The genome sequence of the Dusky Thorn, Ennomos fuscantarius (Haworth, 1809)
Douglas Boyes, Dominic Phillips, Saskia Wutke, Tree of Life Team Sanger, Arjen Van 't Hof
TL;DR
This paper presents the genome sequence of the Dusky Thorn moth, including chromosomal scaffolding and gene annotation.
Contribution
The study provides a high-quality genome assembly and gene annotation for Ennomos fuscantarius.
Findings
The genome assembly spans 444.9 megabases and includes 31 chromosomal pseudomolecules.
Gene annotation identified 12,173 protein coding genes using Ensembl.
Abstract
We present a genome assembly from an individual male Ennomos fuscantarius (the Dusky Thorn; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 444.9 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 15.49 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,173 protein coding genes.
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
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Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | ilEnnFusc2.3 | |
| Species |
| |
| Specimen | ilEnnFusc2 | |
| NCBI taxonomy ID | 722662 | |
| BioProject | PRJEB42951 | |
| BioSample ID | SAMEA7520185 | |
| Isolate information | ilEnnFusc2: male: head and thorax (RNA sequencing), abdomen (Hi-C scaffolding)
| |
| Assembly metrics
|
| |
| Consensus quality (QV) | 56.4 |
|
|
| 99.99% |
|
| BUSCO
| C:98.5%[S:98.1%,D:0.4%],F:0.5%,M:1.1%,n:5,286 |
|
| Percentage of assembly mapped
| 99.95% |
|
| Sex chromosomes | Z chromosome |
|
| Organelles | Mitochondrial genome assembled |
|
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR6560797 | |
| 10X Genomics Illumina | ERR6054405, ERR6054407, ERR6054408, ERR6054406 | |
| Hi-C Illumina | ERR6054409 | |
| PolyA RNA-Seq Illumina | ERR6054410, ERR6787420 | |
| Genome assembly | ||
| Assembly accession | GCA_905220475.3 | |
|
| GCA_905220485.1 | |
| Span (Mb) | 444.9 | |
| Number of contigs | 46 | |
| Contig N50 length (Mb) | 15.5 | |
| Number of scaffolds | 36 | |
| Scaffold N50 length (Mb) | 15.9 | |
| Longest scaffold (Mb) | 19.5 | |
| Genome annotation | ||
| Number of protein-coding genes | 12,173 | |
| Number of non-coding genes | 1,740 | |
| Number of gene transcripts | 23,475 | |
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 18.42 | 37.0 | |
| 2 | 18.21 | 37.0 | |
| 3 | 17.84 | 37.0 | |
| 4 | 17.76 | 37.0 | |
| 5 | 17.17 | 36.5 | |
| 6 | 16.98 | 36.5 | |
| 7 | 16.84 | 36.5 | |
| 8 | 16.68 | 36.5 | |
| 9 | 16.42 | 36.5 | |
| 10 | 16.09 | 37.0 | |
| 11 | 16.07 | 36.5 | |
| 12 | 15.88 | 37.0 | |
| 13 | 15.7 | 36.5 | |
| 14 | 15.53 | 37.0 | |
| 15 | 15.19 | 37.0 | |
| 16 | 15.06 | 37.0 | |
| 17 | 14.83 | 37.0 | |
| 19 | 14.29 | 37.0 | |
| 18 | 14.29 | 37.5 | |
| 20 | 14.18 | 37.0 | |
| 21 | 13.83 | 37.5 | |
| 22 | 11.57 | 37.5 | |
| 23 | 11.43 | 37.0 | |
| 24 | 11.38 | 37.0 | |
| 25 | 11.09 | 37.5 | |
| 26 | 10.57 | 37.5 | |
| 27 | 8.69 | 38.0 | |
| 28 | 7.82 | 38.5 | |
| 29 | 7.77 | 37.5 | |
| 30 | 7.68 | 38.0 | |
| Z | 19.45 | 36.5 | |
| MT | 0.02 | 19.5 |
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.1.7 |
|
| BUSCO | 5.3.2 |
|
| FreeBayes | 1.3.1-17-gaa2ace8 |
|
| gEVAL | N/A |
|
| Hifiasm | 0.12 |
|
| HiGlass | 1.11.6 |
|
| Long Ranger ALIGN | 2.2.2 |
|
| Merqury | MerquryFK |
|
| MitoHiFi | 1 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| SALSA | 2.2 |
|
| sanger-tol/genomenote | v1.0 |
|
| sanger-tol/readmapping | 1.1.0 |
|
- —Wellcome Trust
- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Lepidoptera: Biology and Taxonomy · Genetic diversity and population structure
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; Geometroidea; Geometridae; Ennominae; Ennomos; Ennomos fuscantaria (Haworth, 1809) (NCBI:txid722662).
Background
Ennomos fuscantaria (Dusky Thorn) is a Geometrid moth in the Ennominae subfamily with a forewing length of 17–21 mm; a greyish or mauve-shaded band between the forewing margin and distal cross-line distinguishes it from similar species ( Lewis, 2023; Waring et al., 2017).
E. fuscantaria is a monovoltine species, flying from late July to early October. Though it readily comes to light, it is not often seen out on the wing ( Randle et al., 2019; Waring et al., 2017). The larva feeds predominantly on ash ( Fraxinus excelsior), on which it overwinters as an egg, later to pupate within spun leaves. It can be coaxed to feed on privets ( Ligustrum spp.) ( Waring et al., 2017).
E. fuscantaria can be found in most habitats where ash trees are present; in the UK, it has a widespread distribution, frequently occurring through England and Wales ( Randle et al., 2019). Globally, E. fuscantaria is distributed throughout the western Palearctic, extending from western Europe to eastern Russia and the Mediterranean ( GBIF Secretariat, 2023).
Numbers of E. fuscantaria are prone to fluctuate in the UK and are currently present on the Rothamstead Red List, with suspected population numbers decreasing by of 47% over a 10-year period as of 2019, with earlier research showing a 90% decrease between 1990 and 2001 ( GBIF Secretariat, 2023; Palmer et al., 2015; Waring et al., 2017). This species is highly sensitive to annual variation in climate change, as demonstrate by the large population decrease in population linked to increased rates of ash dieback ( Fox et al., 2021; Palmer et al., 2015). The full genome for E. fuscantaria will provide insights into how we can help to reduce these impacts that changes in climate can cause to occupancy and distribution of these vulnerable species. The full genome can also provide insights into genetic relations between Ennominae moths and the evolution of flightlessness within the genus ( Wahlberg et al., 2010).
The genome of Ennomos fuscantarius was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for Ennomos fuscantarius, based on one specimen collected by Douglas Boyes from Wytham Woods, Oxfordshire.
Genome sequence report
The genome was sequenced from one male Ennomos fuscantarius ( Figure 1) collected from Wytham Woods, UK (51.77, –1.34). A total of 52-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 85-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 12 missing joins or mis-joins, reducing the scaffold number by 13.95%, and increasing the scaffold N50 by 1.61%.
Photograph of the Ennomos fuscantarius (ilEnnFusc2) specimen used for genome sequencing.
The final assembly has a total length of 444.9 Mb in 36 sequence scaffolds with a scaffold N50 of 15.9 Mb ( Table 1). A summary of the assembly statistics is shown in Figure 2, 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 (99.95%) 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 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 Ennomos fuscantarius, ilEnnFusc2.3.
Genome assembly of Ennomos fuscantarius, ilEnnFusc2.3: 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 444,925,525 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 (19,450,406 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (15,884,173 and 11,089,215 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/ilEnnFusc2.3/dataset/CAJMZZ03/snail.
Genome assembly of Ennomos fuscantarius, ilEnnFusc2.3: 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/ilEnnFusc2.3/dataset/CAJMZZ03/blob.
Genome assembly of Ennomos fuscantarius, ilEnnFusc2.3: 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/ilEnnFusc2.3/dataset/CAJMZZ03/cumulative.
Genome assembly of Ennomos fuscantarius, ilEnnFusc2.3: Hi-C contact map of the ilEnnFusc2.3 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=KGN1bRa6S8ahRJaRSp9yrA.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Ennomos fuscantarius, ilEnnFusc2.
The estimated Quality Value (QV) of the final assembly is 56.4 with k-mer completeness of 99.99%, and the assembly has a BUSCO v5.3.2 completeness of 98.5% (single = 98.1%, 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/722662.
Genome annotation report
The Ennomos fuscantarius genome assembly (GCA_905220475.3) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Ennomos_fuscantarius_GCA_905220475.3/Info/Index). The resulting annotation includes 23,475 transcribed mRNAs from 12,173 protein-coding and 1,740 non-coding genes.
Methods
Sample acquisition and nucleic acid extraction
A male Ennomos fuscantarius (specimen ID Ox000200, individual ilEnnFusc2) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.34) on 2019-08-24 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 ilEnnFusc2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Head and thorax 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.
RNA was extracted from whole organism tissue of ilEnnFusc1 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, Illumina HiSeq 4000 (RNA-Seq) and HiSeq X Ten (10X) instruments. Hi-C data were also generated from abdomen tissue of ilEnnFusc2 using the Arima2 kit and sequenced on the HiSeq X Ten 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 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., 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.
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.
Genome annotation
The Ensembl gene annotation system ( Aken et al., 2016) was used to generate annotation for the Ennomos fuscantarius assembly (GCA_905220475.3). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt ( UniProt Consortium, 2019).
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.
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