The genome sequence of the Common Carpet moth, Epirrhoe alternata (Müller, 1764) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Epirrhoe alternata (the Common Carpet; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 358.5 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.99 kilobases in length.


Background
The Common Carpet Epirrhoe alternata is a moth in the family Geometridae with a forewing length of 13-14 mm.It is somewhat variable in appearance, with two subspecies found in the United Kingdom: Epirrhoe alternata alternata across most of the region and Epirrhoe alternata obscurata in the Outer Hebrides (Skinner & Wilson, 2009;Waring et al., 2017).Epirrhoe alternata alternata is quite variable in appearance but has a series of brown and greyish-white bands running across the forewing: E. alternata obscurata is paler with less distinctive markings (Waring et al., 2017).
Epirrhoe alternata overwinters as a pupa and adults are on the wing from April to early October with a peak in numbers occurring in August, presumably as the result of overlapping generations (Waring et al., 2017).The larvae feed on cleavers Galium aparine, lady's bedstraw Galium verum, hedge bedstraw Galium mollugo and other related plants (Skinner & Wilson, 2009;Waring et al., 2017).
In common with many moth species the numbers of Epirrhoe alternata in the British Isles are decreasing (Conrad et al., 2006), but research has shown that restoration of abandoned pastures in Finland did not increase the abundance of this species as it did with some other species of Lepidoptera (Pöyry et al., 2005).
We present a chromosomal-level genome sequence for Epirrhoe alternata, based on one male specimen collected in Wytham Woods, Oxfordshire, for the Darwin Tree of Life Project.

Genome sequence report
The genome was sequenced from one male Epirrhoe alternata (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 52-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. The final assembly has a total length of 358.5 Mb in 39 sequence scaffolds with a scaffold N50 of 13.0 Mb (Table 1).The snail plot 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 (99.88%) 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 5; Table 2).The Z chromosome was identified based on synteny with Epirrhoe tristata (GCA_ 951394285.1).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.
The estimated Quality Value (QV) of the final assembly is 66.2 with k-mer completeness of 100.0%, and the assembly has a BUSCO v completeness of 98.6% (single = 98.1%, duplicated = 0.5%), using the lepidoptera_odb10 reference set (n = 5,286).

Sample acquisition and nucleic acid extraction
A male Epirrhoe alternata (specimen ID Ox000689, ToLID ilEpiAlte1) was collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -1.34) on 2020-07-20 using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
The specimen used for Hi-C sequencing (specimen ID Ox003078, ToLID ilEpiAlte2) was collected from the same location on 2022-07-22 using a light trap.The specimen was collected by Liam Crowley (University of Oxford) and Finley Hutchinson (University of Exeter), and identified by Finley Hutchinson, and then preserved on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the ilEpiAlte1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue of the whole organism was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic 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.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

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 IIe instrument.Hi-C data were also generated from tissue of ilEpiAlte2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly and curation
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 using the TreeVal pipeline (Pointon et al., 2023).
Manual curation was performed using JBrowse2 (Diesh et al., 2023), HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and minimap2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO (Manni et al., 2021).For the three domain-level BUSCO  et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling (Ewels et al., 2020), use MultiQC (Ewels et al., 2016)     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

Figure 2 .
Figure 2. Genome assembly of Epirrhoe alternata, ilEpiAlte1.1:metrics.The BlobToolKit snail plot 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 358,495,576 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 (16,783,756 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (13,027,933 and 8,529,277 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/Epirrhoe_alternata/dataset/GCA_963565295.1/snail.

Figure 3 .
Figure 3. Genome assembly of Epirrhoe alternata, ilEpiAlte1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Epirrhoe_alternata/dataset/GCA_963565295.1/blob.

Figure 4 .
Figure 4. Genome assembly of Epirrhoe alternata, ilEpiAlte1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Epirrhoe_alternata/dataset/GCA_963565295.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Epirrhoe alternata, ilEpiAlte1.1:Hi-C contact map of the ilEpiAlte1.1 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=aQNjFNvqRQ2dZ5-nWk-tyQ.

Table 3
Wellcome Sanger Institute -Legal and GovernanceThe 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

Table 2 . Chromosomal pseudomolecules in the genome assembly of Epirrhoe alternata, ilEpiAlte1. INSDC accession Chromosome Length (Mb) GC%
• 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, Institute), and in some circumstances other Darwin Tree of Life collaborators.