The genome sequence of the Flame Shoulder, Ochropleura plecta (Linnaeus, 1761)

We present a genome assembly from an individual female Ochropleura plecta (the Flame Shoulder; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 643.9 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.34 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,016 protein coding genes.


Background
Widespread and abundant throughout Great Britain, the Isle of Man, Ireland and the Channel Islands (Butterfly Conservation, 2023), Ochropleura plecta, of the Noctuidae family, is a resident and common species of moth found in gardens, farmland, hedgerows, moorland, woodland and wetlands.Adults may be seen between late April through to September, having two generations in southern Britain, while mainly single brooded in the north, flying in June and July.The bright straw-coloured stripe, or flame shoulder, along the leading edge of the forewing, together with the black streak from the base through the oval and kidney markings make it easily recognisable (Waring et al., 2017).The nearest confusion species is Ochropleura leucogaster, Radford's Flame Shoulder, a rare migrant to the south coast, first recorded in 1983 and seen regularly since then (Lewis, 2022).
A completed genome sequence should add evidence to the discussion on evolutionary relationships that are still not completely understood in the noctuid family (Waring et al., 2017).Research by Sisson (2022) on the phylogenetic relationships of noctuid moths from museum specimens highlighted this complexity and found a contradiction of previously documented phylogenetic relationships of Ochropleura plecta.
We present a chromosomally complete genome sequence for Ochropleura plecta based on one female specimen from Wytham Woods as part of the Darwin Tree of Life Project.This project is 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 female Ochropleura plecta (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 46-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 46-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 138 missing joins or mis-joins and removed 34 haplotypic duplications, reducing the assembly length by 0.98% and the scaffold number by 56.41%, and increasing the scaffold N50 by 12.81%. The final assembly has a total length of 643.9 Mb in 34 sequence scaffolds with a scaffold N50 of 21.9 Mb (Table 1).A summary of the assembly statistics is shown in Figure 2, 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 (99.98%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes and the W and Z sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (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/320037.
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 ilOchPlec1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Abdomen tissue was disrupted using a 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.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 head and thorax tissue of ilOchPlec1 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 HiSeq 4000 (RNA-Seq) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from remaining head and thorax tissue of ilOchPlec1 using the Arima2 kit and sequenced on the HiSeq X Ten instrument.et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final 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 Ochropleura plecta assembly (GCA_905475445.1) 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', 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

Huai-Jun Xue
Nankai University, Tianjin, China The authors have assembled the genome of the Flame Shoulder, Ochropleura plecta (Linnaeus, 1761) with Pacific Biosciences HiFi, 10×Genomics and Hi-C data.The genome size is 643.9Mb with a scaffold N50 of 21.9 Mb, and the BUSCO completeness is 98.9% using the lepidoptera_odb10 reference set.This paper provided the information necessary to repeat this work.
Legend of figure 1: If "ilOchPlec1" is a specimen name, it should be put after "specimen" other than the scientific name "Ochropleura plecta".
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: Entomology, adaptation and speciation 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 Ochropleura plecta, ilOchPlec1.1: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 643,962,443 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 (28,980,428 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,894,754 and 14,971,023 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/Ochropleura%20plecta/dataset/CAJQFZ01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Ochropleura plecta, ilOchPlec1.1:Hi-C contact map of the ilOchPlec1.1 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=eAJ5-r_fT3WUaYFQcGactw.

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, however I have significant reservations, as outlined above.
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.
I https://doi.org/10.21956/wellcomeopenres.22350.r72354©2024WengY.Yi-Ming WengDepartment of Entomology, University of Wisconsin Madison, Madison, WI, USA According to the description of nucleic acid extraction and sequencing procedures, the transcriptomic data should have been generated.But the RNA reads were not used in coding gene prediction in BRAKER2 pipeline, I wonder if there is the quality issue or other reasons that the gene prediction did not involve transcriptomic data.ithink it would be better to briefly explain the reasons.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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.