The genome sequence of the Four-dotted Obscure, Oegoconia quadripuncta (Haworth 1829)

TL;DR

This paper presents the genome sequence of the Four-dotted Obscure moth, including a detailed assembly of its chromosomes and mitochondrial DNA.

Contribution

The study provides the first genome assembly for Oegoconia quadripuncta, including scaffolded chromosomal pseudomolecules and the mitochondrial genome.

Findings

The genome assembly spans 622.6 megabases and is scaffolded into 20 chromosomal pseudomolecules.

The mitochondrial genome is 15.39 kilobases in length and has been fully assembled.

The Z sex chromosome is included in the chromosomal scaffolding.

Abstract

We present a genome assembly from an individual male
Oegoconia quadripuncta (the Four-dotted Obscure; Arthropoda; Insecta; Lepidoptera; Autostichidae). The genome sequence is 622.6 megabases in span. Most of the assembly is scaffolded into 20 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.39 kilobases in length.

Figures

Photograph of the Oegoconia quadripuncta (ilOegQuad1) specimen used for genome sequencing. A. Dorsal view, B. Ventral view.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: 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 622,656,778 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 (50,213,132 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (36,190,098 and 20,651,493 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/ilOegQuad1.1/dataset/CASGFV01/snail.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: 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/ilOegQuad1.1/dataset/CASGFV01/blob.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: 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/ilOegQuad1.1/dataset/CASGFV01/cumulative.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: Hi-C contact map of the ilOegQuad1.1 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=WvXNmCQqSuiNwWWPD2TKSg.

Tables

Table 1.. Genome data for Oegoconia quadripuncta, ilOegQuad1.1.

Project accession data
Assembly identifier	ilOegQuad1.1
Species	Oegoconia quadripuncta
Specimen	ilOegQuad1
NCBI taxonomy ID	347754
BioProject	PRJEB59195
BioSample ID	SAMEA111458706
Isolate information	ilOegQuad1, male: whole organism (DNA sequencing and Hi-C scaffolding)
Assembly metrics *		Benchmark
Consensus quality (QV)	62.5	≥ 50
k-mer completeness	100%	≥ 95%
BUSCO **	C:98.4%[S:98.0%,D:0.4%],F:0.5%,M:1.1%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.75%	≥ 95%
Sex chromosomes	Z chromosome	localised homologous pairs
Organelles	Mitochondrial genome assembled	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR10812851
Hi-C Illumina	ERR10818304
Genome assembly
Assembly accession	GCA_949316235.1
Accession of alternate haplotype	GCA_949316225.1
Span (Mb)	622.6
Number of contigs	177
Contig N50 length (Mb)	6.7
Number of scaffolds	44
Scaffold N50 length (Mb)	36.2
Longest scaffold (Mb)	50.2

Table 2.. Chromosomal pseudomolecules in the genome assembly of Oegoconia quadripuncta, ilOegQuad1.

INSDC accession	Chromosome	Length (Mb)	GC%
OX438741.1	1	50.21	37.0
OX438743.1	2	43.64	37.0
OX438744.1	3	41.32	37.0
OX438745.1	4	39.03	37.0
OX438746.1	5	38.66	37.0
OX438747.1	6	36.36	37.0
OX438748.1	7	36.19	37.0
OX438749.1	8	33.42	37.0
OX438750.1	9	31.67	37.5
OX438751.1	10	26.73	37.0
OX438752.1	11	25.63	37.0
OX438753.1	12	24.4	36.5
OX438754.1	13	22.74	36.5
OX438755.1	14	22.2	37.0
OX438756.1	15	21.93	37.0
OX438757.1	16	20.65	37.0
OX438758.1	17	20.61	37.0
OX438759.1	18	20.38	37.5
OX438760.1	19	18.16	37.5
OX438742.1	Z	47.17	36.0
OX438761.1	MT	0.02	25.5

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.1.5	https://github.com/blobtoolkit/blobtoolkit
BUSCO	5.3.2	https://gitlab.com/ezlab/busco
gEVAL	-	https://geval.org.uk/
Hifiasm	0.16.1-r375	https://github.com/chhylp123/hifiasm
HiGlass	1.11.6	https://github.com/higlass/higlass
Merqury	MerquryFK	https://github.com/thegenemyers/MERQURY.FK
MitoHiFi	2	https://github.com/marcelauliano/MitoHiFi
PretextView	0.2	https://github.com/wtsi-hpag/PretextView
purge_dups	1.2.3	https://github.com/dfguan/purge_dups
sanger-tol/ genomenote	v1.0	https://github.com/sanger-tol/genomenote
sanger-tol/ readmapping	1.1.0	https://github.com/sanger-tol/readmapping/tree/1.1.0
YaHS	1.2a	https://github.com/c-zhou/yahs

Full text

Species taxonomy

Eukaryota; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Gelechioidea; Autostichidae; Symmocinae; Oegoconia; Oegoconia quadripuncta (Haworth 1829) (NCBI:txid347754).

Background

Oegoconia quadripuncta, which has fairly recently been given the name ‘Four-dotted Obscure’, is one of only three native species of the family Autostichidae in Britain. Specimens of the genus Oegoconia are distinctive little moths, dark brown with usually three pale yellow stripes across the fore wings, which are held very flat. In Britain and Ireland these were all referred to as Oegoconia quadripuncta until Goddard (1966) recognised O. deauratella as occurring here, and then Agassiz (1982) demonstrated that O. caradjai Popescu-Gorj & Capuse was another previously overlooked resident. All three species are externally very similar and only reliably identified by examination of their genitalia. Genitalia are well illustrated by Sterling et al. (2012) and in resources such as the mothdissection.co.uk website. The sequenced specimen, a male, was identified from the dissected genitalia, particularly the shape of the saccus, confirmed by the COI barcode.

Adults of O. quadripuncta are frequent in light traps in the summer, found throughout much of England and Wales but only recorded locally in Ireland and not in Scotland ( Sterling et al., 2012). Larvae feed on decaying leaves in the litter layer, often below trees and hedgerows, as do the larvae of O. caradjai Popescu-Gorj & Capuse and, it is presumed, O. deauratella. In Britain, O. quadripuncta is invariably described as the most frequently collected species of Oegoconia and is the only one of the three which the first author has found at a couple of regularly sampled sites, but in Belgium it seems that O. caradjai Popescu-Gorj & Capuse is the most common and O. quadripuncta rarely found ( De Prins, 2005).

Autostichidae are part of the species-rich superfamily Gelechioidea ( Wang & Li, 2020), for which very few genome assemblies are currently available, with no previous Autostichidae genome. The fourth species on the British list, Symmoca signatella Herrich-Schäffer, is very different in appearance and probably an occasional accidental import. Interestingly, there are no reports of parasitoid wasps attacking Oegoconia, or any Autostichidae, although some must do so.

Genome sequence report

The genome was sequenced from one male Oegoconia quadripuncta ( Figure 1) collected from Tonbridge, Kent (51.19, 0.29). 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 25 missing joins or misjoins and removed 10 haplotypic duplications, reducing the assembly length by 0.35% and the scaffold number by 4.26%.

Photograph of the Oegoconia quadripuncta (ilOegQuad1) specimen used for genome sequencing. A. Dorsal view, B. Ventral view.

The final assembly has a total length of 622.6 Mb in 44 sequence scaffolds with a scaffold N50 of 36.2 Mb ( Table 1). Most (99.75%) of the assembly sequence was assigned to 20 chromosomal-level scaffolds, representing 19 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.

Table 1.: Genome data for Oegoconia quadripuncta, ilOegQuad1.1.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: 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 622,656,778 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 (50,213,132 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (36,190,098 and 20,651,493 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/ilOegQuad1.1/dataset/CASGFV01/snail.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: 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/ilOegQuad1.1/dataset/CASGFV01/blob.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: 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/ilOegQuad1.1/dataset/CASGFV01/cumulative.

Genome assembly of Oegoconia quadripuncta, ilOegQuad1.1: Hi-C contact map of the ilOegQuad1.1 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=WvXNmCQqSuiNwWWPD2TKSg.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Oegoconia quadripuncta, ilOegQuad1.

The estimated Quality Value (QV) of the final assembly is 62.5 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.4% (single = 98.0%, duplicated = 0.4%), using the lepidoptera_odb10 reference set ( n = 5,286).

Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/347754.

Methods

Sample acquisition and nucleic acid extraction

A male Oegoconia quadripuncta (specimen ID NHMUK014425720, ToLID ilOegQuad1) was collected using a light trap from a garden in Tonbridge, Kent, UK (latitude 51.19, longitude 0.29) on 2021-08-05. The specimen was collected and identified by Gavin Broad (Natural History Museum) and dry-frozen at –80°C.

The sample was prepared for DNA extraction at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilOegQuad1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Tissue from the whole organism was disrupted using a Nippi Powermasher fitted with a BioMasher pestle. DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions.

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 tissue of ilOegQuad1 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 using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, 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.

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 Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). 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 relevant software tool versions and sources.

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

•   Legality of collection, transfer and use (national and international)

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.