The genome sequence of the Ear Moth, Amphipoea oculea (Linnaeus, 1761) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Amphipoea oculea (the Ear Moth; Arthropoda; Insecta; Lepidoptera; Noctuidae)


Background
The Ear Moth Amphipoea oculea (Linnaeus, 1761) is a moth in the Noctuidae family. Adult moths of this species have reddishto purplish-brown coloured forewings, highlighted with a typically white reniform stigma, although this marking can be yellow or orange in some specimens (Leverton, 2001;Skinner & Wilson, 2009). The forewing markings of this species are typically indistinguishable from the other British and Irish Amphipoea (A. crinanensis, fucosa, lucens), but specimens can sometimes be correctly identified by a relatively shorter forewing and darker colouration, amongst other factors such as habitat (Leverton, 2001). This species has little restriction in which habitats it uses, but it does have a preference for damp locales (Skinner & Wilson, 2009).
The species overwinters as an egg from October to March, and upon hatching, the larva feeds internally within the foodplant from April to June (Emmet, 1991). A variety of larval foodplants are used, feeding on grasses, such as Deschampsia cespitosa, and low-growing herbaceous plants and roots such as Petasites hybridus (Emmet, 1991). Pupation occurs in an underground cell (Waring et al., 2017). Adults are on the wing between July and September, any while most often seen at light or sugar after dark, can be found by day nectaring on thistle or ragwort flowers (Skinner & Wilson, 2009).
The genome of Amphipoea oculea was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for Amphipoea oculea, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Amphipoea oculea ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34). A total of 38-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 12 missing joins or mis-joins and removed two haplotypic duplications, reducing the assembly length by 0.44% and the scaffold number by 4.88%, and decreasing the scaffold N50 by 0.69%.
The final assembly has a total length of 669.2 Mb in 39 sequence scaffolds with a scaffold N50 of 23.1 Mb (Table 1). Most (99.99%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z 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/987876.

Sample acquisition and nucleic acid extraction
A male Amphipoea oculea (ilAmpOcul1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2021-07-17. The specimen was taken from woodland habitat using a light trap. The specimen was collected and identified by Douglas Boyes (University of Oxford) and was snap-frozen on dry ice.
The sample was prepared for DNA extraction at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilAmpOcul1 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  RNA was extracted from abdomen tissue of ilAmpOcul1 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  Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from head tissue of ilAmpOcul1 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 purge_dups (Guan 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 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. 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 Nextflow (Di Tommaso et al., 2017) DSL2 pipelines "sanger-tol/readmapping" (Surana et al., 2023a) and "sanger-tol/genomenote" (Surana et al., 2023b). The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated. Table 3 contains a list of relevant software tool versions and sources.   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 The genome sequence is released openly for reuse. The Amphipoea oculea genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data 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 Table 1.