The genome sequence of the Beautiful Hook-tip, Laspeyria flexula (Denis & Schiffermüller, 1775)
Douglas Boyes, Mark L Blaxter, Jose Martinez, Michael Hiller, Rute R da Fonseca
TL;DR
This paper presents the genome sequence of the Beautiful Hook-tip moth, including its chromosomal structure and gene annotations.
Contribution
The study provides a high-quality genome assembly and gene annotations for Laspeyria flexula, including the Z sex chromosome and mitochondrial genome.
Findings
The genome assembly spans 450.9 megabases and includes 31 chromosomal pseudomolecules.
Gene annotation identified 13,281 protein-coding genes using Ensembl.
The mitochondrial genome is 15.58 kilobases in length.
Abstract
We present a genome assembly from an individual male Laspeyria flexula (the Beautiful Hook-tip; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 450.9 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 15.58 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,281 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
Figure 2
Figure 3
Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | ilLasFlex1.1 | |
| Species |
| |
| Specimen | ilLasFlex1 | |
| NCBI taxonomy ID | 938238 | |
| BioProject | PRJEB42131 | |
| BioSample ID | SAMEA7519836 | |
| Isolate information | ilLasFlex1, male: whole organism (DNA sequencing and Hi-C
| |
| Assembly metrics
|
| |
| Consensus quality (QV) | 58.3 |
|
|
| 99.9% |
|
| BUSCO
| C:98.7%[S:98.3%,D:0.4%],
|
|
| Percentage of assembly mapped
| 99.94% |
|
| Sex chromosomes | Z chromosome |
|
| Organelles | Mitochondrial genome assembled |
|
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR6565937 | |
| 10X Genomics Illumina | ERR6003042, ERR6002654, ERR6002655, ERR6002656 | |
| Hi-C Illumina | ERR6002657, ERR6002658, ERR6002659 | |
| PolyA RNA-Seq Illumina | ERR6363259, ERR6787416 | |
| Genome assembly | ||
| Assembly accession | GCA_905147015.1 | |
|
| GCA_905147035.1 | |
| Span (Mb) | 450.9 | |
| Number of contigs | 57 | |
| Contig N50 length (Mb) | 12.2 | |
| Number of scaffolds | 37 | |
| Scaffold N50 length (Mb) | 16.0 | |
| Longest scaffold (Mb) | 24.6 | |
| Genome annotation | ||
| Number of protein-coding genes | 13,281 | |
| Number of non-coding genes | 1,859 | |
| Number of gene transcripts | 23,324 | |
| INSDC
| Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 19.38 | 35.0 | |
| 2 | 18.39 | 35.0 | |
| 3 | 18.1 | 35.0 | |
| 4 | 17.93 | 34.5 | |
| 5 | 17.83 | 35.0 | |
| 6 | 17.36 | 35.0 | |
| 7 | 17.36 | 35.0 | |
| 8 | 16.96 | 35.0 | |
| 9 | 16.93 | 34.5 | |
| 10 | 16.77 | 34.5 | |
| 11 | 16.7 | 34.5 | |
| 12 | 16.0 | 35.0 | |
| 13 | 15.74 | 35.0 | |
| 14 | 15.41 | 34.5 | |
| 15 | 15.22 | 35.0 | |
| 16 | 15.05 | 34.5 | |
| 17 | 14.97 | 35.0 | |
| 18 | 14.12 | 35.5 | |
| 19 | 13.63 | 35.5 | |
| 20 | 13.52 | 35.5 | |
| 21 | 13.45 | 35.0 | |
| 22 | 11.55 | 35.5 | |
| 23 | 11.33 | 35.0 | |
| 24 | 10.96 | 35.5 | |
| 25 | 10.81 | 35.5 | |
| 26 | 9.87 | 35.5 | |
| 27 | 8.37 | 37.0 | |
| 28 | 8.28 | 36.0 | |
| 29 | 7.87 | 36.5 | |
| 30 | 6.15 | 36.5 | |
| Z | 24.6 | 35.0 | |
| MT | 0.02 | 20.0 |
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.0.7 |
|
| 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/
| v1.0 |
|
| sanger-tol/
| 1.1.0 |
|
- —Wellcome Trust
- —Wellcome Trust
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Taxonomy
TopicsLepidoptera: Biology and Taxonomy · Plant and animal studies · Insect and Arachnid Ecology and Behavior
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; Erebidae; Erebinae; Laspeyria; Laspeyria flexula (Denis & Schiffermüller, 1775) (NCBI:txid938238).
Background
The Darwin Tree of Life project, as part of our goals of sequencing all eukaryotic species in Ireland and Britain ( Blaxter et al., 2022), is generating high quality reference genomes for many lepidoptera. Here we present a chromosomally-complete genome sequence for the Beautiful Hook-tip Laspeyria flexula ( [Denis & Schiffermüller], 1775), a member of subfamily Boletobiinae within the noctuid family Erebidae (see NBN Atlas Partnership, 2023). Erebid moth species form approximately 4% of the Lepidoptera fauna of Britain and Ireland, including representatives of 63 genera (UK Species Inventory; Natural History Museum, 2023). Erebid moths are important herbivores, including invasive pests of native and agricultural ecosystems, but some species also act as sentinels for the impacts of anthropogenic impacts such as pesticide use and pollution and of the impacts of climate change ( Fox, 2013).
While larvae of most moths feed on vascular plant material, some, including L. flexula, feed on lichens. The well-camouflaged caterpillars of L. flexula feed on foliose and crustose lichens that grow on the bark of both deciduous and coniferous trees. Lichenivorous moths are threatened not only through generalised habitat loss but also especially through the impacts of atmospheric pollution on their food sources. It is well recognised that atmospheric pollution, most notably SO 2, resulting from the burning of fossil fuels, led to massive declines in many lichen species in Ireland and Britain, and more widely. Following acceptance of emission controls many lichens have rebounded, and lichenivorous species such as L. flexula are also showing recovery (albeit against a general background of declines in moth abundances ( Fox et al., 2013), especially in areas that had, historically, the highest atmospheric SO 2 levels ( Pescott et al., 2015). We hope that this genome sequence will assist in analysis of the population history and recovery of L. flexula, and of its adaptations to lichenivory.
Genome sequence report
The genome was sequenced from one male Laspeyria flexula ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.34). A total of 48-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 84-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 13 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the assembly length by 0.23% and the scaffold number by 9.76%.
Photograph of the Laspeyria flexula (ilLasFlex1) specimen used for genome sequencing
The final assembly has a total length of 450.9 Mb in 37 sequence scaffolds with a scaffold N50 of 16.0 Mb ( Table 1). Most (99.94%) 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 Laspeyria flexula, ilLasFlex1.1.
Genome assembly of Laspeyria flexula, ilLasFlex1.1: 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 450,897,266 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 (24,595,128 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (16,004,308 and 10,809,102 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/Laspeyria%20flexula/dataset/CAJHUK01.1/snail.
Genome assembly of Laspeyria flexula, ilLasFlex1.1: 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/Laspeyria%20flexula/dataset/CAJHUK01.1/blob.
Genome assembly of Laspeyria flexula, ilLasFlex1.1: 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/Laspeyria%20flexula/dataset/CAJHUK01.1/cumulative.
Genome assembly of Laspeyria flexula, ilLasFlex1.1: Hi-C contact map of the ilLasFlex1.1 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=czjwYVChQ92r5isvbpie4g.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Laspeyria flexula, ilLasFlex1.
The estimated Quality Value (QV) of the final assembly is 58.3 with k-mer completeness of 99.9%, and the assembly has a BUSCO v5.3.2 completeness of 98.7% (single = 98.3%, 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/938238.
Genome annotation report
The Laspeyria flexula genome assembly (GCA_905147015.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Laspeyria_flexula_GCA_905147015.1/Info/Index). The resulting annotation includes 23,324 transcribed mRNAs from 13,281 protein-coding and 1,859 non-coding genes.
Methods
Sample acquisition and nucleic acid extraction
A male Laspeyria flexula (specimen ID Ox000046, individual ilLasFlex1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.34) on 2019-06-29 using a light trap. A second specimen (ilLasFlex2, ToLID ilLasFlex2), collected from the same location on 2020-06-13, was used for RNA sequencing. Both specimens were 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 ilLasFlex1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Tissue from the whole organism was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. 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 abdomen tissue of ilLasFlex2 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.
Protocols developed by 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 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 (RNA-Seq) and HiSeq X Ten (10X) instruments. Hi-C data were also generated from remaining tissue of ilLasFlex1 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 Laspeyria flexula assembly (GCA_905147015.1). 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.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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