The genome sequence of the Little Grey, Eudonia lacustrata (Panzer, 1804)

We present a genome assembly from an individual male Eudonia lacustrata (the Little Grey; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 699.5 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.29 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,652 protein coding genes.


Background
Eudonia lacustrata (Little Grey) is a common micro-moth in the family Crambidae.The moth is common and widespread throughout most of Britain.The species occurs throughout Europe, apart from the far north, and east to western China (Leraut, 2012) In the UK, the adult moth is on the wing from mid-May to August and is single-brooded.The moth is found in open habitats, woodland, parks and gardens.E. lacustrata is a small moth with a forewing length of 8-9 mm.The light grey forewing has dark speckling, and a pattern of darker grey cross lines, and spots.The adult rests in the day on tree-trunks and walls, and flies at night, when it readily comes to light.The larvae, which are contained within a silk tube, feed on mosses usually growing on tree-trunks and walls (Sterling & Parsons, 2018).
A genome sequence from E. lacustrata will be useful for comparative studies across the Lepidoptera.The genome of E. lacustrata 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. lacustrata based on male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Eudonia lacustrata (Figure 1) collected from Wytham Woods, Oxfordshire (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 30 missing joins or mis-joins and removed 9 haplotypic duplications, reducing the assembly length by 0.26% and the scaffold number by 17.91%, and increasing the scaffold N50 by 0.54%.
The final assembly has a total length of 699.5 Mb in 54 sequence scaffolds with a scaffold N50 of 24.5 Mb (Table 1).Most (99.67%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 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/1100991.

Genome annotation report
The Eudonia lacustrata genome assembly (GCA_947562085.1)was annotated using the Ensembl rapid annotation pipeline (Table 1).The resulting annotation includes 21,845 transcribed mRNAs from 21,652 protein-coding genes.

Sample acquisition and nucleic acid extraction
Two Eudonia lacustrata (specimen IDs Ox0576 and Ox0577) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-07-05, using a light trap.Douglas Boyes (University of Oxford) collected and identified the specimens.The specimens were snap-frozen on dry ice.Specimen Ox0577 (ToLID ilEudLacu3) was used for DNA sequencing and specimen Ox0576 (ToLID ilEudLacu4) was used for Hi-C scaffolding.
A specimen (specimen ID Ox002244, individual ilEudLacu5), collected from the same location on 2022-06-21 by Liam Crowley (University of Oxford), was used for RNA sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilEudLacu3 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Whole organism tissue was disrupted using a

Number of scaffolds 54
Scaffold N50 length (Mb) 24.5 Longest scaffold (Mb) 65.0  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.

Genome annotation
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 whole organism tissue of ilEudLacu5 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 a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud 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), Illumina NovaSeq 6000 (RNA-Seq) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from whole organism tissue of ilEudLacu4 using the Arimav2 kit and sequenced on the Illumina NovaSeq 6000 instrument.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 BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Eudonia lacustrata assembly (GCA_947562085.1) in Ensembl Rapid Release.

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',

Version
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

Jerome H L Hui
The Chinese University of Hong Kong, Hong Kong, Hong Kong In this Data Note, Boyes and colleagues sequenced and assembled the genome of a male crambid moth Eudonia lacustrata (Panzer, 1804), or commonly known as little grey.According to the NatureSpot, this species has a distribution over most of Great Britain.Molecular data of this species are limited to mitochondrial cytochrome oxidase subunit 1 (COI) sequences and a few other gene sequences deposited to the NCBI database.Therefore, this new genome resource will be useful for further studies, ranging from revealing their population structures, revealing the climate change impact on them, to understanding their evolution with other insects.
This genome resource is excellent from the summary statistics, with high BUSCO scores, high sequence continuity (scaffold N50), and majority of sequences contained on the 29 pseudochromosomes (plus sex chromosome and mitochondrion).To sum up, this is another valuable contribution from the Darwin Tree of Life Consortium.

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: I have published with Peter Holland more than three years ago, and confirm that this potential conflict of interest did not affect my ability to write an objective and unbiased

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: Bioinformatics, Animal Genomics 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 Eudonia lacustrata, ilEudLacu3.1: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 699,510,583 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 (65,033,386 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (24,461,152 and 15,798,145 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/ilEudLacu3.1/dataset/CANOAT01/snail.
Genome assembly, curation and evaluationAssembly was carried out withHifiasm (Cheng et al., 2021)   and haplotypic duplication was identified and removed with purge_dups(Guan et al., 2020).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS.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) andPretext (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

Figure 5 .
Figure 5. Genome assembly of Eudonia lacustrata, ilEudLacu3.1:Hi-C contact map of the ilEudLacu3.1 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=Sep94sigTu2RzJYWR6TH2A.

Open Peer Review Current Peer Review Status: Version 1
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.