The genome sequence of the Brown Rustic, Charanyca ferruginea (Esper, 1785)

We present a genome assembly from an individual female Charanyca ferruginea (the Brown Rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 854.6 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.36 kilobases in length. Gene annotation of this assembly on Ensembl identified 23,126 protein coding genes.


Background
The Brown Rustic Charanyca ferruginea (Esper, 1785) (syn.Rusina ferruginea) is a medium-sized moth of the family Noctuidae.Despite its drab appearance it is usually fairly distinctive, especially the males which have five or six whitish spots along the leading edge of the relatively broad forewings, a faint dark cross-band halfway down the forewing, and a few lighter crossbands.The species is sexually dimorphic: the females lack the strongly feathered antennae of the males, are smaller, have narrower forewings and are generally more obscurely marked.This occasionally leads to confusion with dark forms of the Square-spot Rustic Xestia xanthographa (Denis & Schiffermüller, 1775), although that species lacks the whitish spots along the forewing edge (Waring et al., 2017).
C. ferruginea is widespread across much of Europe, and is a common species across most of Britain, occurring in a range of habitats, while in Ireland and the Channel Islands it is similarly widespread but more localised.Despite this, the species' abundance has shown a significant decrease in recent years, dropping by 45% between 1970 and 2016 according to data collected by Rothamsted Research (Randle et al., 2019).The larvae occur on various herbaceous plants from August to May, feeding at night and overwintering almost fully-grown before pupating in a cocoon underground in Spring (Henwood et al., 2020).Larvae are equally drab, being yellowish-or orangey-brown and increasingly pale along the abdominal segments.A white dorsal line along the thorax is reduced to dots or dashes, or entirely absent, along the abdomen, and there is a faint subdorsal line on each side of the body.Spiracles are black, separating it from similar species Dypterygia scabriuscula (Linnaeus, 1758) and Hyppa rectilinea (Esper, 1796) which have white or orange spiracles (Henwood et al., 2020).Adults fly in a single generation in May or June-July, and come to light, sugar and flowers (Waring et al., 2017).
We present a chromosomally complete genome sequence for Charanyca ferruginea, based on one female specimen from Wytham Woods, Oxfordshire, 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 Charanyca ferruginea (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 46-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 99 missing joins or mis-joins and removed 4 haplotypic duplications, increasing the assembly length by 1.7% and reducing the scaffold number by 42.31%, and increasing the scaffold N50 by 1.09%. The final assembly has a total length of 854.6 Mb in 44 sequence scaffolds with a scaffold N50 of 27.8 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics.The distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3, and the cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.97%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 29 autosomes and the Z and W sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The Hi-C data indicate some collinearity between the Z and W chromosomes.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/987475.
The specimen was collected and identified by Douglas Boyes (University of Oxford) and was snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilChaFerr1 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.

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 a Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from thorax tissue of ilChaFerr1 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).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was 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, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using 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 Charanyca ferruginea assembly (GCA_947361185.1) 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  The introduction to the species itself is very nicely written and interesting.It would benefit from an explanation of what having a genome assembly will bring to the study of this species.
In the "sequencing" section of the Methods it is not stated how the 10X library was sequenced and indeed what this data was used for, as it is not listed in Table 1, despite being included in the sequencing BioProject (I am having to guess a bit, but it looks like this is the following runs: ERR10149553, ERR10149554, ERR10149555) and apparently one of these experiments was used to generate Figure 3.

Figure 3:
It is not clear from the figure legend what the y axis corresponds to.In fact only by searching for the accession number listed can one find out that this corresponds to a WGS dataset which is not listed within the rest of the article.Given that in the "Genome sequence report" section it is stated that 46-fold HiFi coverage was generated, I feel it would make more sense to generate the blobplot using this dataset, rather than the one used, which looks to only be at 20-fold coverage.

Table 1:
The stated 100% kmer completeness must correspond to the value obtained when combining the primary and alternate assemblies together.Is this also true for the QV value?The BUSCO and % assigned to chromosome values must be from the primary assembly only.I find this a little bit confusing and has to potential to mislead people and should be made much clearer.
As I have brought up in a previous Data Note from the same consortium, I find the reporting of the genome annotation incomplete.Stating the number of genes and transcripts alone does not give the reader sufficient information to establish the quality and usefulness of the annotation alongside the genome assembly.As a minimum, I would like to see the BUSCO completeness scores as well as OMArk completeness and consistency scores of the annotated sequences.This should also be coupled with a report of the number of exons per gene on average, the number of single-exon genes.
The methods section for the genome annotation is not informative or complete.While Braker2 is listed as the annotation tool, there is no indication of whether the genome was repeat masked (and how) and what supporting evidences were used to generate the gene model predictions -i.e which set of protein sequences were used in the "default protein mode" as is stated in the text.

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? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genome assembly, genome annotation 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, however I have significant reservations, as outlined above.

Doga Cedden
Department of Evolutionary Developmental Genetics, Johann-Friedrich-Blumenbach Institute, Göttingen, Germany Boyes & Hutchinson report a high-quality genome assembly from Charanyca ferruginea.The authors provided adequate background information regarding the C. ferruginea.The methodology is clearly described and appropriate.Sufficient quality control is provided regarding the genome assembly.The provided data depository contains the necessary data.
I only have one concern regarding the mitochondrial genome assembly.The reported GC% is 18.5 for the mitochondrial genome assembly.This seems low, but similar or even lower mitochondrial GC% contents are also observed mitochondrial genome GC% is observed in other insects.This could be at least briefly mentioned in the text for unfamiliar readers.
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 work on coleopteran pests mainly using RNA-seq and RNA interference methods.
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.The data note " The genome sequence of the Brown Rustic, Charanyca ferruginea" by Douglas Boyes and Finley Hutchinson describes the sequencing and genome assembly of Charanyca ferruginea.The authors utilized long-read sequencing technology (PacBio) to produce an initial assembly that was subsequently scaffolded using Hi-C, resulting in a genome assembly at the chromosome level with nearly all scaffolds assigned to chromosomes.The genome annotation includes 23,326 transcribed mRNAs.The presentation of the results are clear, but the aims should be more clear why this species was selected and I would also expect to see the results of genome masking.
Below are some more specific comments and suggestions: Keywords: The keywords need to be different from the title otherwise are useless.

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Species identification: the author should cite the literature used.

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Mitochondrial genome: I strongly recommend submitting the genome separated and with ○ an independent accession number.Moreover, the authors described the methods but did not provide any information about the mitochondrial genome annotation (coding genes, rRNAs, and tRNAs).I would expect that the authors submit the annotations obtained by MITOS separately.

Annotation:
The authors should identify and quantify the transposable elements.

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Command lines: It is common practice to include the command lines used with all software.I believe this information can be very useful for the readers.Reviewer Expertise: Genomic, transcriptomic and proteomic 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, however I have significant reservations, as outlined above.
I have no knowledge of the species taxonomy reported here.So I could not comment on it.The photograph provided as figure 1 is of good quality.The authors clearly explained the morphology features and appearance of the insect.It would have been better if they had shown a photo of live insect along with the specimen sample.
The authors have done 46 fold coverage sequencing in long read sequencing technology.The contig N50 value was sufficiently high.BUSCO analysis gives an idea of completeness of the genome sequence.As the results indicate 98% completeness we can assume the genome sequence is a near complete one.
Not much research work was done using this insect as evident from the literature search using the insect name.The genome sequence reported here with might form basis for designing molecular biology works in the future.
There is an unwanted symbol trailing the words 'BioMasher pestle].' in Methods section.

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: Bioinformatics, Genomics 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 Charanyca ferruginea, ilChaFerr1.1: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 854,663,949 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 (61,979,575 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (27,761,565 and 18,037,562 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/Charanyca%20ferruginea/dataset/CANAIE01/snail.

Figure 3 .
Figure 3. Genome assembly of Charanyca ferruginea, ilChaFerr1.1:BlobToolKit GC-coverage plot.Scaffolds 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/Charanyca%20ferruginea/dataset/CANAIE01/blob.

Figure 4 .
Figure 4. Genome assembly of Charanyca ferruginea, ilChaFerr1.1: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/Charanyca%20ferruginea/dataset/CANAIE01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Charanyca ferruginea, ilChaFerr1.1:Hi-C contact map of the ilChaFerr1.1 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=Rr_u06gvQG65l-FLLKvYow.

©
2024 Brown T. 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.Thomas Brown 1 Leibniz IZW (Berlin) & ERGA/BGP, Leibniz, Germany 2 Berlin Center for Genomics in Biodiversity Research, Berlin, Berlin, Germany Boyes et al. present a chromosome-scale genome assembly of the Brown Rustic, a moth from the British Isles.The genome is of high quality and the methodology describing the sampling and DNA extraction are well described.There are a few issues I wish to highlight to ensure that the process is truly transparent and can be interpreted fully by the readers.

Reviewer Report 09
August 2024 https://doi.org/10.21956/wellcomeopenres.22299.r90360© 2024 Cedden D. 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.

Reviewer Report 09
August 2024 https://doi.org/10.21956/wellcomeopenres.22299.r90369© 2024 Ragionieri L. 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.Lapo Ragionieri 1 University of Cologne, Cologne, Germany 2 Libera Universita di Bolzano (Ringgold ID: 18956), Bolzano, Trentino-Alto Adige/South Tyrol, Italy

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Is the rationale for creating the dataset(s) clearly described?PartlyAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.

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.
https://doi.org/10.21956/wellcomeopenres.22299.r90366