The genome sequence of the Mouse Moth, Amphipyra tragopoginis (Clerck 1759)

We present a genome assembly from an individual male Amphipyra tragopoginis (the Mouse Moth; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 806 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.3 kilobases in length. Gene annotation of this assembly on Ensembl has identified 13,359 protein coding genes.


Background
The Mouse moth, Amphipyra tragopoginis (Clerck 1759), is a moth in the family Noctuidae found across central and northern Europe, with scattered records from Asia (GBIF Secretariat, 2021). The species has also been recorded in the United States and Canada, where it is has probably been accidentally introduced (Forbes, 1954;Lotts & Naberhaus, 2021). The forewings of the adult are a uniform shiny grey marked with three small black dots forming an elongated triangle; this simple pattern is distinctive among moths in the UK. The adult moths have an unusual behavioural trait that suits the common name well: when disturbed the moth often scuttles away, instead of flying. Despite the aversion to flight as an escape response, A tragopoginis was abundant in a study of aerial insects sampled using suction traps at a height of 12 metres and is clearly a strong flyer (Wood et al., 2009). The moth seems to fly mostly in the early part of the night, just after darkness, and favours warm nights up to 21°C (Taylor, 1963).
In the UK, the adult moth is on the wing from July to September, and although sometimes caught at light it is more reliably attracted to sugary baits. Since most moth recording uses light traps, it is therefore possible that the moth is under recorded. Even so, standardised trapping methods conducted annually across the UK suggest the moth is declining in abundance, with a decrease of over 80% from 1970 to 2016 (Randle et al., 2019). The decline is unlikely to be connected to food plant availability, since the larvae are polyphagous and feed on the leaves of a large range of trees, shrubs and herbaceous plants.
A genome sequence from Amphipyra tragopoginis will be useful for research into wing pattern evolution and behavioural adaptations, and more generally for comparative studies across the Lepidoptera.

Genome sequence report
The genome was sequenced from one male Amphipyra tragopoginis ( Figure 1) collected from Wytham Woods, UK (latitude 51.77, longitude -1.34). A total of 41-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 49-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 23 missing or mis-joins and removed four haplotypic duplications, reducing the assembly length by 1.36% and the scaffold number by 23.81%, and increasing the scaffold N50 by 4.17%.
The final assembly has a total length of 805.7 Mb in 32 sequence scaffolds with a scaffold N50 of 28.3 Mb (Table 1). Most (99.99%) 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 2- Figure 5; Table 2). The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.8% using the lepidoptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Genome annotation report
The A. tragopoginis GCA_905220435.1 assembly was generated using the Ensembl rapid annotation pipeline (Table 1; https:// rapid.ensembl.org/Amphipyra_tragopoginis_GCA_905220435.1/). The resulting annotation includes 13359 protein coding genes with an average length of 20,732.64 bp and an average coding length of 1,473.70 bp, and 2680 non-protein coding genes. There is an average of 7.05 exons and 6.05 introns per canonical protein coding transcript, with an average intron length of 2,884.07. A total of 4,772 gene loci have more than one associated transcript.

Sample acquisition and nucleic acid extraction
Two A. tragopoginis specimens (ilAmpTrag1 and ilAmpTrag2) were collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 24 August 2019 using a light trap. The specimens were collected and identified by Douglas Boyes (University of Oxford) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilAmpTrag2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Head and 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. Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 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. 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 abdomen tissue of ilAmpTrag1 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  and HiSeq X Ten (10X) instruments. Hi-C data were also generated from abdomen tissue of ilAmpTrag2 using the Arima v2 kit and sequenced on the HiSeq X Ten instrument.

Genome assembly
Assembly was carried out with Hifiasm (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 SALSA2 (Ghurye et al., 2019). The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano- Silva et al., 2022), which  performed annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate. The genome sequence is released openly for reuse. The Amphipyra tragopoginis 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. Raw data and assembly accession identifiers are reported in Table 1.