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. 2025 Mar 18;10:141. [Version 1] doi: 10.12688/wellcomeopenres.23796.1

The genome sequence of the Maple Pug moth, Eupithecia inturbata (Hübner, 1817)

Finley Hutchinson 1, Liam M Crowley 2; University of Oxford and Wytham Woods Genome Acquisition Lab; Darwin Tree of Life Barcoding collective; Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team; Wellcome Sanger Institute Scientific Operations: Sequencing Operations; Wellcome Sanger Institute Tree of Life Core Informatics team; Tree of Life Core Informatics collective; Darwin Tree of Life Consortiuma
PMCID: PMC11992513  PMID: 40225904

Abstract

We present a genome assembly from a female Eupithecia inturbata (Maple Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence has a total length of 427.76 megabases. Most of the assembly (99.94%) is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled, with a length of 15.33 kilobases. Gene annotation of this assembly on Ensembl identified 12,386 protein-coding genes.

Keywords: Eupithecia inturbata, Maple Pug moth, genome sequence, chromosomal, Lepidoptera

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; Larentiinae; Eupithecia; Eupithecia inturbata (Hübner) (NCBI:txid934853)

Background

The genome of the Maple Pug, Eupithecia inturbata, 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 chromosome-level genome sequence for Eupithecia inturbata, based on a female specimen from Wytham Woods, Oxfordshire, United Kingdom ( Figure 1).

Figure 1. Photograph of the Eupithecia inturbata (ilEupIntu1) specimen used for genome sequencing.

Figure 1.

Genome sequence report

Sequencing data

The genome of a specimen of Eupithecia inturbata ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 22.05 Gb from 3.09 million reads. GenomeScope analysis of the PacBio HiFi data estimated the haploid genome size at 422.93 Mb, with a heterozygosity of 0.47% and repeat content of 23.00%. These values provide an initial assessment of genome complexity and the challenges anticipated during assembly. Based on this estimated genome size, the sequencing data provided approximately 50.0x coverage of the genome. Chromosome conformation Hi-C sequencing produced 110.68 Gb from 732.96 million reads.

Table 1 summarises the specimen and sequencing information, including the BioProject, study name, BioSample numbers, and sequencing data for each technology.

Table 1. Specimen and sequencing data for Eupithecia inturbata.

Project information
Study title Eupithecia inturbata (maple pug)
Umbrella BioProject PRJEB67611
Species Eupithecia inturbata
BioSpecimen SAMEA113425663
NCBI taxonomy ID 934853
Specimen information
Technology ToLID BioSample accession Organism part
PacBio long read sequencing ilEupIntu1 SAMEA113426987 whole organism
Hi-C sequencing ilEupIntu1 SAMEA113426987 whole organism
Sequencing information
Platform Run accession Read count Base count (Gb)
Hi-C Illumina NovaSeq 6000 ERR12144000 7.33e+08 110.68
PacBio Revio ERR12205265 3.09e+06 22.05

Assembly statistics

The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The assembly was improved by manual curation, which corrected 14 misjoins or missing joins and removed 7 haplotypic duplications. These interventions decreased the scaffold count by 18.18%. The final assembly has a total length of 427.76 Mb in 35 scaffolds, with 53 gaps, and a scaffold N50 of 14.97 Mb ( Table 2).

Table 2. Genome assembly data for Eupithecia inturbata.

Genome assembly
Assembly name ilEupIntu1.1
Assembly accession GCA_963662085.1
Alternate haplotype accession GCA_963662135.1
Assembly level for primary assembly chromosome
Span (Mb) 427.76
Number of contigs 88
Number of scaffolds 35
Longest scaffold (Mb) 20.78
Assembly metric Measure Benchmark
Contig N50 length 9.91 Mb ≥ 1 Mb
Scaffold N50 length 14.97 Mb = chromosome N50
Consensus quality (QV) Primary: 62.7; alternate:
61.8; combined 62.2
≥ 40
k-mer completeness Primary: 90.00%; alternate:
83.95%; combined: 99.23%
≥ 95%
BUSCO * C:98.1%[S:97.6%,D:0.6%],
F:0.3%,M:1.5%,n:5,286
S > 90%; D < 5%
Percentage of assembly mapped to
chromosomes
99.95% ≥ 90%
Sex chromosomes Z localised homologous pairs
Organelles Mitochondrial genome:
15.33 kb
complete single alleles
Genome annotation of assembly GCA_963662085.1 at Ensembl
Number of protein-coding genes 12,386
Number of non-coding genes 1,799
Number of gene transcripts 23,139

* BUSCO scores based on the lepidoptera_odb10 BUSCO set using version 5.5.0. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison.

The snail plot in Figure 2 provides a summary of the assembly statistics, indicating the distribution of scaffold lengths and other assembly metrics. Figure 3 shows the distribution of scaffolds by GC proportion and coverage. Figure 4 presents a cumulative assembly plot, with separate curves representing different scaffold subsets assigned to various phyla, illustrating the completeness of the assembly.

Figure 2. Genome assembly of Eupithecia inturbata, ilEupIntu1.1: metrics.

Figure 2.

The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the lepidoptera_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963662085.1/dataset/GCA_963662085.1/snail.

Figure 3. Genome assembly of Eupithecia inturbata, ilEupIntu1.1: BlobToolKit GC-coverage plot.

Figure 3.

Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963662085.1/blob.

Figure 4. Genome assembly of Eupithecia inturbata, ilEupIntu1.1: BlobToolKit cumulative sequence plot.

Figure 4.

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/GCA_963662085.1/dataset/GCA_963662085.1/cumulative.

Most of the assembly sequence (99.95%) was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 5; Table 3). The specimen is heterogametic, therefore female. During curation, chromosome Z was assigned based on read coverage statistics, but no W chromosome could be identified.

Figure 5. Genome assembly of Eupithecia inturbata: Hi-C contact map of the ilEupIntu1.1 assembly, visualised using HiGlass.

Figure 5.

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=FFENAPbrRG-iKuz10tLzmA.

Table 3. Chromosomal pseudomolecules in the genome assembly of Eupithecia inturbata, ilEupIntu1.

INSDC accession Name Length (Mb) GC%
OY759177.1 1 17.26 37.5
OY759178.1 2 17.15 37.5
OY759179.1 3 16.94 37.5
OY759180.1 4 16.73 37.5
OY759181.1 5 16.45 38
OY759182.1 6 15.94 37.5
OY759183.1 7 15.52 37.5
OY759184.1 8 15.39 37.5
OY759185.1 9 15.28 37.5
OY759186.1 10 15.27 37
OY759187.1 11 15.0 37
OY759188.1 12 14.99 37
OY759189.1 13 14.97 37.5
OY759190.1 14 14.95 37.5
OY759191.1 15 14.73 37.5
OY759192.1 16 14.53 38
OY759193.1 17 14.23 37.5
OY759194.1 18 14.21 37.5
OY759195.1 19 13.88 37.5
OY759196.1 20 13.42 37.5
OY759197.1 21 12.82 37.5
OY759198.1 22 11.42 37.5
OY759199.1 23 11.01 37.5
OY759200.1 24 10.96 37
OY759201.1 25 10.25 37.5
OY759202.1 26 9.89 37
OY759203.1 27 8.57 37.5
OY759204.1 28 8.51 38
OY759205.1 29 8.46 38.5
OY759206.1 30 8.01 37.5
OY759176.1 Z 20.78 37.5
OY759207.1 MT 0.02 20

The mitochondrial genome was also assembled. This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record in GenBank.

Assembly quality metrics

The estimated Quality Value (QV) and k-mer completeness metrics, along with BUSCO completeness scores, were calculated for each haplotype and the combined assembly. The QV reflects the base-level accuracy of the assembly, while k-mer completeness indicates the proportion of expected k-mers identified in the assembly. BUSCO scores provide a measure of completeness based on benchmarking universal single-copy orthologues.

The primary haplotype has a QV of 62.7, and the combined primary and alternate assemblies achieve an estimated QV of 62.2. The k-mer completeness for the primary haplotype is 90.00%, and for the alternate haplotype it is 83.95%. The combined primary and alternate assemblies achieve a k-mer completeness of 99.23%. BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5,286) gave a completeness score of 98.1% (single = 97.6%, duplicated = 0.6%).

Table 2 provides assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The assembly achieves the EBP reference standard of 6.C.Q62.

Genome annotation report

The Eupithecia inturbata genome assembly (GCA_963662085.1) was annotated at the European Bioinformatics Institute (EBI) on Ensembl Rapid Release. The resulting annotation includes 23,139 transcribed mRNAs from 12,386 protein-coding and 1,799 non-coding genes ( Table 2; https://rapid.ensembl.org/Eupithecia_inturbata_GCA_963662085.1/Info/Index). The average transcript length is 15,332.34. There are 1.63 coding transcripts per gene and 7.30 exons per transcript.

Methods

Sample acquisition and DNA barcoding

An adult male Eupithecia inturbata (specimen ID Ox003039, ToLID ilEupIntu1) was collected from Wytham Woods, Oxfordshire, United Kingdom (latitude 51.77, longitude –1.34) on 2022-07-22 by light trap. The specimen was collected by Finley Hutchinson and Liam Crowley (University of Oxford), identified by Finley Hutchinson and preserved on dry ice.

The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024). A small sample was dissected from the specimen and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI) ( Pereira et al., 2022). The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification ( Crowley et al., 2023). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al., 2024). The standard operating procedures for Darwin Tree of Life barcoding have been deposited on protocols.io ( Beasley et al., 2023).

Metadata collection for samples adhered to the Darwin Tree of Life project standards described by Lawniczak et al. (2022).

Nucleic acid extraction

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023b). The ilEupIntu1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).

HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). The DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Hi-C sample preparation

Tissue from the whole organism of the ilEupIntu1 sample was processed for Hi-C sequencing at the WSI Scientific Operations core, using the Arima-HiC v2 kit. In brief, 20–50 mg of frozen tissue (stored at –80 °C) was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde concentration. After crosslinking, the tissue was homogenised using the Diagnocine Power Masher-II and BioMasher-II tubes and pestles. Following the Arima-HiC v2 kit manufacturer's instructions, crosslinked DNA was digested using a restriction enzyme master mix. The 5’-overhangs were filled in and labelled with biotinylated nucleotides and proximally ligated. An overnight incubation was carried out for enzymes to digest remaining proteins and for crosslinks to reverse. A clean up was performed with SPRIselect beads prior to library preparation. Additionally, the biotinylation percentage was estimated using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and Qubit HS Assay Kit and Arima-HiC v2 QC beads.

Library preparation and sequencing

Library preparation and sequencing were performed at the WSI Scientific Operations core.

PacBio HiFi

At a minimum, samples were required to have an average fragment size exceeding 8 kb and a total mass over 400 ng to proceed to the low input SMRTbell Prep Kit 3.0 protocol (Pacific Biosciences, California, USA), depending on genome size and sequencing depth required. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA) as per the manufacturer's instructions. The kit includes the reagents required for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead cleanup, and nuclease treatment. Following the manufacturer’s instructions, size selection and clean up was carried out using diluted AMPure PB beads (Pacific Biosciences, California, USA). DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) with Qubit 1X dsDNA HS assay kit and the final library fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) and gDNA 55kb BAC analysis kit.

Samples were sequenced using the Sequel IIe system (Pacific Biosciences, California, USA). The concentration of the library loaded onto the Sequel IIe was in the range 40–135 pM. The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, as well as perform primary and secondary analysis of the data upon completion.

Samples were sequenced on a Revio instrument (Pacific Biosciences, California, USA). Prepared libraries were normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases were hybridised to create circularised complexes according to manufacturer’s instructions. The complexes were purified with the 1.2X clean up with SMRTbell beads. The purified complexes were then diluted to the Revio loading concentration (in the range 200–300 pM), and spiked with a Revio sequencing internal control. Samples were sequenced on Revio 25M SMRT cells (Pacific Biosciences, California, USA). The SMRT link software, a PacBio web-based end-to-end workflow manager, was used to set-up and monitor the run, as well as perform primary and secondary analysis of the data upon completion.

Hi-C

For Hi-C library preparation, DNA was fragmented using the Covaris E220 sonicator (Covaris) and size selected using SPRISelect beads to 400 to 600 bp. The DNA was then enriched using the Arima-HiC v2 kit Enrichment beads. Using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) for end repair, A-tailing, and adapter ligation. This uses a custom protocol which resembles the standard NEBNext Ultra II DNA Library Prep protocol but where library preparation occurs while DNA is bound to the Enrichment beads. For library amplification, 10 to 16 PCR cycles were required, determined by the sample biotinylation percentage. The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on an Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation

Assembly

Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.

The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019). The contigs were further scaffolded using the provided Hi-C data ( Rao et al., 2014) in YaHS ( Zhou et al., 2023) using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al., 2022), BUSCO ( Manni et al., 2021) and MERQURY.FK ( Rhie et al., 2020).

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation). Flat files and maps used in curation were generated in TreeVal ( Pointon et al., 2023). Manual curation was primarily conducted using PretextView ( Harry, 2022), with additional insights provided by JBrowse2 ( Diesh et al., 2023) and HiGlass ( Kerpedjiev et al., 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were corrected, and duplicate sequences were tagged and removed. Sex chromosomes were identified by read coverage statistics. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Assembly quality assessment

The Merqury.FK tool ( Rhie et al., 2020), run in a Singularity container ( Kurtzer et al., 2017), was used to evaluate k-mer completeness and assembly quality for the primary and alternate haplotypes using the k-mer databases ( k = 31) that were computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.

A Hi-C contact map was produced for the final version of the assembly. The Hi-C reads were aligned using bwa-mem2 ( Vasimuddin et al., 2019) and the alignment files were combined using SAMtools ( Danecek et al., 2021). The Hi-C alignments were converted into a contact map using BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020). The contact map is visualised in HiGlass ( Kerpedjiev et al., 2018).

The blobtoolkit pipeline is a Nextflow ( Di Tommaso et al., 2017) port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in 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 lineages, the pipeline aligns the BUSCO genes to the UniProt Reference Proteomes database ( Bateman et al., 2023) with DIAMOND blastp ( Buchfink et al., 2021). The genome is also divided into chunks according to the density of the BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database using DIAMOND blastx. Genome sequences without a hit are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The blobtools suite combines all these outputs into a blobdir for visualisation.

The blobtoolkit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), relying on the Conda package manager, the Bioconda initiative ( Grüning et al., 2018), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), as well as the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.

Table 4 contains a list of relevant software tool versions and sources.

Table 4. Software tools: versions and sources.

Software tool Version Source
BEDTools 2.30.0 https://github.com/arq5x/bedtools2
BLAST 2.14.0 ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/
BlobToolKit 4.3.9 https://github.com/blobtoolkit/blobtoolkit
BUSCO 5.5.0 https://gitlab.com/ezlab/busco
bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2
Cooler 0.8.11 https://github.com/open2c/cooler
DIAMOND 2.1.8 https://github.com/bbuchfink/diamond
fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows
FastK 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c https://github.com/thegenemyers/FASTK
Gfastats 1.3.6 https://github.com/vgl-hub/gfastats
GoaT CLI 0.2.5 https://github.com/genomehubs/goat-cli
Hifiasm 0.19.5-r587 https://github.com/chhylp123/hifiasm
HiGlass 44086069ee7d4d3f6f3f0012569789ec138f42b84
aa44357826c0b6753eb28de
https://github.com/higlass/higlass
MerquryFK d00d98157618f4e8d1a9190026b19b471055b22e https://github.com/thegenemyers/MERQURY.FK
Minimap2 2.24-r1122 https://github.com/lh3/minimap2
MitoHiFi 3 https://github.com/marcelauliano/MitoHiFi
MultiQC 1.14, 1.17, and 1.18 https://github.com/MultiQC/MultiQC
NCBI Datasets 15.12.0 https://github.com/ncbi/datasets
Nextflow 23.04.1 https://github.com/nextflow-io/nextflow
PretextView 0.2.5 https://github.com/sanger-tol/PretextView
purge_dups 1.2.5 https://github.com/dfguan/purge_dups
samtools 1.19.2 https://github.com/samtools/samtools
sanger-tol/ascc - https://github.com/sanger-tol/ascc
sanger-tol/blobtoolkit 0.5.1 https://github.com/sanger-tol/blobtoolkit
Seqtk 1.3 https://github.com/lh3/seqtk
Singularity 3.9.0 https://github.com/sylabs/singularity
TreeVal 1.2.0 https://github.com/sanger-tol/treeval
YaHS 1.2a.2 https://github.com/c-zhou/yahs

Genome annotation

The Ensembl Genebuild annotation system ( Aken et al., 2016) was used to generate annotation for the Eupithecia inturbata assembly (GCA_963662085.1) in Ensembl Rapid Release at the EBI. 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.

Funding Statement

This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540) and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].

[version 1; peer review: 2 approved, 2 approved with reservations]

Data availability

European Nucleotide Archive: Eupithecia inturbata (maple pug). Accession number PRJEB67611; https://identifiers.org/ena.embl/PRJEB67611. The genome sequence is released openly for reuse. The Eupithecia inturbata 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 and Table 2.

Author information

Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.12157525.

Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.12158331.

Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.12162482.

Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.12165051.

Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.12160324.

Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.12205391.

Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.

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Wellcome Open Res. 2025 Apr 25. doi: 10.21956/wellcomeopenres.26246.r121415

Reviewer response for version 1

Maríndia Deprá 1

The paper titled “The genome sequence of the Maple Pug moth, Eupithecia inturbata (Hübner, 1817)” is a Data Note presenting the nuclear and mitochondrial genome assemblies of Eupithecia inturbata. The nuclear genome spans 427.76 Mb and has been scaffolded into 31 chromosomal pseudomolecules, representing 30 autosomes and the Z sex chromosome. The mitochondrial genome is 15.33 Kb in length.

The methods employed follow standard practices for bioinformatics analyses, utilizing well-established pipelines such as Nextflow, BUSCO, and DIAMOND. The manuscript provides detailed descriptions of the software tools, databases, and parameters used, along with explanation of each step in the data processing workflow.

Although the manuscript does not include direct links to the datasets, the accession numbers are provided, ensuring data accessibility and enabling potential reuse.

Overall, the manuscript is well structured and technically sound. However, it would benefit from the inclusion of additional information about the species, such as aspects of its biology and habitat.

One methodological point that could be clarified is the identification of the Z chromosome: the manuscript states that this was based on read coverage statistics, but it is unclear which metrics were used. Wouldn’t homology-based identification be more appropriate? Furthermore, identification of the W chromosome would also be desirable.

Finally, I suggest revising the sections that refer to the sex of the sequenced specimen. In some parts of the manuscript (e.g., the abstract and page 3), it is described as female, while in others it is referred to as male.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

No

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

genetics and genomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Wellcome Open Res. 2025 Apr 11. doi: 10.21956/wellcomeopenres.26246.r121412

Reviewer response for version 1

Panagiotis Ioannidis 1

The present manuscript presents the genome assembly and initial annotation of a lepidopteran species.

I would like to see a more detailed overview of the biology of this insect in the Background section. Currently the authors only mention the site from where they collected the sample.

Other than this the manuscript is very well-written with many technical details explaining all the methodology. Moreover, the figure legends are among the best I've seen!

Like other genome reports in this journal, a gene set was generated with the Ensembl Rapid Release pipeline. My first comment on this is that it would be nice for the reader to have at least a few details on the methodology followed for gene prediction (e.g. was RNAseq used as evidence, which proteins were used as evidence, etc). I guess that a detailed report can be found at Ensembl's web site, and that's why I'm not asking for details; a few basic things would be enough. My second comment is one that I have mentioned in my other reviews for this journal, but I should mention it again here; it is almost obligatory (!) to have the BUSCO scores for the generated gene set and put them side-by-side with the BUSCO scores from the corresponding genome. This way the reader can see how good is the gene prediction pipeline (normally, the gene set score should be higher than the genome score).

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

bioinformatics, insect genomics, insect transcriptomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Wellcome Open Res. 2025 Apr 11. doi: 10.21956/wellcomeopenres.26246.r121416

Reviewer response for version 1

Annabel Whibley 1

This Data Note reports the genome assembly and Ensembl gene annotation of the Maple Pug moth Eupithecia inturbata. 

Please note that there is an error in the methods: the specimen is recorded as male in the “Sample acquisition” section. This should be corrected to avoid confusion.

The assembly is of high quality, with clear reporting of metadata and methods and use of appropriate bioinformatics tools. The accession links are functional. The report follows DToL templating with  clear presentation of relevant information. 

The apparent absence of the W chromosome from this female is interesting. The reduced dosage of the Z chromosome compared to autosomes is consistent with the female identification of the specimen. Atypical sex chromatin has been described in Geometrids, including in the family Larentiinae (e.g. Cells 2021, 10(9), 2230; https://doi.org/10.3390/cells10092230).  It would be interesting to explore this finding further (and to compare to closely related species).

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Genomics, Bioinformatics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Wellcome Open Res. 2025 Apr 5. doi: 10.21956/wellcomeopenres.26246.r120962

Reviewer response for version 1

Sivasankaran Kuppusamy 1

The authors have done chromosome-level genome assembly of the Maple Pug moth,  Eupithecia inturbata (Hubner, 1817).  The species' genome size was estimated at 427.76 Mb. They have extracted and sequenced DNA using the correct methodology. Furthermore, they assembled and annotated using the appropriate software. The comprehensive data will be necessary for the phylogenomic studies of the lepidopteran moths.  

Comments on the manuscript:

  • The first sentence of the Background can include the author’s name of the species. The sentence may change like this “The genome of the Maple Pug,  Eupithecia inturbata (Hubner, 1817)”….

  • The authors consistently included the complete name of the genus,  Eupithecia inturbata, throughout the article. If at all possible, start with the full genus name and then abbreviate it to  E. inturbata.

  • Authors have mentioned the sequence was done based on a female specimen in the Abstract, Background and Genome sequence report. But in the Methods authors have mentioned “An adult male  E. inturbata was collected from Wytham Woods”. I think there is an error. Authors can correct it. 

Above all, I confirm that the manuscript meets the necessary scientific standard and is suitable for indexing

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Phylogeny of Lepidoptera

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    European Nucleotide Archive: Eupithecia inturbata (maple pug). Accession number PRJEB67611; https://identifiers.org/ena.embl/PRJEB67611. The genome sequence is released openly for reuse. The Eupithecia inturbata 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 and Table 2.


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