The genome sequence of a ground beetle, Ophonus ardosiacus (Lutshnik, 1922)

TL;DR

This paper presents the genome sequence of a ground beetle species, including chromosomal scaffolds and gene annotations.

Contribution

The study provides a high-quality genome assembly and gene annotation for the ground beetle Ophonus ardosiacus.

Findings

The genome assembly is 911.9 megabases long and includes 19 chromosomal pseudomolecules.

The mitochondrial genome is 16.61 kilobases in length and has been assembled.

Gene annotation identified 40,995 protein coding genes using Ensembl.

Abstract

We present a genome assembly from an individual female
Ophonus ardosiacus (a ground beetle; Arthropoda; Insecta; Coleoptera; Carabidae). The genome sequence is 911.9 megabases in span. Most of the assembly is scaffolded into 19 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.61 kilobases in length. Gene annotation of this assembly on Ensembl identified 40,995 protein coding genes.

Figures

Photograph of the Ophonus ardosiacus (icOphArdo1) specimen used for genome sequencing.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: 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 911,917,119 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 (89,420,107 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (52,059,326 and 31,939,453 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 endopterygota_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/icOphArdo1.1/dataset/CALPBS01/snail.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: 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/icOphArdo1.1/dataset/CALPBS01/blob.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: 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/icOphArdo1.1/dataset/CALPBS01/cumulative.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: Hi-C contact map of the icOphArdo1.1 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=HVSPI2KAS8WA2aeR28Wf2w.

Tables

Table 1.. Genome data for Ophonus ardosiacus, icOphArdo1.1.

Project accession data
Assembly identifier	icOphArdo1.1
Species	Ophonus ardosiacus
Specimen	icOphArdo1
NCBI taxonomy ID	247415
BioProject	PRJEB52573
BioSample ID	SAMEA7746467
Isolate information	icOphArdo1, female: abdomen (DNA sequencing), head and thorax (Hi-C scaffolding)
Assembly metrics *		Benchmark
Consensus quality (QV)	58.8	≥ 50
k-mer completeness	100%	≥ 95%
BUSCO **	C:98.1%[S:96.8%,D:1.2%], F:0.4%,M:1.5%,n:2,124	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.69%	≥ 95%
Sex chromosomes	X chromosome	localised homologous pairs
Organelles	Mitochondrial genome assembled	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR9709324, ERR9709325
10X Genomics Illumina	ERR9682486–ERR9682489
Hi-C Illumina	ERR9682485
Genome assembly
Assembly accession	GCA_943142095.1
Accession of alternate haplotype	GCA_943138245.1
Span (Mb)	911.9
Number of contigs	302
Contig N50 length (Mb)	8.3
Number of scaffolds	73
Scaffold N50 length (Mb)	52.1
Longest scaffold (Mb)	89.4
Genome annotation
Number of protein-coding genes	40,995
Number of gene transcripts	41,360

Table 2.. Chromosomal pseudomolecules in the genome assembly of Ophonus ardosiacus, icOphArdo1.

INSDC accession	Chromosome	Length (Mb)	GC%
OW964823.1	1	89.42	30.5
OW964824.1	2	64.89	30.5
OW964825.1	3	64.2	30.5
OW964826.1	4	57.89	31.0
OW964827.1	5	57.09	30.5
OW964828.1	6	56.14	30.5
OW964829.1	7	52.06	31.0
OW964830.1	8	52.11	30.5
OW964831.1	9	51.22	31.0
OW964832.1	10	50.42	30.5
OW964834.1	11	45.49	31.0
OW964835.1	12	43.56	31.0
OW964836.1	13	40.65	31.0
OW964837.1	14	39.02	31.0
OW964838.1	15	31.94	31.0
OW964839.1	16	28.01	31.0
OW964840.1	17	20.71	30.5
OW964841.1	18	15.72	31.0
OW964833.1	X	47.7	30.5
OW964842.1	MT	0.02	19.0

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.1.5	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; Coleoptera; Adephaga; Caraboidea; Carabidae; Harpalinae; Harpalini; Ophonus; Ophonus ardosiacus (Lutshnik, 1922) (NCBI:txid247415).

Background

Ophonus ardosiacus is a ground beetle of the family Carabidae, order Coleoptera. Adults grow to 14 mm in length, and the grooved elytra vary in colour from dark brown to black, with a metallic blue sheen, and reddish-brown appendages ( UK Beetles, no date).

Ophonus ardosiacus has a wide Palearctic distribution, and is common across central and southern Europe, as far east as Czechia, and also in northern Africa ( GBIF Secretariat, 2022), with rare records in Czechia and the Netherlands. It is regarded as nationally scarce (category B) in Britain ( Hyman, 1992; Telfer, 2016). However, O. ardosiacus has become more common here over the past few decades, occurring more widely inland. This shift may be a response to climate change, or to an increase in favourable habitats ( Harvey, 2004). It occurs from the midlands of England to the south ( Giglio et al., 2008).

The typical habitats of O. ardosiacus include open grassland and some agricultural or coastal areas in areas with chalky, limestone or clay soils ( Harvey, 2004). These ground beetles consume the seeds of many annual plants, especially carrot seed heads. Both the larvae and adults are phytophagous ( UK Beetles, no date).

The genome of O. ardosiacus has now been 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 O. ardosiacus, based on one female specimen from Wytham Woods, Oxfordshire. The genome sequence will prove useful in studies assessing invertebrate communities for agricultural areas as part of integrated pest management.

Genome sequence report

The genome was sequenced from one female Ophonus ardosiacus ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, –1.33). A total of 25-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 13-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 94 missing joins or mis-joins and removed 20 haplotypic duplications, reducing the assembly length by 0.95%, and the scaffold number by 45.52%, and increasing the scaffold N50 by 96.53%.

Photograph of the Ophonus ardosiacus (icOphArdo1) specimen used for genome sequencing.

The final assembly has a total length of 911.9 Mb in 73 sequence scaffolds with a scaffold N50 of 52.1 Mb ( Table 1). Most (99.69%) of the assembly sequence was assigned to 19 chromosomal-level scaffolds, representing 18 autosomes and the X 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 Ophonus ardosiacus, icOphArdo1.1.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: 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 911,917,119 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 (89,420,107 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (52,059,326 and 31,939,453 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 endopterygota_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/icOphArdo1.1/dataset/CALPBS01/snail.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: 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/icOphArdo1.1/dataset/CALPBS01/blob.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: 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/icOphArdo1.1/dataset/CALPBS01/cumulative.

Genome assembly of Ophonus ardosiacus, icOphArdo1.1: Hi-C contact map of the icOphArdo1.1 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=HVSPI2KAS8WA2aeR28Wf2w.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Ophonus ardosiacus, icOphArdo1.

The estimated Quality Value (QV) of the final assembly is 58.8 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.1% (single =96.8%, duplicated = 1.2%), using the endopterygota_odb10 reference set ( n = 2,124).

Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/247415.

Genome annotation report

The Ophonus ardosiacus genome assembly (GCA_943142095.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Ophonus_ardosiacus_GCA_943142095.1/Info/Index). The resulting annotation includes 41,360 transcribed mRNAs from 40,995 protein-coding genes.

Methods

Sample acquisition and nucleic acid extraction

The specimen selected for genome sequencing was a female Ophonus ardosiacus (specimen ID Ox000754, individual icOphArdo1) collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude –1.33) on 2020-08-04 by potting. Liam Crowley (University of Oxford) collected and identified the specimen. The specimen was snap-frozen on dry ice.

DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The icOphArdo1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Abdomen tissue 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.

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 icOphArdo1 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 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., 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 Ophonus ardosiacus assembly (GCA_943142095.1) 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.