 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 17.4 kilobases in length.


Background
The European plaice Pleuronectes platessa (Linnaeus, 1758), is common species of flatfish belonging to the family Pleuronectidae.Pleuronectidae have compressed, oval shaped bodies with both eyes on the right side and are widely distributed in cool temperate waters.P. platessa is a bottom-living fish found on sand, mud, and gravel substrates down to 200 m, but they are more common at depths ranging from 10 to 50 m.Juveniles live in shallow water, frequenting estuaries and the intertidal zone and will move into deeper water when they attain a greater size at approximately 2 years.The species identified by its conspicuous red/orange spots on the dorsal side of its body and has a series of bony knobs that run in a curved line from the eyes back to the lateral line.Adult fish can grow up to 90 cm long, with most adults reaching lengths of 50 to 60 cm (Wheeler, 1978).P. platessa occur from the Barents Sea and Iceland to southern Spain and the western Mediterranean, but are most common in the seas surrounding the British Isles (Dipper, 2022).It is a commercially important species in the north-east Atlantic region and is the most economically important species of flatfish for European fisheries (Madsen et al., 2013;Prellezo & Carvahlo, 2020) with several stocks around the UK including the eastern English Channel, western English Channel, North Sea, Celtic Sea, and Irish Sea (Ellis et al., 2012).
Unsustainable levels of exploitation during the 1970s and 1980s reduced P. platessa spawning stock biomass to critical levels.Since then, fishing pressure has reduced, allowing spawning stock biomass to increase over the last 5-10 years in all stocks and is now listed as of Least Concern on the IUCN Red List due to the population trend increasing (Freyhof, 2014).

Genome sequence report
The genome was sequenced from one Pleuronectes platessa (Figure 1) collected from Looe Ground, Cornwall, UK (latitude 50.28, longitude -4.24).A total of 35-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 103 missing joins or mis-joins and removed 32 haplotypic duplications, reducing the scaffold number by 17.97%, and increasing the scaffold N50 by 0.82%.
The final assembly has a total length of 687.4 Mb in 356 sequence scaffolds with a scaffold N50 of 26.6 Mb (Table 1).Most (91.24%) of the assembly sequence was assigned to 24 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/8262.

Sample acquisition and nucleic acid extraction
A Pleuronectes platessa specimen (specimen no.MBA-210513-002A, ToLID fPlePla1) was collected from Looe Ground, The fish was first anesthetised 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.Samples taken from the animal were preserved in liquid nitrogen.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The fPlePla1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.
Muscle 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.
RNA was extracted from muscle tissue of fPlePla1 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 and 10X Genomics read cloud 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 were 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 fPlePla1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
The assembly was checked for contamination as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020).The genome was analysed within the BlobToolKit environment (Challis et al., 2020) andBUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.Table 3 contains a list of 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 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   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

University of Gothenburg, Strömstad, Sweden
This article presents a reference genome for the European plaice (Pleuronectes platessa).This genome is spited across 24 chromosome (91% of the total genome) and 332 unplaced contigs.This new genomic resource will be relevant for many researchers working on this commercially important species.On a side note: There is already published work on population genomics of the European plaice showing that two large chromosomal rearrangements (two putative inversions) segregate in northern plaice populations (North Sea, Baltic Sea, Barents Sea, and Iceland) and distinguish different plaice populations (see [1] and [2]).I cannot see evidence for these inversions being polymorphic in the individual sequenced for the current assembly.However, I think this information would be worth mentioning somewhere in the note in case people uses this genome in future genomic work.
I also noticed one surprising result that could benefit from some clarification in the text.In the BlobToolKit GC-coverage plot, there is a quite large variation of GC content across the unplaced contig that is somewhat surprising.I was wondering to what extent this could be driven by contamination?I don't think other genomes have this wide range of variation the other notes.
All of my comments are very minor details, and otherwise, the note is very well written and presented.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: phylogenetics, phylogeography, population genetics of a wide range of taxa 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.

Figure 1 .
Figure 1.Photographs of the Pleuronectes platessa (fPlePla1) specimen used for genome sequencing.A. right side, B. left side.

Figure 2 .
Figure 2. Genome assembly of Pleuronectes platessa, fPlePla1.1: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 687,405,470 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 (33,765,178 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (26,648,505 and 17,067,268 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/fPlePla1.1/dataset/CANAFI01/snail.

Figure 3 .
Figure 3. Genome assembly of Pleuronectes platessa, fPlePla1.1:GC coverage.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/fPlePla1.1/dataset/CANAFI01/blob.

Figure 4 .
Figure 4. Genome assembly of Pleuronectes platessa, fPlePla1.1:cumulative sequence.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/fPlePla1.1/dataset/CANAFI01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Pleuronectes platessa, fPlePla1.1:Hi-C contact map.Hi-C contact map of the fPlePla1.1 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=MfSPpN31RZmJxGNO9eKGuQ.

Table 1 . Genome data for Pleuronectes platessa, fPlePla1.1. Project accession data
* Assembly metric benchmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" from(Rhie et al., 2021).** BUSCO scores based on the actinopterygii_odb10 BUSCO set using v5.3.2.C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison.A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/fPlePla1.1/dataset/CANAFI01/cumulative.The specimen was taken from its habitat of broken shell and muddy sand by Sean Mctierney, Rachel Brittain and Mitchell Brenen (Marine Biological Association) using an otter trawl from the MV Sepia.The specimen was identified by Robert Mrowicki, Patrick Adkins, Joanna Harley and Rachel Brittain (Marine Biological Association) based on gross morphology.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Pleuronectes platessa, fPlePla1.
and the assembly have been deposited in INSDC databases.The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute.Raw data and assembly accession identifiers are reported in Table1.Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558. data