The genome sequence of the Powdered Quaker, Orthosia gracilis (Schiffermüller, 1775)

We present a genome assembly from an individual male Orthosia gracilis (the powdered quaker; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 715.5 megabases in span. Most of the assembly is scaffolded into 14 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.43 kilobases in length.


Background
Orthosia gracilis (Powdered Quaker) is a common and widespread Noctuid moth found throughout Europe and eastwards to China (Leraut, 2019). Its distribution and abundance in Great Britain and Ireland decreased significantly over the last 50 years, although it has shown some recovery in the last 20 years (Randle et al., 2019).
The common name Powdered Quaker is a reference to the plain clothing traditionally worn by Quakers (Marren, 2019). In this species, the wings have a dusting of tiny black flecks which distinguish it from other spring-flying species in the genus. There is significant variation in the ground colour of the moth with dark forms usually occurring on bogs where the larvae feed on bog-myrtle. These dark forms can occur in the same locations as paler forms, but the two forms do not mix as the dark form occurs earlier in all of its life stages (Heath & Emmet, 1983).
The Powdered Quaker has one generation a year, flying between March and May. It is found in habitats that include damp woodland and gardens, but it favours marshland. The adult moth regularly comes to light, is attracted to sugar, and feeds on sallow catkins and blackthorn flowers. The larvae feed at night on a range of herbaceous and woody plants (Waring et al., 2017).
A genome sequence from O. gracilis will be useful for research into colour variation in moths, and more generally for comparative studies across the Lepidoptera. The genome of O. gracilis was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all the named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for O. gracilis based on a male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Orthosia gracilis ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34). A total of 36-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 six missing joins or mis-joins and removed two haplotypic duplications, reducing the assembly length by 0.29% and increasing the scaffold count by one.
The final assembly has a total length of 715.5 Mb in 19 sequence scaffolds with a scaffold N50 of 52.0 Mb (Table 1). Most (99.98%) of the assembly sequence was assigned to 14 chromosomal-level scaffolds, representing13 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/997542.

Sample acquisition and nucleic acid extraction
A male Orthosia gracilis (specimen ID Ox001092, individual ilOrtGrac1) 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 identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilOrtGrac1 sample was weighed and dissected on dry ice with head tissue set aside for Hi-C  sequencing. Thorax tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. 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 solidphase 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.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II (HiFi) instrument. Hi-C data were also generated from head tissue of ilOrtGrac1 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 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., 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.   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   The genome sequence is released openly for reuse. The Orthosia gracilis 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.