The genome sequence of the Small Angle Shades, Euplexia lucipara (Linnaeus, 1758)

TL;DR

This paper presents the genome sequence of the Small Angle Shades moth, including chromosomal scaffolding and gene annotation.

Contribution

The study provides a high-quality genome assembly and gene annotation for Euplexia lucipara, including the Z sex chromosome and mitochondrial genome.

Findings

The genome assembly spans 661.8 megabases and is scaffolded into 31 chromosomal pseudomolecules.

The mitochondrial genome is 15.37 kilobases long and was successfully assembled.

Gene annotation identified 20,395 protein coding genes using Ensembl.

Abstract

We present a genome assembly from an individual male
Euplexia lucipara (the Small Angle Shades; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 661.8 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 15.37 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,395 protein coding genes.

Figures

Photograph of the Euplexia lucipara (ilEupLuci1) specimen used for genome sequencing.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: 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 661,825,765 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 (37,151,478 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (22,669,833 and 15,555,094 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/Euplexia%20lucipara/dataset/CAKLHH01.1/snail.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: 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/Euplexia%20lucipara/dataset/CAKLHH01.1/blob.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: 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/Euplexia%20lucipara/dataset/CAKLHH01.1/cumulative.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: Hi-C contact map of the ilEupLuci1.1 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=e6psxBA4RJqdiQYZcvsoiw.

Tables

Table 1.. Genome data for Euplexia lucipara, ilEupLuci1.1.

Project accession data
Assembly identifier	ilEupLuci1.1
Assembly release date	2021-12-17
Species	Euplexia lucipara
Specimen	ilEupLuci1
NCBI taxonomy ID	987933
BioProject	PRJEB46851
BioSample ID	SAMEA7701470
Isolate information	ilEupLuci1, male: abdomen (DNA sequencing), head and thorax (Hi-C scaffolding)
Assembly metrics *		Benchmark
Consensus quality (QV)	57.5	≥ 50
k-mer completeness	99.99%	≥ 95%
BUSCO **	C:99.0%[S:98.5%,D:0.5%], F:0.3%,M:0.7%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.62%	≥ 95%
Sex chromosomes	Z chromosome	localised homologous pairs
Organelles	Mitochondrial genome assembled	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR6907906
10X Genomics Illumina	ERR6688636, ERR6688637, ERR6688638, ERR6688639
Hi-C Illumina	ERR6688635
Genome assembly
Assembly accession	GCA_921972225.1
Accession of alternate haplotype	GCA_921956305.1
Span (Mb)	661.8
Number of contigs	128
Contig N50 length (Mb)	14.9
Number of scaffolds	71
Scaffold N50 length (Mb)	22.7
Longest scaffold (Mb)	37.2
Genome annotation
Number of protein-coding genes	20,395
Number of gene transcripts	20,590

Table 2.. Chromosomal pseudomolecules in the genome assembly of Euplexia lucipara, ilEupLuci1.

INSDC accession	Chromosome	Length (Mb)	GC%
OV179143.1	1	26.32	36.5
OV179144.1	2	25.29	36.5
OV179145.1	3	25.21	37.0
OV179146.1	4	24.67	37.0
OV179147.1	5	24.66	36.5
OV179148.1	6	24.57	36.5
OV179149.1	7	24.25	37.0
OV179150.1	8	24.24	36.5
OV179151.1	9	23.91	37.0
OV179152.1	10	23.1	36.5
OV179153.1	11	22.99	36.5
OV179154.1	12	22.98	36.5
OV179155.1	13	22.67	37.0
OV179156.1	14	22.58	37.0
OV179157.1	15	22.44	36.5
OV179158.1	16	22.42	36.5
OV179159.1	17	22.13	37.0
OV179160.1	18	21.56	37.0
OV179161.1	19	20.97	37.0
OV179162.1	20	20.56	37.0
OV179163.1	21	20.53	37.0
OV179164.1	22	20.26	36.5
OV179165.1	23	18.4	37.0
OV179166.1	24	17.64	37.0
OV179167.1	25	15.56	37.0
OV179168.1	26	15.09	37.0
OV179169.1	27	12.58	37.5
OV179170.1	28	11.85	37.5
OV179171.1	29	11.15	38.0
OV179172.1	30	11.11	38.0
OV179142.1	Z	37.15	36.5
OV179173.1	MT	0.02	18.5

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.1.7	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

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; Obtectomera; Noctuoidea; Noctuidae; Hadeninae; Euplexia; Euplexia lucipara (Linnaeus, 1758) (NCBI:txid987933).

Background

The Small Angle Shades ( Euplexia lucipara) is a noctuid moth with a wide distributed in a variety of habitats across Britain and Ireland ( Randle et al., 2019). Globally, its range extends across temperate Europe and Asia to Japan ( GBIF Secretariat, 2023). In appearance, it varies little across its range, and has a distinct resting posture with folder forewings that resemble a dead leaf ( Waring et al., 2017).

This is one of a relatively small set of British and Irish insects that feeds frequently on bracken ( Pteridium aquilinum (L.) Kuhn) and other ferns ( Lawton, 1982); its larvae also feed on a wide variety of angiosperms ( Henwood et al., 2020). This species has a single generation in the UK, over-wintering as a pupa.

The genome of the Small Angle Shades, Euplexia lucipara, 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 Euplexia lucipara, based on one male specimen from Wytham Woods, Oxfordshire.

Genome sequence report

The genome was sequenced from one male Euplexia lucipara ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.34). A total of 26-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 51-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 27 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the assembly length by 0.46% and the scaffold number by 22.83%, and increasing the scaffold N50 by 1.13%.

Photograph of the Euplexia lucipara (ilEupLuci1) specimen used for genome sequencing.

The final assembly has a total length of 661.8 Mb in 71 sequence scaffolds with a scaffold N50 of 22.7 Mb ( Table 1). Most (99.62%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome. 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. 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 Euplexia lucipara, ilEupLuci1.1.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: 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 661,825,765 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 (37,151,478 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (22,669,833 and 15,555,094 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/Euplexia%20lucipara/dataset/CAKLHH01.1/snail.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: 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/Euplexia%20lucipara/dataset/CAKLHH01.1/blob.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: 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/Euplexia%20lucipara/dataset/CAKLHH01.1/cumulative.

Genome assembly of Euplexia lucipara, ilEupLuci1.1: Hi-C contact map of the ilEupLuci1.1 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=e6psxBA4RJqdiQYZcvsoiw.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Euplexia lucipara, ilEupLuci1.

The estimated Quality Value (QV) of the final assembly is 57.5 with k-mer completeness of 99.99%, and the assembly has a BUSCO v5.3.2 completeness of 99.0% (single = 98.5%, duplicated = 0.5%), 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/987933.

Genome annotation report

The Euplexia lucipara genome assembly (GCA_921972225.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Euplexia_lucipara_GCA_921972225.1/Info/Index). The resulting annotation includes 20,590 transcribed mRNAs from 20,395 protein-coding genes.

Methods

Sample acquisition and nucleic acid extraction

A male Euplexia lucipara (specimen ID Ox000606, ToLID ilEupLuci1) 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.

DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilEupLuci1 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 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 head and thorax tissue of ilEupLuci1 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., 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 BRAKER2 pipeline ( Brůna et al., 2021) was used in the default protein mode to generate annotation for the Euplexia lucipara assembly (GCA_921972225.1) 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.