The genome sequence of the Common Flat-body moth, Agonopterix heracliana Linnaeus, 1758 [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Agonopterix heracliana (the Common Flat-body; Arthropoda; Insecta; Lepidoptera; Depressariidae). The genome sequence is 539.1 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 15.36 kilobases in length.


Background
Moths in the genus Agonopterix, family Depressariidae, have a characteristic resting shape with overlapping rounded wings giving an oval outline.Many species in the genus have distinctive wing markings although Agonopterix heracliana can be difficult to distinguish from A. ciliella, with both species having a row of three pale marks on the specked grey forewings.Fine markings on the hindwings and genitalia differences can be used to distinguish the two species.There is a complex history concerning the naming of A. heracliana, including changes of genus, several misidentifications and a mix-up of historic specimens in Linnaeus' collection; this taxonomic history is described by Karsholt et al. (2006).
A. heracliana has been recorded across much of Europe, with a high concentration of records from the Netherlands, United Kingdom, Denmark and southern regions of Norway, Sweden and Finland (GBIF Secretariat, 2024).In Britain, the species is commonest in East Anglia, the Thames valley, south Wales and the Wales/England border (NBN Atlas Partnership, 2024).The adult moth is active in the colder months, from autumn to early spring, with the larvae developing during early summer.Scattered records from around Europe suggest that adults take measures to avoid extremes of heat or cold, for example by sheltering in caves or military bunkers (Moog et al., 2021).The preferred larval foodplants are variety of umbellifers (family Apiaceae), with larvae using silk to spin a tube or fold in a leaf.
A genome sequence of Agonopterix heracliana was determined as part of the Darwin Tree of Life project.The genome sequence will facilitate research into larval food plant adaptations and will contribute to the growing set of resources for studying evolution in the Lepidoptera.

Genome sequence report
The genome was sequenced from an adult Agonopterix heracliana (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 40-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 21 missing joins or mis-joins and removed 12 haplotypic duplications, reducing the assembly length by 2.70%, and decreasing the scaffold N50 by 2.83%. The final assembly has a total length of 539.1 Mb in 47 sequence scaffolds with a scaffold N50 of 19.6 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 30 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).Chromosome Z was assigned by synteny to Agriphila straminella (GCA_950108535.1).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.

Sample acquisition and nucleic acid extraction
An adult Agonopterix heracliana (specimen ID Ox000652, ToLID ilAgoHera1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), 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 Ox003081, ToLID ilAgoHera2) was collected in a light trap

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 ilAgoHera2 using the Arima v2 kit.The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.(Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow (Di  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 lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database (Bateman et al., 2023) with DIAMOND (Buchfink et al., 2021) blastp.
The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx.Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn (Altschul et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.Software tool Version (Ewels et al., 2020), use MultiQC (Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative (Grüning et al., 2018), the Biocontainers infrastructure (da Veiga Leprevost et al., 2017), and the Docker (Merkel, 2014) and Singularity (Kurtzer et al., 2017) containerisation solutions.
Table 3 contains a list of relevant software tool versions and sources.

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', 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 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, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

Figure 2 .
Figure 2. Genome assembly of Agonopterix heracliana, ilAgoHera1.1: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 539,129,159 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 (29,756,636 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,568,409 and 12,633,633 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/Agonopterix_heracliana/dataset/GCA_963693445.1/snail.

Figure 3 .
Figure 3. Genome assembly of Agonopterix heracliana, ilAgoHera1.1: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/Agonopterix_heracliana/dataset/GCA_963693445.1/blob.

Figure 4 .
Figure 4. Genome assembly of Agonopterix heracliana, ilAgoHera1.1: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/Agonopterix_heracliana/dataset/GCA_963693445.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Agonopterix heracliana, ilAgoHera1.1:Hi-C contact map of the ilAgoHera1.1 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=Ry4u_F2cR---JG_dTYrjKQ.

Table 1 . Genome data for Agonopterix heracliana, ilAgoHera1.1. Project accession data
at the same location on 2022-07-22.The specimen was collected by Liam Crowley (University of Oxford) and Finley Hutchinson (University of Essex) and identified by Finley Hutchinson and 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,