The genome sequence of the Cloaked Minor, Mesoligia furuncula (Denis & Schiffermüller, 1775)

We present a genome assembly from an individual female Mesoligia furuncula (the Cloaked Minor; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 889.6 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 15.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,903 protein coding genes.


Background
The Cloaked Minor Mesoligia furuncula is a small moth in the family Noctuidae (wingspan 22-28 mm) found widely across central and northern Europe, with scattered records from Russia, Kyrgyzstan, Uzbekistan, Kazakhstan, China and Japan (GBIF Secretariat, 2022).The moth is common across southern England, northern France, Belgium and the Netherlands, where it is found in grassland, scrub, open woodland and suburban areas (De Prins & Steeman, 2022;Randle et al., 2019).In Scotland, Northern Ireland, Wales and Ireland the species is more coastal in its distribution, being primarily associated with dunes and coastal grassland (GBIF Secretariat, 2022).
The forewing colouration is variable, but a consistent feature is a division of the ground colour into two near equal regions: a darker basal region (the 'cloak') and a paler cream, buff or brown distal region.Colour variants were originally considered different species before conspecificity was recognized (Newman, 1869).The adult moth is on the wing in July and August, with the larvae feeding on grasses including sheep'sfescue Festuca ovina, tufted hair-grass Deschampsia cespitosa and false oat-grass Arrhenatherum elatius (De Prins & Steeman, 2022;Waring et al., 2017).There is a single generation per year in northern Europe; the larva overwinters before recommencing feeding in spring and pupating in a chamber at the base of the food plant.
The genome of M. furuncula was sequenced as part of the Darwin Tree of Life project.The assembled sequence will be useful in understanding adaptations to grass feeding and the genetic basis of colour polymorphism, and contribute to comparative genomic studies across Lepidoptera.

Genome sequence report
The genome was sequenced from one female Mesoligia furuncula (Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -1.33).A total of 24-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 62-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 284 missing joins or mis-joins and removed 27 haplotypic duplications, reducing the assembly length by 1.44% and the scaffold number by 67.25%, and increasing the scaffold N50 by 62.83%.
The final assembly has a total length of 889.6 Mb in 93 sequence scaffolds with a scaffold N50 of 30.3 Mb (Table 1).Most (99.64%) 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 sex was designated as female due to half coverage of the Z chromosome.Linkage has been observed in the Hi-C map between chromosomes 8 and 25.This linkage is specific, such that an alternative karyotype could be constructed {8A:8B,25A:25B} and {8B:25A,8A,25B}.No support for this fusion between chromosome 8 and 25 can be seen in the PacBio reads which derive from a different sample to the Hi-C.
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/997551.
The resulting annotation includes 22,079 transcribed mRNAs from 21,903 protein-coding genes.

Sample acquisition and nucleic acid extraction
Two adult M. furuncula were collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude   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 whole organism tissue of ilMesFuru3 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 Illumina NovaSeq 6000 (10X) instruments.
Hi-C data were also generated from head tissue of ilMesFuru2, 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).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes    et al., 2019).The assembly was checked for contamination as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which runs MitoFinder (Allio 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.
To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020).The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.Table 3 contains a list of software tool versions and sources.

Frederik Hendrickx
Royal Belgian Institute of Natural Sciences, Brussels, Belgium The paper clearly describes the quality and completeness of the genome, which is also of very high quality.I have two suggestions for improvement: 1.Within the abstract, I suggest to replace 'Most of the assembly is scaffolded..." directly by the exact percentage i.e. "99.64% of the assembly is scaffolded..." 2. Within the part "Genome sequence report", there is one paragraph that is less clear and concerns the linkage between chromosomes 8 and 25.First, it is unclear what is meant with "This linkage is specific..." and it would be helpful to elaborate a bit more what is exactly meant with "specific".Second, the HiC contact map (Fig. 5) does not show this linkage.Third, the authors describe that PacBio reads do not support this linkage and attribute this to the fact that another individual was used.However, I wonder how reasonable it is that the PacBio assembly would reveal this linkage?In sum, some more explanation about this linkage and what it potentially entails would be welcome.
3. I suggest to add the chromosome numbers in Fig. 5 (also helpful to interpret the previous point).

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.
Under the subheading "Genome sequence report" first paragraph seventh line" with chromosome conformation" It can be changed as "with chromosome confirmation" The manuscript is well prepared, and it can be accepted for indexing.

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: Analyzing the phylogenetic relationship of the lepidopteran Noctuoid moths using mitochondrial genome sequence 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 Mesoligia furuncula, ilMesFuru1.1: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 889,627,388 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 (41,648,309 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (30,263,258 and 20,641,088 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/ilMesFuru1.1/dataset/CAKAJR01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Mesoligia furuncula, ilMesFuru1.1:Hi-C contact map of the ilMesFuru1.1 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=HtJsaWOeRfirYpmZg7BeWQ.

Peer Review Current Peer Review Status: Version 1
was used in the default protein mode to generate annotation for the Mesoligia furuncula assembly (GCA_916614155.1) in Ensembl Rapid Release.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.All efforts are undertaken to minimise the suffering of animals used for sequencing.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.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.