Abstract
We present a genome assembly from an individual male Eilema sororcula (the Orange Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 729.4 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.46 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,093 protein coding genes.
Keywords: Eilema sororcula, Orange Footman, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Obtectomera; Noctuoidea; Erebidae; Arctiinae; Lithosiini; Eilema; Eilema sororcula (Hufnagel, 1766) (NCBI:txid987424).
Background
The Orange Footman Eilema sororcula had a local distribution in the south and east of the UK until the late 20th century, but like many related lichen-feeding Footman species, it has recently spread northwards and increased spectacularly in both its distribution and abundance ( Randle et al., 2019). At the time of writing there has been only a handful of records from Ireland ( Moths Ireland, 2023), but it has been recorded across much of Europe and east across Eurasia to Korea and China ( GBIF Secretariat, 2023).
The preferred habitats for E. sororcula are mature woodlands where its larvae feed on algae and lichens growing on trees ( Henwood et al., 2020). In Britain and Ireland, the adult moth is mostly observed during May and June, with the peak period of observations occurring a few weeks earlier in the year than in the 1970s ( Randle et al., 2019).
Here we present a chromosomally complete genome sequence for E. sororcula based on one male specimen from Wytham Woods, Oxfordshire, UK. A genome sequence for E. sororcula will facilitate studies into molecular adaptations to lichen-feeding and contribute to a growing data set of resources for understanding lepidopteran biology.
Genome sequence report
The genome was sequenced from one male Eilema sororcula ( Figure 1) collected from| Wytham Woods, Oxfordshire, UK (51.77, –1.34). A total of 43-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 93-fold coverage in 10X Genomics read clouds was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 81 missing joins or mis-joins and removed 7 haplotypic duplications, reducing the assembly length by 0.15% and the scaffold number by 60.4%, and increasing the scaffold N50 by 5.25%.
Figure 1. Photograph of the Eilema sororcula (ilEilSoro1) specimen used for genome sequencing.
The final assembly has a total length of 729.4 Mb in 40 sequence scaffolds with a scaffold N50 of 25.3 Mb ( Table 1). Most (99.94%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 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.
Figure 2. Genome assembly of Eilema sororcula, ilEilSoro1.1: 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 729,417,332 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 (51,764,148 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,298,203 and 16,955,058 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/ilEilSoro1.1/dataset/CAJZCU01.1/snail.
Figure 5. Genome assembly of Eilema sororcula, ilEilSoro1.1: Hi-C contact map of the ilEilSoro1.1 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=IiePitoAQqCad3afW9wBMg.
Table 1. Genome data for Eilema sororcula, ilEilSoro1.1.
| Project accession data | ||
|---|---|---|
| Assembly identifier | ilEilSoro1.1 | |
| Species | Eilema sororcula | |
| Specimen | ilEilSoro1 | |
| NCBI taxonomy ID | 987424 | |
| BioProject | PRJEB46305 | |
| BioSample ID | SAMEA7631555 | |
| Isolate information | ilEilSoro1, male: whole organism (DNA sequencing and Hi-C scaffolding) | |
| Assembly metrics * | Benchmark | |
| Consensus quality (QV) | 61.3 | ≥ 50 |
| k-mer completeness | 100% | ≥ 95% |
| BUSCO ** | C:98.5%[S:97.8%,D:0.7%],F:0.3%,M:1.2%,n:5,286 | C ≥ 95% |
| Percentage of assembly mapped
to chromosomes |
99.94% | ≥ 95% |
| Sex chromosomes | Z chromosome | localised homologous pairs |
| Organelles | Mitochondrial genome assembled | complete single alleles |
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR6807993, ERR6939231 | |
| 10X Genomics Illumina | ERR6688449–ERR6688452 | |
| Hi-C Illumina | ERR6688453 | |
| Genome assembly | ||
| Assembly accession | GCA_914829495.1 | |
| Accession of alternate haplotype | GCA_914829255.1 | |
| Span (Mb) | 729.4 | |
| Number of contigs | 157 | |
| Contig N50 length (Mb) | 11.8 | |
| Number of scaffolds | 40 | |
| Scaffold N50 length (Mb) | 25.3 | |
| Longest scaffold (Mb) | 51.8 | |
| Genome annotation | ||
| Number of protein-coding genes | 21,093 | |
| Number of gene transcripts | 21,274 | |
* Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from ( Rhie et al., 2021).
** BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.3.2. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/ilEilSoro1.1/dataset/CAJZCU01.1/busco.
Figure 3. Genome assembly of Eilema sororcula, ilEilSoro1.1: 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/ilEilSoro1.1/dataset/CAJZCU01.1/blob.
Figure 4. Genome assembly of Eilema sororcula, ilEilSoro1.1: 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/ilEilSoro1.1/dataset/CAJZCU01.1/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Eilema sororcula, ilEilSoro1.
| INSDC accession | Name | Size (Mb) | GC% |
|---|---|---|---|
| OU618533.1 | 1 | 30.01 | 38.5 |
| OU618534.1 | 2 | 28.88 | 38.6 |
| OU618535.1 | 3 | 27.72 | 39.1 |
| OU618536.1 | 4 | 27.46 | 39.5 |
| OU618537.1 | 5 | 27.37 | 38.5 |
| OU618538.1 | 6 | 27.23 | 38.4 |
| OU618539.1 | 7 | 27.15 | 38.4 |
| OU618540.1 | 8 | 27.12 | 38.5 |
| OU618541.1 | 9 | 26.1 | 38.7 |
| OU618542.1 | 10 | 25.84 | 38.5 |
| OU618543.1 | 11 | 25.59 | 38.2 |
| OU618544.1 | 12 | 25.3 | 38.7 |
| OU618545.1 | 13 | 25.05 | 38.2 |
| OU618546.1 | 14 | 24.98 | 38.6 |
| OU618547.1 | 15 | 24.1 | 38.7 |
| OU618548.1 | 16 | 23.88 | 38.8 |
| OU618549.1 | 17 | 23.65 | 38.9 |
| OU618550.1 | 18 | 23.21 | 38.7 |
| OU618551.1 | 19 | 23.2 | 39.8 |
| OU618552.1 | 20 | 23.01 | 38.7 |
| OU618553.1 | 21 | 22.79 | 38.9 |
| OU618554.1 | 22 | 20.73 | 39.4 |
| OU618555.1 | 23 | 20.53 | 39.5 |
| OU618556.1 | 24 | 19.97 | 38.7 |
| OU618557.1 | 25 | 16.96 | 39.4 |
| OU618558.1 | 26 | 16.45 | 39.2 |
| OU618559.1 | 27 | 16.3 | 40.2 |
| OU618560.1 | 28 | 13.71 | 40.2 |
| OU618561.1 | 29 | 12.97 | 40.8 |
| OU618532.1 | Z | 51.76 | 38.4 |
| OU618562.1 | MT | 0.02 | 19.5 |
| - | - | 0.4 | 45.8 |
The estimated Quality Value (QV) of the final assembly is 61.3 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.5% (single = 97.8%, duplicated = 0.7%), 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/987424.
Genome annotation report
The Eilema sororcula genome assembly (GCA_914829495.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Eilema_sororculum_GCA_914829495.1/Info/Index). The resulting annotation includes 21,274 transcribed mRNAs from 21,093 protein-coding genes.
Methods
Sample acquisition and nucleic acid extraction
A male Eilema sororcula (specimen ID Ox000399, individual ilEilSoro1) was collected in Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.34) on 2020-05-22, using a light trap. 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 ilEilSoro1 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. 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 and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (10X) instruments. Hi-C data were also generated from tissue of ilEilSoro1 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 ( Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using SALSA2 ( Ghurye et al., 2019). 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 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.
Table 3. Software tools: versions and sources.
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.0.7 | https://github.com/blobtoolkit/blobtoolkit |
| BUSCO | 5.3.2 | https://gitlab.com/ezlab/busco |
| FreeBayes | 1.3.1-17-gaa2ace8 | https://github.com/freebayes/freebayes |
| Hifiasm | 0.15.3 | https://github.com/chhylp123/hifiasm |
| HiGlass | 1.11.6 | https://github.com/higlass/higlass |
| Long Ranger ALIGN | 2.2.2 |
https://support.10xgenomics.com/genome-exome/
software/pipelines/latest/advanced/other-pipelines |
| 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 |
| SALSA | 2.2 | https://github.com/salsa-rs/salsa |
| 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 |
Genome annotation
The BRAKER2 pipeline ( Brůna et al., 2021) was used in the default protein mode to generate annotation for the Eilema sororcula assembly (GCA_914829495.1) 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
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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved, 1 approved with reservations]
Data availability
European Nucleotide Archive: Eilema sororcula (orange footman). Accession number PRJEB46305; https://identifiers.org/ena.embl/PRJEB46305. ( Wellcome Sanger Institute, 2021)
The genome sequence is released openly for reuse. The Eilema sororcula genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1.
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4789928.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.4783585.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.4790455.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.
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