The genome sequence of the September Thorn moth, Ennomos erosaria (Denis & Schiffermüller), 1775
Douglas Boyes, Liam M. Crowley, Denise C. Wawman, Jason Charamis, James Mallet
TL;DR
This paper provides the genome sequence of the September Thorn moth, including its chromosomal structure and mitochondrial genome.
Contribution
The study presents a high-quality genome assembly of the September Thorn moth with scaffolded chromosomes and mitochondrial DNA.
Findings
The genome assembly spans 423.1 megabases and includes 32 chromosomal pseudomolecules.
The mitochondrial genome is 16.3 kilobases long and has been assembled.
Abstract
We present a genome assembly from an individual female Ennomos erosaria (the September Thorn moth; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 423.1 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 16.3 kilobases in length.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | ilEnnEroa1.1 | |
| Species |
| |
| Specimen | ilEnnEroa1 | |
| NCBI taxonomy ID | 934814 | |
| BioProject | PRJEB66772 | |
| BioSample ID | SAMEA7701438 | |
| Isolate information | ilEnnEroa1, female: abdomen (PacBio DNA sequencing)
| |
| Assembly metrics
|
| |
| Consensus quality (QV) | 60.4 |
|
|
| 100.0% |
|
| BUSCO
| C:98.7%[S:98.3%,D:0.4%],
|
|
| Percentage of assembly mapped
| 99.65% |
|
| Sex chromosomes | ZW |
|
| Organelles | Mitochondrial genome: 16.3 kb |
|
| Raw data accessions | ||
| PacificBiosciences Sequel IIe | ERR12102463, ERR12102462 | |
| Hi-C Illumina | ERR12102445 | |
| PolyA RNA-Seq Illumina | ERR12102444 | |
| Genome assembly | ||
| Assembly accession | GCA_963580505.1 | |
|
| GCA_963580315.1 | |
| Span (Mb) | 423.1 | |
| Number of contigs | 97 | |
| Contig N50 length (Mb) | 9.2 | |
| Number of scaffolds | 55 | |
| Scaffold N50 length (Mb) | 14.6 | |
| Longest scaffold (Mb) | 19.26 | |
| INSDC
| Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 17.52 | 37.5 | |
| 2 | 17.41 | 37.5 | |
| 3 | 17.29 | 37.5 | |
| 4 | 17.05 | 37.5 | |
| 5 | 16.28 | 37.5 | |
| 6 | 15.87 | 37.0 | |
| 7 | 15.71 | 37.5 | |
| 8 | 15.59 | 37.5 | |
| 9 | 15.57 | 37.0 | |
| 10 | 14.11 | 37.5 | |
| 11 | 14.86 | 37.5 | |
| 12 | 14.67 | 37.5 | |
| 13 | 14.59 | 37.5 | |
| 14 | 14.54 | 37.5 | |
| 15 | 14.4 | 37.5 | |
| 16 | 13.63 | 37.5 | |
| 17 | 13.62 | 37.5 | |
| 18 | 13.48 | 38.0 | |
| 19 | 13.46 | 38.0 | |
| 20 | 13.02 | 37.5 | |
| 21 | 12.85 | 38.0 | |
| 22 | 11.39 | 38.0 | |
| 23 | 10.85 | 37.5 | |
| 24 | 10.72 | 38.0 | |
| 25 | 9.9 | 38.0 | |
| 26 | 9.42 | 38.0 | |
| 27 | 8.45 | 38.0 | |
| 28 | 8.29 | 39.0 | |
| 29 | 7.68 | 38.5 | |
| 30 | 7.44 | 38.0 | |
| W | 1.58 | 34.0 | |
| Z | 19.26 | 37.5 | |
| MT | 0.02 | 18.0 |
| Software tool | Version | Source |
|---|---|---|
| BEDTools | 2.30.0 |
|
| Blast | 2.14.0 |
|
| BlobToolKit | 4.3.7 |
|
| BUSCO | 5.4.3 and 5.5.0 |
|
| bwa-mem2 | 2.2.1 |
|
| Cooler | 0.8.11 |
|
| DIAMOND | 2.1.8 |
|
| fasta_windows | 0.2.4 |
|
| FastK | 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c |
|
| GoaT CLI | 0.2.5 |
|
| Hifiasm | 0.16.1-r375 |
|
| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84a
|
|
| MerquryFK | d00d98157618f4e8d1a9190026b19b471055b22e |
|
| MitoHiFi | 2 |
|
| MultiQC | 1.14, 1.17, and 1.18 |
|
| NCBI Datasets | 15.12.0 |
|
| Nextflow | 23.04.0-5857 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| samtools | 1.16.1, 1.17, and 1.18 |
|
| sanger-tol/genomenote | 1.1.1 |
|
| sanger-tol/readmapping | 1.2.1 |
|
| Seqtk | 1.3 |
|
| Singularity | 3.9.0 |
|
| TreeVal | 1.0.0 |
|
| YaHS | yahs-1.1.91eebc2 |
|
- —Wellcome Trust
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Taxonomy
TopicsLepidoptera: Biology and Taxonomy · Insect Resistance and Genetics · Insect and Arachnid Ecology and Behavior
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Obtectomera; Geometroidea; Geometridae; Ennominae; Ennomos; Ennomos erosaria (Denis & Schiffermüller), 1775 (NCBI:txid934814).
Background
The September Thorn Ennomos erosaria is one of a group of superficially similar-looking moths in the family Geometridae. It is distinguished from the other Thorns by its plain or only very slightly speckled wings, that range in colour from yellow to dark brown, and cross-lines, which, although they are variable, usually converge near the trailing edge of the wing ( Waring et al., 2017).
Although its common name might suggest that it has a limited flight season that does not overlap with other thorns, the single generation of Ennomos erosaria, is on the wing from July to early October. It overwinters as an egg on the foodplant and the larva feed from April to early July before pupating between the leaves ( Waring et al., 2017). The main foodplants are oaks Quercus spp., birches Betula spp., limes Tilia spp. and Beech Fagus sylvatica ( Skinner & Wilson, 2009; Waring et al., 2017).
Ennomos erosaria is currently endangered in the United Kingdom (UK), its numbers having declined by 87% in the 40 years to 2007 ( Conrad et al., 2006; Fox et al., 2013). Its range in the UK is mostly limited to England and Wales and lowland Scotland, but while numbers have been decreasing more rapidly in the south of the country since 2000, it remains common in most areas ( Waring et al., 2017).
Here we present a chromosomal-level whole genome sequence for Ennomos erosaria, based on one female specimen from Wytham Woods, Oxfordshire, UK.
Genome sequence report
The genome was sequenced from a female Ennomos erosaria ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.34). A total of 30-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 13 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the scaffold N50 by 1.80%.
Photograph of the Ennomos erosaria (ilEnnEroa1) specimen used for genome sequencing.
The final assembly has a total length of 423.1 Mb in 55 sequence scaffolds with a scaffold N50 of 14.6 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (99.65%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 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). Chromosome W and Chromosome Z were assigned by read coverage statistics. 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 Ennomos erosaria, ilEnnEroa1.1.
Genome assembly of Ennomos erosaria, ilEnnEroa1.1: metrics.The BlobToolKit snail plot 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 423,092,927 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 (19,264,547 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (14,589,179 and 9,417,489 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/Ennomos_erosarius/dataset/GCA_963580505.1/snail.
Genome assembly of Ennomos erosaria, ilEnnEroa1.1: BlobToolKit GC-coverage plot.Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Ennomos_erosarius/dataset/GCA_963580505.1/blob.
Genome assembly of Ennomos erosaria, ilEnnEroa1.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Ennomos_erosarius/dataset/GCA_963580505.1/cumulative.
Genome assembly of Ennomos erosaria, ilEnnEroa1.1: Hi-C contact map of the ilEnnEroa1.1 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=LIUdIc4KQbe53ZQ-lOb7xA.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Ennomos erosaria, ilEnnEroa1.
The estimated Quality Value (QV) of the final assembly is 60.4 with k-mer completeness of 100.0%, and the assembly has a BUSCO v completeness of 98.7% (single = 98.3%, duplicated = 0.4%), using the lepidoptera_odb10 reference set ( n = 5,286).
Metadata for specimens, BOLD barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/934814.
Methods
Sample acquisition and nucleic acid extraction
A female Ennomos erosaria (specimen ID Ox000574, ToLID ilEnnEroa1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.34) on 2020-07-05 using a light trap. The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice. The specimen used for Hi-C and RNA sequencing (specimen ID Ox003092, ToLID ilEnnEroa2) was collected from the same location on 2022-07-22 using a light trap. The specimen was collected and identified by Liam Crowley (University of Oxford) and preserved on dry ice.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the ilEnnEroa1 sample was weighed and dissected on dry ice ( Jay et al., 2023).
Tissue from the abdomen was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted using the Automated MagAttract v1 protocol ( Sheerin et al., 2023). DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 30 ( Todorovic et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. 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 head and thorax tissue of ilEnnEroa2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol ( do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).
Sequencing
Pacific Biosciences HiFi circular consensus 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 IIe (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from $HIC_TISSUE tissue of ilEnnEroa2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Genome assembly and curation
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 TreeVal pipeline ( Pointon et al., 2023). Manual curation was performed using JBrowse2 ( Diesh et al., 2023), HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), 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.
Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a), “sanger-tol/genomenote” ( Surana et al., 2023b), and “sanger-tol/blobtoolkit” ( Muffato et al., 2024). The pipeline sanger-tol/readmapping aligns the Hi-C reads with bwa-mem2 ( Vasimuddin et al., 2019) and combines the alignment files with SAMtools ( Danecek et al., 2021). The sanger-tol/genomenote pipeline transforms the Hi-C alignments into a contact map with BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020), which i ( Kerpedjiev et al., 2018) Glass ( Kerpedjiev et al., 2018). It also provides statistics about the assembly with the NCBI datasets ( Sayers et al., 2024) report, computes k-mer completeness and QV consensus quality values with FastK and MerquryFK, and a completeness assessment with BUSCO ( Manni et al., 2021).
The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads with SAMtools and minimap2 ( Li, 2018) and generates coverage tracks for regions of fixed size. In parallel, it queries the GoaT database ( Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO ( Manni et al., 2021). For the three domain-level BUSCO lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database ( Bateman et al., 2023) with DIAMOND ( Buchfink et al., 2021) blastp. The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx. Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling ( Ewels et al., 2020), use MultiQC ( Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), and the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.
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.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Abdennur N Mirny LA : Cooler: scalable storage for Hi-C data and other genomically labeled arrays. Bioinformatics. 2020;36(1):311–316. 10.1093/bioinformatics/btz 540 31290943 PMC 8205516 · doi ↗ · pubmed ↗
- 2Allio R Schomaker-Bastos A Romiguier J : Mito Finder: efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 32243090 PMC 7497042 · doi ↗ · pubmed ↗
- 3Altschul SF Gish W Miller W : Basic Local Alignment Search Tool. J Mol Biol. 1990;215(3):403–410. 10.1016/S 0022-2836(05)80360-2 2231712 · doi ↗ · pubmed ↗
- 4Bateman A Martin MJ Orchard S : Uni Prot: the universal protein knowledgebase in 2023. Nucleic Acids Res. 2023;51(D 1):D 523–D 531. 10.1093/nar/gkac 1052 36408920 PMC 9825514 · doi ↗ · pubmed ↗
- 5Bernt M Donath A Jühling F : MITOS: improved de novo metazoan mitochondrial genome annotation. Mol Phylogenet Evol. 2013;69(2):313–319. 10.1016/j.ympev.2012.08.023 22982435 · doi ↗ · pubmed ↗
- 6Buchfink B Reuter K Drost HG : Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods. 2021;18(4):366–368. 10.1038/s 41592-021-01101-x 33828273 PMC 8026399 · doi ↗ · pubmed ↗
- 7Challis R Kumar S Sotero-Caio C : Genomes on a Tree (Goa T): a versatile, scalable search engine for genomic and sequencing project metadata across the eukaryotic tree of life [version 1; peer review: 2 approved]. Wellcome Open Res. 2023;8:24. 10.12688/wellcomeopenres.18658.1 36864925 PMC 9971660 · doi ↗ · pubmed ↗
- 8Challis R Richards E Rajan J : Blob Tool Kit – interactive quality assessment of genome assemblies. G 3 (Bethesda). 2020;10(4):1361–1374. 10.1534/g 3.119.400908 32071071 PMC 7144090 · doi ↗ · pubmed ↗