An adult Solea solea (specimen ID ERGA_FV_BE_019, ToLID fSolSol10) was collected from Kwintebank (North Sea; latitude 51.28, longitude 2.65) on 2021-08-19. The specimen was collected, identified and preserved by Filip A.M. Volckaert (KU Leuven). This specimen was used for genome sequencing and Hi-C data for scaffolding.

The specimen used for RNA sequencing (ToLID fSolSol7) was a juvenile specimen collected from Wenduinebank (W03) (North Sea; latitude 51.28, longitude 2.95) on 2021-07-21. The specimen was collected, identified and preserved by Filip A.M. Volckaert (KU Leuven).

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023). The fSolSol10 sample was weighed and dissected on dry ice ( Jay et al., 2023) and gonad tissue was cryogenically disrupted using the Covaris cryoPREP ^® Automated Dry Pulverizer ( Narváez-Gómez et al., 2023).

HMW DNA was extracted using the Automated MagAttract v1 protocol ( Sheerin et al., 2023). DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system ( Todorovic et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

RNA was extracted from gonad tissue of fSolSol7 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol ( do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Tissue from the gill of the fSolSol10 sample was processed at the WSI Scientific Operations core, using the Arima-HiC v2 kit. Tissue (stored at –80 °C) was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde. After crosslinking, the tissue was homogenised using the Diagnocine Power Masher-II and BioMasher-II tubes and pestles. Following the kit manufacturer's instructions, crosslinked DNA was digested using a restriction enzyme master mix. The 5’-overhangs were then filled in and labelled with biotinylated nucleotides and proximally ligated. An overnight incubation was carried out for enzymes to digest remaining proteins and for crosslinks to reverse. A clean up was performed with SPRIselect beads prior to library preparation.

Pacific Biosciences SMRTbell libraries were constructed using the Revio HiFi prep kit, according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Revio instrument.

For Hi-C library preparation, DNA was fragmented to a size of 400 to 600 bp using a Covaris E220 sonicator. The DNA was then enriched, barcoded, and amplified using the NEBNext Ultra II DNA Library Prep Kit following manufacturers’ instructions. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on an Illumina NovaSeq 6000 instrument.

Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit, following the manufacturer’s instructions. RNA sequencing was performed on the Illumina NovaSeq 6000 instrument.

Assembly

The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). The contigs were further scaffolded using the provided Hi-C data ( Rao et al., 2014) in YaHS ( Zhou et al., 2023) using the --break option. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation). Manual curation was primarily conducted using PretextView ( Harry, 2022), with additional insights provided by JBrowse2 ( Diesh et al., 2023) and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Evaluation of the final assembly

A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using the “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b) pipelines. The genome readmapping pipelines were developed using the nf-core tooling ( Ewels et al., 2020), use MultiQC ( Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), and the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions. The genome was also analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021) were calculated.

Table 4 contains a list of relevant software tool versions and sources.

The Ensembl Genebuild annotation system ( Aken et al., 2016) was used to generate annotation for the Solea solea assembly (GCA_958295425.1) in Ensembl Rapid Release at the EBI. Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt ( UniProt Consortium, 2019).

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 best practice wherever possible.

The overarching areas of consideration are:

•   Ethical review of provenance and sourcing of the material

•   Legality of collection, transfer and use (national and international)

Each transfer of samples is undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Tree of Life collaborator, Genome Research Limited (operating as the Wellcome Sanger Institute) and in some circumstances other Tree of Life collaborators.