The genome sequence of a carabid beetle, Nebria salina (Fairmaire & Laboulbène, 1854)

We present a genome assembly from an individual female Nebria salina (a carabid beetle; Arthropoda; Insecta; Coleoptera; Carabidae). The genome sequence is 256.7 megabases in span. Most of the assembly is scaffolded into 21 chromosomal pseudomolecules, including the assembled X sex chromosome. The mitochondrial genome has also been assembled and is 24.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 10,671 protein coding genes.

Nebria salina prefers drier, less productive soils such as sand dunes and heaths, or upland grasslands and moraines (Anderson et al., 2000;Duff, 2012;Luff, 1998).It may occur together with the more eurytopic N. brevicollis in some marginal habitats such as woodland edge (Lindroth, 1985).James (2018) advises that records of N. salina from nutrient-rich soils that lack voucher specimens should be treated with caution.
Nebria salina adults are nocturnal and prey on a variety of invertebrates of suitable size such as beetles, flies, spiders, mites and springtails (Telfer & Butterfield, 2004).The larvae are also active surface predators (Lindroth, 1974).Nebria salina has an annual life cycle, spending the winter as a larva and emerging as an adult in the spring.After a period of spring activity, the adults enter a summer diapause but become active again in the autumn when they mate, producing the generation of larvae that will live through the next winter (Lindroth, 1985;Telfer & Butterfield, 2004).
The high-quality genome sequence described here is the first one reported for Nebria salina.It has been generated as part of the Darwin Tree of Life project.It will aid research into the taxonomy, biology and ecology of the species.

Genome sequence report
The genome was sequenced from one female Nebria salina (Figure 1) collected from Wigmore Park, Luton,England (51.88,.A total of 45-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 51-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 172 missing joins or mis-joins, and removed two haplotypic duplications, reducing the assembly length by 0.18% and the scaffold number by 19.56%, and increasing the scaffold N50 by 147.22%. The final assembly has a total length of 256.7 Mb in 551 sequence scaffolds with a scaffold N50 of 11.3 Mb (Table 1).Most (91.74%) of the assembly sequence was assigned to 21 chromosomal-level scaffolds, representing 20 autosomes, and the X sex chromosome.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/878211.

Sample acquisition and nucleic acid extraction
One adult Nebria salina (icNebSali1) was hand-picked from Wigmore Park, Luton, England (51.88, -0.37) on 20 May 2020 by Olga Sivell.It was identified by Duncan Sivell following Luff (2007) and Walters (2010).The specimen was snap-frozen using dry ice.The tissue samples taken from it were stored in a CoolRack prior to genome sequencing.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The icNebSali1 sample was weighed and dissected on dry ice with head tissue set aside for Hi-C sequencing.Thorax 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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.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.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).One round of polishing  et al., 2021;Simão et al., 2015) were calculated.Table 3 contains a list of software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Nebria salina assembly (GCA_944039245.1).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).

XiaoLong Liu
Hubei University, Wuhan, China In this work, the methods part includes DNA extraction, sequencing, genome assembly, genome annotation, and so on, and the work is appropriate, and the details are sufficient.The genome sequence is released openly and clearly presented.The raw sequence and assembly data have been deposited in INSDC databases and listed in the manuscript.
During the review process, I identified one issue, The genome sequence of Nebria salina, used one female.Readers who are not proficient in this field, may have a question, why not use the male and the differences in the genomes of male and female.In the "Background" part, it is better to add the pattern of sexual differentiation and mention the X chromosome.

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.To enhance the overall understanding of Nebria's taxonomic position, it is recommended that the authors provide a more detailed discussion, even if it means sacrificing some aspects of bionomics and species-level comparisons.Additionally, it would be beneficial to include information on other available genomes of the Adephaga suborder to provide a broader context for the findings.

Reviewer
The research presentation is clear and important for future advancements in the field.

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: Entomology, phylogeny 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 2 .
Figure 2. Genome assembly of Nebria salina, icNebSali1.1: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 256,740,106 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 (30,902,303 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (11,282,846 and 183,590 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 endopterygota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/icNebSali1.1/dataset/ CALUEK01/snail.

Figure 3 .
Figure 3. Genome assembly of Nebria salina, icNebSali1.1: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/icNebSali1.1/dataset/CALUEK01/blob.

Figure 4 .
Figure 4. Genome assembly of Nebria salina, icNebSali1.1: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/icNebSali1.1/dataset/ CALUEK01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Nebria salina, icNebSali1.1:Hi-C contact map.Hi-C contact map of the icNebSali1.1 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=eQJxQM3JQcKLF5KpyGKRZA.
Expertise: Insect biochemistry and physiology, Transcriptomics 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.Reviewer Report 10 July 2023 https://doi.org/10.21956/wellcomeopenres.21459.r60416© 2023 Bocak L. 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.Ladislav Bocak Biodiversity & Molecular Evolution, Czech Advanced Technology and Research Institute, Olomouc, Czech Republic Sivell & Sivell have presented a chromosome-level genome assembly of an individual female Nebria salina, a carabid beetle from the Carabidae family in Adephaga.The genome sequence data has been scaffolded into 21 chromosomal pseudomolecules, including the assembled X sex chromosome.While some assembly metrics values fall slightly below the benchmarks, the study significantly contributes to the exploration of the earliest evolution of beetles, which represent the largest radiation of animals on Earth.