The genome sequence of the Mottled Pug, Eupithecia exiguata (Hübner, 1813)

TL;DR

This paper provides the genome sequence of the Mottled Pug moth, including chromosomal scaffolding and gene annotations.

Contribution

The study presents a high-quality genome assembly and annotation for Eupithecia exiguata, including chromosomal pseudomolecules and mitochondrial DNA.

Findings

The genome assembly spans 372.9 megabases and includes 31 chromosomal pseudomolecules.

Gene annotation identified 11,194 protein coding genes using Ensembl.

The mitochondrial genome is 16.39 kilobases in length.

Abstract

We present a genome assembly from an individual male
Eupithecia exiguata (the Mottled Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 372.9 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 16.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,194 protein coding genes.

Figures

Photograph of the Eupithecia exiguata (ilEupExig1) specimen used for genome sequencing.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: 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 372,887,712 bp assembly. The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (19,876,806 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (13,143,396 and 8,794,744 bp), respectively. The pale grey spiral shows the cumulative sequence 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/Eupithecia%20exiguata/dataset/CAMTYU01/snail.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: 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/Eupithecia%20exiguata/dataset/CAMTYU01/blob.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: 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/Eupithecia%20exiguata/dataset/CAMTYU01/cumulative.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: Hi-C contact map of the ilEupExig1.1 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=PURr_tyLQ6Obof4vztyYVA.

Tables

Table 1.. Genome data for Eupithecia exiguata, ilEupExig1.1.

Project accession data
Assembly identifier	ilEupExig1.1
Species	Eupithecia exiguata
Specimen	ilEupExig1
NCBI taxonomy ID	934847
BioProject	PRJEB55723
BioSample ID	SAMEA10979157
Isolate information	ilEupExig1, male: whole organism (DNA sequencing and Hi-C scaffolding)
Assembly metrics *		Benchmark
Consensus quality (QV)	66.5	≥ 50
k-mer completeness	100%	≥ 95%
BUSCO **	C:97.9%[S:97.4%,D:0.5%], F:0.6%,M:1.5%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.99%	≥ 95%
Sex chromosomes	Z chromosome	localised homologous pairs
Organelles	Mitochondrial genome assembly	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR10168716
Hi-C Illumina	ERR10149546
Genome assembly
Assembly accession	GCA_947086465.1
Accession of alternate haplotype	GCA_947086475.1
Span (Mb)	372.9
Number of contigs	118
Contig N50 length (Mb)	5.4
Number of scaffolds	32
Scaffold N50 length (Mb)	13.1
Longest scaffold (Mb)	19.9
Genome annotation
Number of protein-coding genes	11,194
Number of non-coding genes	1,243
Number of gene transcripts	19,529

Table 2.. Chromosomal pseudomolecules in the genome assembly of Eupithecia exiguata, ilEupExig1.

INSDC accession	Chromosome	Length (Mb)	GC%
OX352227.1	1	15.5	37.5
OX352228.1	2	15.45	37.5
OX352229.1	3	15.33	38.0
OX352230.1	4	14.83	38.0
OX352231.1	5	14.09	37.5
OX352232.1	6	14.08	37.5
OX352233.1	7	13.77	37.5
OX352234.1	8	13.74	37.5
OX352235.1	9	13.61	37.5
OX352236.1	10	13.55	37.5
OX352237.1	11	13.48	37.0
OX352238.1	12	13.14	37.5
OX352239.1	13	13.06	37.5
OX352240.1	14	13.01	37.0
OX352241.1	15	12.44	37.5
OX352242.1	16	12.38	38.0
OX352243.1	17	12.33	37.5
OX352244.1	18	12.16	37.5
OX352245.1	19	12.15	37.5
OX352246.1	20	11.5	37.5
OX352247.1	21	10.88	37.5
OX352248.1	22	9.62	37.5
OX352249.1	23	9.26	38.0
OX352250.1	24	9.14	37.5
OX352251.1	25	8.79	37.5
OX352252.1	26	8.64	37.5
OX352253.1	27	7.28	37.5
OX352254.1	28	6.9	37.5
OX352255.1	29	6.48	38.0
OX352256.1	30	6.38	38.5
OX352226.1	Z	19.88	37.5
OX352257.1	MT	0.02	19.0

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
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	yahs-1.1.91eebc2	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; Obtectomera; Geometroidea; Geometridae; Larentiinae; Eupithecia; Eupithecia exiguata (Hübner, 1813) (NCBI:txid934847).

Background

The Mottled Pug ( Eupithecia exiguata) is a small geometrid moth. Its larvae feed on Hawthorn, Blackthorn and other shrubs ( Henwood et al., 2020; Waring et al., 2017). It occurs in woodland, hedgerow and garden habitats and is common and widespread across much of England and Wales. It is also widespread in Ireland but there are fewer records from Scotland, where it is spreading; its distribution overall has increased markedly since 1970 ( Randle et al., 2019). Globally, The Mottled Pug occurs across Europe and Asia to the Pacific coast of Russia and China ( GBIF Secretariat, 2023).

The genome of the mottled pug, Eupithecia exiguata, 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 Eupithecia exiguata, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report

The genome was sequenced from one male Eupithecia exiguata ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.32). A total of 56-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 20 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 15.38%.

Photograph of the Eupithecia exiguata (ilEupExig1) specimen used for genome sequencing.

The final assembly has a total length of 372.9 Mb in 32 sequence scaffolds with a scaffold N50 of 13.1 Mb ( Table 1). Most (99.99%) 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 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 Eupithecia exiguata, ilEupExig1.1.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: 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 372,887,712 bp assembly. The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (19,876,806 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (13,143,396 and 8,794,744 bp), respectively. The pale grey spiral shows the cumulative sequence 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/Eupithecia%20exiguata/dataset/CAMTYU01/snail.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: 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/Eupithecia%20exiguata/dataset/CAMTYU01/blob.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: 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/Eupithecia%20exiguata/dataset/CAMTYU01/cumulative.

Genome assembly of Eupithecia exiguata, ilEupExig1.1: Hi-C contact map of the ilEupExig1.1 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=PURr_tyLQ6Obof4vztyYVA.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Eupithecia exiguata, ilEupExig1.

The estimated Quality Value (QV) of the final assembly is 66.5 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 97.9% (single = 97.4%, 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/934847.

Genome annotation report

The Eupithecia exiguata genome assembly (GCA_947086465.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Eupithecia_exiguata_GCA_947086465.1/Info/Index). The resulting annotation includes 19,529 transcribed mRNAs from 11,194 protein-coding and 1,243 non-coding genes.

Methods

Sample acquisition and nucleic acid extraction

A male Eupithecia exiguata (specimen ID Ox001895, individual ilEupExig1) was collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude –1.32) on 2021-05-28 using a light trap. The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation; DNA extraction; HMW DNA fragmentation; and fragmented DNA clean-up. The sample was prepared for DNA extraction at the WSI Tree of Life laboratory: the ilEupExig1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing ( https://dx.doi.org/10.17504/protocols.io.x54v9prmqg3e/v1). Tissue from the whole organism was disrupted using a Nippi Powermasher fitted with a BioMasher pestle ( https://dx.doi.org/10.17504/protocols.io.5qpvo3r19v4o/v1). DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions.

Protocols developed in the Tree of Life laboratory are publicly available on protocols.io ( https://dx.doi.org/10.17504/protocols.io.8epv5xxy6g1b/v1).

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 instrument. Hi-C data were also generated from remaining tissue of ilEupExig1 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 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., 2023b) and “sanger-tol/genomenote” ( Surana et al., 2023a). 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 Eupithecia exiguata assembly (GCA_947086465.1). 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.