The genome sequence of the Dark Spectacle, Abrostola triplasia (Linnaeus, 1758)

We present a genome assembly from an individual male Abrostola triplasia (the Dark Spectacle; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 362.7 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 15.34 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,532 protein coding genes.


Background
The Dark Spectacle (Abrostola triplasia) is a widespread and common moth species in the UK and Ireland, where it is most frequent and abundant in the west (Randle et al., 2019;Waring et al., 2017).It especially favours areas with acidic soils, but unlike the closely-related Spectacle, Abrostola tripartita (Hufnagel, 1766) there are relatively few records from Scotland, most of them from southern Scotland.Distribution trends for this species in the UK and Ireland show a long term decline of 38%, in contrast to A. tripartita which has increased its distribution over the same period (Randle et al., 2019).Abrostola triplasia has been recorded across much of temperate Europe and Asia to China and Japan (GBIF Secretariat, 2023).
The main larval foodplants are nettle (Urtica dioica) and hop (Humulus lupulus).The distinctive larva with its two yellow "eye spots" feeds mostly at night and it has been suggested that it is a snake mimic (Henwood et al., 2020).
Here we present a chromosomally complete genome sequence for Abrostola triplasia based on one male specimen from Wytham Woods, Oxfordshire, UK.A genome sequence for Abrostola triplasia will contribute to a growing data set of resources for understanding lepidopteran biology.The genome of Abrostola triplasia 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.

Genome sequence report
The genome was sequenced from one male Abrostola triplasia (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 39-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 one mis-join. The final assembly has a total length of 362.7 Mb in 35 sequence scaffolds with a scaffold N50 of 12.8 Mb (Table 1).Most (99.96%) 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).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/254365.

Sample acquisition and nucleic acid extraction
A male Abrostola triplasia (specimen ID Ox001901, individual ilAbrTril1) was 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 specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilAbrTril1 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.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.
RNA was extracted from abdomen tissue of ilAbrTril1 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 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) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from ilAbrTril1 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).
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,

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Abrostola triplasia assembly (GCA_946251915.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

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

Software tool Version
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 5 needs axis labels and a color legend.While I understand that the axes denote chromosomes and positions along them, labels would be helpful.

3.
There is at least one position shown on Figure 5 that seems to have a globally high interaction frequencies to the rest of the genome.This should probably be addressed briefly in the text.

4.
The assembly section thoroughly describes the tools used, but should probably more fully describe the parameter choices and options used in each step.If this would make the text too long for publication here, these details could be relegated to a supplementary document.Alternatively (and probably preferably), the scripts used to conduct the analysis could be made available.

5.
The second sentence of the Background section reads a bit awkwardly.Perhaps something like "... there are relatively few records from Scotland, mostly from southern parts of the 6.
region."?As a side-note, I very much appreciate the careful documentation of tool versions in Table 3.

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: I am a population and ecological geneticist specializing on methodological approaches.My major study species include Monarch Butterflies.I am not a specialist on genome assemblies, and so am not the best person to dive deeply into the technicalities of that aspect.However, the overall quality of the genome produced seems excellent in comparison to what I typically work with.
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.

Is the rationale for creating the dataset(s) clearly described? Partly
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: Population genetics, molecular evolution
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Abrostola triplasia, ilAbrTril1.1: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 362,722,748 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 (21,208,463 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (12,758,438 and 8,625,622 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/ilAbrTril1.1/dataset/ CAMIUI01/snail.

Figure 5 .
Figure 5. Genome assembly of Abrostola triplasia, ilAbrTril1.1:Hi-C contact map of the ilAbrTril1.1 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=XXUZC-xDR-uMmH0_Av5lzg.

Table 3
contains a list of relevant software tool versions and sources.