The genome sequence of the Rustic Shoulder-knot, Apamea sordens (Hufnagel, 1766)

We present a genome assembly from an individual male Apamea sordens (the Rustic Shoulder-knot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 614 megabases in span. The whole assembly is scaffolded into 31 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 16.3 kilobases in length.


Background
Apamea sordens (the Rustic Shoulder-knot) is a moth in the family Noctuidae found commonly in grasslands, gardens, farmland and woodland rides in the UK.The moth is found across the Palaearctic as far east as China and Japan, and also occurs in North America (National Biodiversity Atlas).The pattern and position of cross lines, marginal marks and circles (orbicular and reniform stigma) on the adult forewings are very similar to those of several closely related species, which can make distinguishing members of the genus Apamea difficult.The consistency of the markings suggests these moths share conserved patterning mechanisms during wing development, in an analogous way to the well-studied nymphalid ground plan of many butterflies (Nijhout, 1994;Schwanwitsch, 1929).A. sordens is distinguished from its close relatives by its pale brown, silvery appearance, and a diagnostic forked black streak at the base of each forewing (the 'shoulder-knot').The specific name sordens, meaning 'dirty', does not do justice to this delicately patterned moth (Maitland Emmet, 1991).
In the south of the UK, the adult moth is usually on the wing one to two weeks earlier than other Apamea species, commencing in mid-May and seen until mid-June (PWHH, pers.obs.).Larvae of A. sordens feed on various grasses, overwintering as a larva in the UK and pupating in early spring (Robinson et al., 2010;Waring & Townsend, 2017).The species has been reported as an agricultural pest in several countries including Kazakhstan, Iran and Georgia, with larvae continuing to feed on cereal crops after harvest (Hashemi et al., 1995;Keburia et al., 2010;Shek & Evdokimov, 1972).Availability of a genome sequence would facilitate future research into pest control strategies and into fundamental biological questions such as the molecular basis of wing patterning.
The genome of A. sordens 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.Here we present a chromosomally complete genome sequence for A. sordens, based on the ilApaSord1 specimen from Wytham Woods, Berkshire, UK.

Genome sequence report
The genome was sequenced from one male A. sordens specimen (Figure 1) collected from Wytham Woods, Berkshire, UK (latitude 51.77, longitude -1.32).A total of 38-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 five missing or mis-joins and removed two haplotypic duplications, reducing the scaffold number by 8.82%.
The final assembly has a total length of 614.3 Mb in 31 sequence scaffolds with a scaffold N50 of 21.3 Mb (Table 1).The whole assembly sequence was assigned to 31 chromosomallevel 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 mitochondrial genome was also assembled.The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.4% using the lepidoptera_odb10 reference set.Evaluation of the assembly shows a consensus quality value (QV) of 72.1 and k-mer completeness of 100%.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male A. sordens specimen (ilApaSord1) was collected using a light trap from Wytham Woods, Berkshire, UK (latitude 51.77, longitude -1.32) by Douglas Boyes (University of Oxford).The sample was identified by Douglas Boyes and snapfrozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute.The ilApaSord1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.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 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.

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 the Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from head tissue of ilApaSord1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Gabriela Montejo-Kovacevich
University of Cambridge, Cambridge, England, UK This is a report detailing the sequencing assembly and annotation of Apamea sordens or "Rustic Shoulder-Knot", a UK grassland moth.This assembly appears to be of very high quality and the analysis and reporting of the data is well done.It is a very valuable resource for pest research and evolutionary biology.
The manuscript should be accepted.I only have a couple of small comments: It would be helpful if the term 'moth' appeared on the title and probably Lepidoptera as well

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Remove colloquial terms that reduce clarity, especially for international readers (e.g. Background "on the wing" to refer to when adults fly)

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The date when the specimen was collected should be specified in the methods, at least the month/year

Figure 2 .
Figure 2. Genome assembly of Apamea sordens, ilApaSord1.1: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 614,329,545 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (32,512,628 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (21,259,203 and 15,914,481 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/ilApaSord1.1/dataset/ilApaSord1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Apamea sordens, ilApaSord1.1:GC coverage.BlobToolKit GC-coverage plot.Chromosomes are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilApaSord1.1/dataset/ilApaSord1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Apamea sordens, ilApaSord1.1:cumulative sequence.BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilApaSord1.1/dataset/ ilApaSord1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Apamea sordens, ilApaSord1.1:Hi-C contact map.Hi-C contact map of the ilApaSord1.1 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=UY8hNZtsRzKBeffW2HEOow.

Open Peer Review Current Peer Review Status: Version 1
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.Liu Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Guangzhou Key Laboratory of Insect Development Regulation and Applied Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, ChinaThe genome sequence of Apamea sordens was describe which is 614 megabases in span.The whole assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome.The mitochondrial genome has also been assembled.The research method and data analysis are reasonable.Information is basically clear.Only one point on page 6 figure 3 "Chromosomes are coloured by phylum."Here, in figure3, I can't get information of chromosomes.May the author provide more description in the legend.

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
Reviewer Expertise: Genomics; function of genesI 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.
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.

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? Partly Competing Interests:
No competing interests were disclosed.
○ I can't see any files under the genome assembly accession on ENA https://www.ebi.ac.uk/ena/browser/view/PRJEB54051 ○ Is Reviewer Expertise: Evolutionary biology and genetics I