The genome sequence of the Hebrew Character, Orthosia gothica (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Orthosia gothica (the Hebrew character; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 1,065.1 megabases in span. Most of the assembly is scaffolded into 37 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.38 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,691 protein coding genes


Background
The Hebrew Character moth Orthosia gothica is a spring-flying moth found across Eurasia from Portugal and Ireland to Japan, and from the north of Scandinavia to the south of Italy (GBIF Secretariat, 2023).The moth gets its common name from a distinctive black or brown mark on each forewing in the shape of the letter ‫נ‬ (nun) in the Hebrew alphabet.The species name gothica derives from the same marking, likening the shape to a Gothic arch.In Britain and Ireland the flight period spans from March to May, with the species recorded from all regions and almost all habitats, sometimes abundantly (MothsIreland, 2023;NBN Atlas Partnership, 2023).The adults are attracted to light and can also be found feeding on nectar at sallow catkins (South, 1961).
The larvae of O. gothica are polyphagous, feeding on leaves of herbaceous plants and deciduous trees, and the species has been recorded as a minor pest in orchards and forestry (Bues et al., 1994).Steps taken toward potential pest control include a synthetic male-attractant chemical blend (Tòth et al., 1992) and bisexual lures based on floral compounds or chemicals from rotting fruit (Szanyi et al., 2022).The species has been used as a model system to study biochemical responses of lepidopteran larvae to insecticides (Egaas, 2000;Egaas et al., 1992).
A genome sequence of the Hebrew Character Orthosia gothica was determined as part of the Darwin Tree of Life project.The genome sequence will facilitate research into adaptations to polyphagy and the biochemical basis of insecticide response, and will contribute to the growing set of resources for studying molecular evolution in the Lepidoptera.

Genome sequence report
The genome was sequenced from one female Orthosia gothica (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 21-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 172 missing joins or mis-joins and removed 85 haplotypic duplications, reducing the assembly length by 4.93% and the scaffold number by 20.14%. The final assembly has a total length of 1,065.1 Mb in 337 sequence scaffolds with a scaffold N50 of 32.4 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (97.61%) of the assembly sequence was assigned to 37 chromosomal-level scaffolds, representing 36 autosomes and the Z sex chromosome.The Z chromosome was identified based on coverage; a W chromosome was not identified.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (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.

Sample acquisition and nucleic acid extraction
A female Orthosia gothica (specimen ID Ox001095, ToLID ilOrtGoth1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2021-03-31 using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.size of 12-20 kb in a Megaruptor 3 system with speed setting 31 (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.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 abdomen tissue of ilOrtGoth1 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.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023).

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 head tissue of ilOrtGoth1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with     et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected 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., 2023), 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.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Orthosia gothica assembly (GCA_949775005.1).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).

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 Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, 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 further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

Figure 2 .
Figure 2. Genome assembly of Orthosia gothica, ilOrtGoth1.1: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 1,065,129,911 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 (64,640,039 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (32,428,790 and 17,961,581 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 lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilOrtGoth1_1/dataset/ilOrtGoth1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Orthosia gothica, ilOrtGoth1.1: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/ilOrtGoth1_1/dataset/ilOrtGoth1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Orthosia gothica, ilOrtGoth1.1: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/ilOrtGoth1_1/dataset/ilOrtGoth1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Orthosia gothica, ilOrtGoth1.1:Hi-C contact map of the ilOrtGoth1.1 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=YA-Wg3v2T3qyxzkeuf2aZA.