The genome sequence of the Thicket Knot-horn, Acrobasis suavella (Zincken, 1818)

TL;DR

This paper presents the genome sequence of the Thicket Knot-horn moth, including chromosomal scaffolding and gene annotations.

Contribution

The study provides a high-quality genome assembly and annotation for Acrobasis suavella, including chromosomal pseudomolecules and mitochondrial DNA.

Findings

The genome assembly spans 647.3 megabases and includes 30 chromosomal pseudomolecules.

The mitochondrial genome is 15.31 kilobases long and fully assembled.

Gene annotation identified 19,101 protein-coding genes using Ensembl.

Abstract

We present a genome assembly from an individual male
Acrobasis suavella (the Thicket Knot-horn; Arthropoda; Insecta; Lepidoptera; Pyralidae). The genome sequence is 647.3 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.31 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,101 protein coding genes.

Figures

Photograph of the Acrobasis suavella (ilAcrSuav1) specimen used for genome sequencing.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: 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 647,282,432 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 (51,000,710 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (23,584,496 and 14,517,006 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/ilAcrSuav1.1/dataset/CALPDP01.1/snail.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: 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/ilAcrSuav1.1/dataset/CALPDP01.1/blob.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: 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/ilAcrSuav1.1/dataset/CALPDP01.1/cumulative.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: Hi-C contact map of the ilAcrSuav1.1 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=W69aQoBuSxGnPnFq02aE5Q.

Tables

Table 1.. Genome data for Acrobasis suavella, ilAcrSuav1.1.

Project accession data
Assembly identifier	ilAcrSuav1.1
Species	Acrobasis suavella
Specimen	ilAcrSuav1
NCBI taxonomy ID	1857951
BioProject	PRJEB52024
BioSample ID	SAMEA10979088
Isolate information	ilAcrSuav1, male: whole organism (DNA sequencing and HiC scaffolding) ilAcrSuav3: whole organism (RNA sequencing)
Assembly metrics *		Benchmark
Consensus quality (QV)	63.6	≥ 50
k-mer completeness	100%	≥ 95%
BUSCO **	C:98.8%[S:98.4%,D:0.4%], F:0.5%,M:0.7%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.99%	≥ 95%
Sex chromosomes	Z chromosome	localised homologous pairs
Organelles	Mitochondrial genome assembled	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR9745002
Hi-C Illumina	ERR9503461
PolyA RNA-Seq Illumina	ERR10123692
Genome assembly
Assembly accession	GCA_943193695.1
Accession of alternate haplotype	GCA_943193685.1
Span (Mb)	647.3
Number of contigs	54
Contig N50 length (Mb)	22.0
Number of scaffolds	31
Scaffold N50 length (Mb)	23.6
Longest scaffold (Mb)	51.0
Genome annotation
Number of protein-coding genes	19,101
Number of gene transcripts	19,275

Table 2.. Chromosomal pseudomolecules in the genome assembly of Acrobasis suavella, ilAcrSuav1.

INSDC accession	Chromosome	Length (Mb)	GC%
OW971929.1	1	28.4	36.5
OW971930.1	2	28.2	36.5
OW971931.1	3	28.13	36.5
OW971932.1	4	26.65	36.5
OW971933.1	5	26.64	36.5
OW971934.1	6	25.35	36.0
OW971935.1	7	24.43	36.5
OW971936.1	8	23.91	36.5
OW971937.1	9	23.71	36.5
OW971938.1	10	23.6	36.0
OW971939.1	11	23.58	36.5
OW971940.1	12	23.58	36.0
OW971941.1	13	23.45	36.5
OW971942.1	14	22.38	36.5
OW971943.1	15	21.95	36.0
OW971944.1	16	21.48	36.5
OW971945.1	17	21.05	36.5
OW971946.1	18	20.46	36.5
OW971947.1	19	20.43	37.0
OW971948.1	20	17.55	36.5
OW971949.1	21	16.84	36.5
OW971950.1	22	15.74	36.5
OW971951.1	23	14.92	37.0
OW971952.1	24	14.52	36.5
OW971953.1	25	13.66	37.0
OW971954.1	26	13.43	38.5
OW971955.1	27	11.18	37.0
OW971956.1	28	10.95	37.5
OW971957.1	29	10.06	37.0
OW971928.1	Z	51.0	36.5
OW971958.1	MT	0.02	19.5

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.1.3	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; Pyraloidea; Pyralidae; Phycitinae; Acrobasis; Acrobasis suavella (Zincken, 1818) (NCBI:txid1857951).

Background

Acrobasis suavella (Zincken, 1818) is a moth of the Pyralidae family. The adult moths of this species are marked with a mixture of ruddy purple and grey on the forewings, and in some specimens the intensity of these markings can create a handsome burgundy and silver appearance to the moth. The adults of this species are on the wing in Britain and Ireland between June and August, flying at night. The adult moth is seldom seen by day but comes readily to light ( Goater et al., 1986; Parsons & Davis, 2018)

The most frequently recorded larval foodplant for the species in Britain and Ireland is Prunus spinosa, but larvae have been found on Cotoneaster, Crataegus, and Sorbus ( Parsons & Davis, 2018). The species reportedly prefers stunted and isolated P. spinosa plants, and open habitats such as downland where such plants occur ( Goater et al., 1986; Parsons & Davis, 2018). The larva feeds from September to June within a thick silken tube coated with leaf fragments and larval frass, and pupation occurs within, or adjacent to, the larval gallery ( Parsons & Davis, 2018).

In Britain, the moth is most widespread across southern England and Wales, but there is also a record from Shetland ( Langmaid & Young, 2004), possibly indicating vagrancy. Globally the species is found across Europe east to the Caucasus ( Streltzov et al., 2022), and appears to have become established in North America, around Vancouver, British Columbia, since at least the early 20th century, feeding on Cotoneaster ( Heinrich, 1939; Neunzig, 1990). It is therefore possible the species may expand its range in the future via the ornamental plants trade.

The genome of Acrobasis suavella 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 Acrobasis suavella, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report

The genome was sequenced from one male Acrobasis suavella ( 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 was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 7 missing joins or mis-joins and removed one haplotypic duplication, reducing the assembly length by 0.13%% and the scaffold number by 11.43%.

Photograph of the Acrobasis suavella (ilAcrSuav1) specimen used for genome sequencing.

The final assembly has a total length of 647.3 Mb in 31 sequence scaffolds with a scaffold N50 of 23.6 Mb ( Table 1). Most (99.99%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 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 Acrobasis suavella, ilAcrSuav1.1.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: 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 647,282,432 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 (51,000,710 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (23,584,496 and 14,517,006 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/ilAcrSuav1.1/dataset/CALPDP01.1/snail.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: 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/ilAcrSuav1.1/dataset/CALPDP01.1/blob.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: 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/ilAcrSuav1.1/dataset/CALPDP01.1/cumulative.

Genome assembly of Acrobasis suavella, ilAcrSuav1.1: Hi-C contact map of the ilAcrSuav1.1 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=W69aQoBuSxGnPnFq02aE5Q.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Acrobasis suavella, ilAcrSuav1.

The estimated Quality Value (QV) of the final assembly is 63.6 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.8% (single = 98.4%, duplicated = 0.4%), using the lepidoptera_odb10reference 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/1857951.

Genome annotation report

The Acrobasis suavella genome assembly (GCA_943193695.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Acrobasis_suavella_GCA_943193695.1/Info/Index). The resulting annotation includes 19,275 transcribed mRNAs from 19,101 protein-coding genes.

Methods

Sample acquisition and nucleic acid extraction

Two Acrobasis suavella specimens (ilAcrSuav1 and ilAcrSuav3) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.34) on 2021-07-24. The specimens were taken from a grassland habitat using a light trap. The specimens were collected and identified by Douglas Boyes (University of Oxford) and were snap-frozen on dry ice.

DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilAcrSuav1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Whole organism 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. 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 ilAcrSuav3 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 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) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from tissue of ilAcrSuav1 that had been set aside, 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 scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination 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., 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 Acrobasis suavella assembly (GCA_943193695.1) 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 materialLegality 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.