The genome sequence of the Birch Bell, Epinotia demarniana (Fischer von Röslerstamm, 1839) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Epinotia demarniana (the Birch Bell; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 735.8 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.46 kilobases in length.


Background
Epinotia demarniana (Fischer von Röslerstamm, 1839) is a moth of the Tortricidae family. The adult moths have an attractive pied appearance, showing little variation in colour or pattern (Bradley et al., 1979). In Great Britain, the adults are at large between June and July, flying high around treetops in the evening. By day the adult moths rest high up amongst foliage and can be caught by shaking or kicking suitable trees, then catching the adult moths as they flutter to the ground (Bradley et al., 1979;Elliott et al., 2018). After oviposition larvae begin feeding in September. The larval stages feed within the catkins of Alnus, Salix caprea, and Betula until May, subsequently pupating in a cocoon amongst leaf litter until June (Bradley et al., 1979;Elliott et al., 2018).
This species frequents heaths and open woodland, as well as wetter habitats such as fens, river banks, or boggy moorland (Bradley et al., 1979). The species is broadly distributed across England and into Wales, but is absent from Scotland and Ireland (Elliott et al., 2018). The species is regarded within the British Isles as being local or scarce1. Globally the species occurs across northern Eurasia in a possibly continuous distribution stretching from Great Britain at its western extremity to Hokkaido in the east (GBIF Secretariat, 2022;Hancock et al., 2015). The species has also been recorded from eastern Russia and north-eastern China (Byun et al., 2008), suggesting a broad distribution at the eastern edge of its range.
The genome of Epinotia demarniana 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 E. demarniana, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Epinotia demarniana ( Figure 1) collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (51.77, -1.34). A total of 30-fold coverage in Pacific Biosciences singlemolecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 22 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the assembly length by 0.2% and the scaffold number by 12.5%.
The final assembly has a total length of 735.8 Mb in 42 sequence scaffolds with a scaffold N50 of 26.9 Mb (Table 1). Most (99.85%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 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/1594294.

Sample acquisition and nucleic acid extraction
Two Epinotia demarniana specimens (specimen ID Ox001904, ilEpiDema1 and specimen ID Ox001904, ilEpiDema2) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2021-06-16 using a light trap. The specimens were collected and identified by Douglas Boyes (University of Oxford) and were then preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilEpiDema1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Whole organism 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. 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 and 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 whole organism tissue of ilEpiDema2 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.  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    The genome sequence is released openly for reuse. The Epinotia demarniana 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.