On 07 September 2023, Matjaž Gregorič hand collected six specimens of Leviellus thorelli (five females, one male), at Kremenica, Slovenia (lat = 45.941472, lon = 14.548028) and Ig, Slovenia (lat = 45.964847, lon = 14.519679), which were determined based on morphology from primary taxonomic literature ( Levi, 1974). The specimens were identified by Matjaž Gregorič in Slovenia. No permit was required for the collection of the here used specimens, as communicated and confirmed by the Slovenian Ministry of Natural Resources and Spatial Planning (communicated on 16. October 2023). Specimens were hand collected, euthanized by being put alive at -80 °C, and until DNA extraction, they were preserved at -80 °C.

Physical reference materials for the here sequenced specimen have been deposited in the Slovenian Museum of Natural History, Ljubljana, Slovenia https://www.pms-lj.si/en/ under the accession number ARA8125-ARA8128.

Tissues from prosoma and from four whole organisms (proxies), as well as residual DNA and RNA, were deposited in the Leibniz Institute for the Analysis of Biodiversity Change (LIB) Biobank, Museum Koenig, Bonn, Germany, under the collection IDs ZFMK-TIS-102871, ZFMK-TIS-102868, ZFMK-TIS-102869, ZFMK-TIS-102870, ZFMK-TIS-102872, ZFMK-DNA-FD19596994, and ZFMK-RNA-FD19597045, respectively. Genitals used for future specimen identification are deposited at the Slovenian Museum of Natural History, Ljubljana, Slovenia https://www.pms-lj.si/en/.

The estimated genome size, estimated by Genomes on a Tree (GoaT) ( Challis et al., 2023) by ancestral state reconstruction is 2.18 Gb. This estimated genome size corresponds to a diploid genome with a haploid number of 12 chromosomes (2n=24). All information for this species was retrieved from Genomes on a Tree ( Challis et al., 2023).

DNA was extracted from the prosoma of an adult female (qqLevThor3) using the Blood & Cell Culture DNA Mini Kit (Qiagen) following the manufacturer’s instructions. DNA quantification was performed using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific), and DNA integrity was assessed using a Genomic DNA 165 Kb Kit (Agilent) on the Femto Pulse system (Agilent). The DNA was stored at +4 °C until sequenced.

RNA was extracted from prosoma using a RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA quantification was performed using the Qubit RNA BR kit, and RNA integrity was assessed using a Bioanalyzer 2100 system (Agilent) RNA 6000 Nano Kit (Agilent). The RNA was stored at -80 °C until sequenced.

For long-read whole genome sequencing, a library was prepared using the SQK-LSK114 Kit (Oxford Nanopore Technologies, ONT), which was then sequenced across two R10.4.1 flow cells on a PromethION 24 A Series instrument (ONT). A short-read whole-genome sequencing library was prepared using the KAPA Hyper Prep Kit (Roche). A Hi-C library was prepared from the prosoma of a different adult female (qqLevThor2) using the Dovetail Omni-C Kit (Cantata Bio), followed by the KAPA Hyper Prep Kit for Illumina sequencing (Roche). The RNA library was prepared using the KAPA mRNA Hyper prep kit (Roche). All short-read libraries were sequenced on a NovaSeq 6000 instrument (2x150bp, Illumina). In total 40x Oxford Nanopore, 62x Illumina WGS shotgun, and 125x HiC data were sequenced to generate the assembly.

The genome was assembled using the CNAG CLAWS pipeline ( Gomez-Garrido, 2024). Briefly, reads were preprocessed for quality and length using Trim Galore v0.6.7 ( http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and Filtlong v0.2.1 ( https://github.com/rrwick/Filtlong), and initial contigs were assembled using NextDenovo v2.5.0 ( Hu et al., 2024), followed by polishing of the assembled contigs using HyPo v1.0.3 ( Kundu et al., 2019), removal of retained haplotigs using purge-dups v1.2.6 ( Guan et al., 2020) and scaffolding with YaHS v1.2a ( Zhou et al., 2024). Finally, assembled scaffolds were curated via manual inspection using Pretext v0.2.5 with the Rapid Curation Toolkit ( https://gitlab.com/wtsi-grit/rapid-curation ) to remove any false joins and incorporate any sequences not automatically scaffolded into their respective locations in the chromosomal pseudomolecules (or super-scaffolds). Summary analysis of the released assembly was performed using the ERGA-BGE Genome Report ASM Galaxy workflow ( 10.48546/workflowhub.workflow.1103.2).

The genome assembly has a total length of 2,190,932,837 bp in 13 superscaffolds and 17 additional unlocalized and 100 unplaced scaffolds ( Figure 1 & Figure 2), with a GC content of 34.3%. The assembly has a contig N50 of 5,371,768 bp and L50 of 119 and a scaffold N50 of 167,117,025 bp and L50 of 7. The assembly has a total of 809 gaps, totalling 161.8 kb in cumulative size. The single-copy gene content analysis using the Arthropoda database with BUSCO ( Manni et al., 2021) resulted in 96.8% completeness (93.1% single and 3.8% duplicated). 93.2% of reads k-mers were present in the assembly and the assembly has a base accuracy Quality Value (QV) of 47.5 as calculated by Merqury ( Rhie et al., 2020).

The main plot is divided into 1,000 size-ordered bins around the circumference, with each bin representing 0.1% of the 2,190,932,837 bp assembly. The distribution of sequence lengths is shown in dark grey, with the plot radius scaled to the longest sequence present in the assembly (193 Mb, shown in red). Orange and pale-orange arcs show the scaffold N50 and N90 sequence lengths (167,117,025 and 154,116,833 bp), respectively. The pale grey spiral shows the cumulative sequence 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 found in the assembled genome from the Arthropoda database (odb10) is shown in the top right.

The diagonal corresponds to intra-chromosomal contacts, depicting chromosome boundaries. The frequency of contacts is shown on a logarithmic heatmap scale. Hi-C matrix bins were merged into a 200 kb bin size for plotting.