The genome sequence of the Ruddy Darter, Sympetrum sanguineum (Müller, 1764)
Liam M. Crowley, Denise C. Wawman, Cinta Pegueroles, Ainhoa López, Andrea Galimberti, Jason Charamis
TL;DR
This paper presents the genome sequence of the Ruddy Darter, a dragonfly species, with detailed haplotype-resolved assemblies.
Contribution
The study provides a high-quality, haplotype-resolved genome assembly for Sympetrum sanguineum.
Findings
The genome assembly includes two haplotypes with total lengths of 1,500.53 and 1,304.05 megabases.
Haplotype 1 is scaffolded into 13 chromosomal pseudomolecules, including the X sex chromosome.
Haplotype 2 is scaffolded into 12 autosomes.
Abstract
We present a genome assembly from a male specimen of Sympetrum sanguineum (Ruddy Darter; Arthropoda; Insecta; Odonata; Libellulidae). The haplotype-resolved assembly contains two haplotypes with total lengths of 1,500.53 megabases and 1,304.05 megabases. Most of haplotype 1 is scaffolded into 13 chromosomal pseudomolecules, including the X sex chromosome, while haplotype 2 is scaffolded into 12 autosomes.
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
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Figure 4
Figure 5| Project information | |||
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| Sympetrum sanguineum (ruddy darter) | ||
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| PRJEB60309 | ||
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| SAMEA110451513 | ||
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| 197176 | ||
| Specimen information | |||
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| ioSymSang1 | SAMEA110451696 | thorax |
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| ioSymSang1 | SAMEA110451696 | thorax |
| Sequencing information | |||
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| ERR12737259 | 1.58e+09 | 238.79 |
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| ERR10968291 | 7.23e+08 | 109.2 |
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| ERR10962207 | 5.90e+05 | 7.63 |
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| ERR10962206 | 2.11e+06 | 25.47 |
| Genome assembly | Haplotype 1 | Haplotype 2 |
|---|---|---|
| Assembly name | ioSymSang1.hap1.1 | ioSymSang1.hap2.1 |
| Assembly accession | GCA_964198025.1 | GCA_964198035.1 |
| Assembly level | chromosome | chromosome |
| Span (Mb) | 1,500.53 | 1,304.05 |
| Number of contigs | 2,015 | 1,242 |
| Number of scaffolds | 1,387 | 672 |
| Longest scaffold (Mb) | 135.43 | 144.71 |
| Assembly metrics (benchmark)
| Haplotype 1 | Haplotype 2 |
| Contig N50 length
| 2.52 Mb | 2.62 Mb |
| Scaffold N50 length
| 96.0 Mb | 96.02 Mb |
| Consensus quality (QV) (≥ 40) | 62.7 | 63.5 |
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| 80.59% (combined 98.66%) | 75.16% |
| BUSCO
| C:95.9%[S:94.3%,D:1.6%],
| C:91.6%[S:90.3%,D:1.3%],
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| Percentage of assembly mapped
| 82.06% | 86.66% |
| Sex chromosomes (localised
| X | - |
| Organelles
| Mitochondrial genome: 17.27 kb | |
| Haplotype 1 | Haplotype 2 | ||||||
|---|---|---|---|---|---|---|---|
| INSDC accession | Name | Length (Mb) | GC% | INSDC accession | Name | Length (Mb) | GC% |
| 1 | 135.43 | 35.5 | 1 | 144.71 | 35.5 | ||
| 2 | 128.59 | 36 | 2 | 129.28 | 36 | ||
| 3 | 108.6 | 35.5 | 3 | 104.81 | 35.5 | ||
| 4 | 108.06 | 35.5 | 4 | 105.49 | 35.5 | ||
| 5 | 101.93 | 35.5 | 5 | 104.18 | 35.5 | ||
| 6 | 99.38 | 35.5 | 6 | 96.02 | 35.5 | ||
| 7 | 96.0 | 35 | 7 | 85.92 | 35.5 | ||
| 8 | 92.25 | 35.5 | 8 | 93.44 | 35.5 | ||
| 9 | 90.99 | 35.5 | 9 | 90.72 | 35.5 | ||
| 10 | 79.96 | 35 | 10 | 80.82 | 35 | ||
| 11 | 79.07 | 35.5 | 11 | 74.63 | 35 | ||
| 12 | 20.64 | 35 | 12 | 20.09 | 35 | ||
| X | 90.43 | 35.5 | |||||
| MT | 0.02 | 26.5 | |||||
| Software tool | Version | Source |
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| BEDTools | 2.30.0 |
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| BLAST | 2.14.0 |
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| BlobToolKit | 4.3.7 |
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| BUSCO | 5.4.3 and 5.5.0 |
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| bwa-mem2 | 2.2.1 |
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| Cooler | 0.8.11 |
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| DIAMOND | 2.1.8 |
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| fasta_windows | 0.2.4 |
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| FastK | 427104ea91c78c3b8b8b49f1a7d6bbeaa869ba1c |
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| Gfastats | 1.3.6 |
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| GoaT CLI | 0.2.5 |
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| Hifiasm | 0.19.8-r603 |
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| HiGlass | 44086069ee7d4d3f6f3f0012569789ec138f42b84
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| Merqury.FK | d00d98157618f4e8d1a9190026b19b471055b22e |
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| MitoHiFi | 3 |
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| MultiQC | 1.14, 1.17, and 1.18 |
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| NCBI Datasets | 15.12.0 |
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| Nextflow | 23.04.0-5857 |
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| PretextView | 0.2 |
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| purge_dups | 1.2.5 |
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| samtools | 1.16.1, 1.17, and 1.18 |
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| sanger-tol/ascc | - |
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| sanger-tol/genomenote | 1.1.1 |
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| sanger-tol/readmapping | 1.2.1 |
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| Seqtk | 1.3 |
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| Singularity | 3.9.0 |
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| TreeVal | 1.0.0 |
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| YaHS | 1.2a.2 |
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- —Wellcome Trust
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Taxonomy
TopicsGenomics and Phylogenetic Studies · Environmental DNA in Biodiversity Studies · Genetic diversity and population structure
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Palaeoptera; Odonata; Epiprocta; Anisoptera; Cavilabiata; Libellulidae; Sympetrum; Sympetrum sanguineum (Müller, 1764) (NCBI:txid)
Background
The Ruddy Darter Sympetrum sanguineum is a small dragonfly in the family Libellulidae. It is 20–26 mm long with a hind wing measuring 23–29 mm. Mature males have a blood-red abdomen with darker sides and females are yellowish but may turn red as they age. Both sexes have a black line running down their sides from the frons to the hind part of the abdomen. Unlike other darters, they have entirely black legs, and unlike the Common Darter Sympetrum striolatum which has a straight abdomen, S. sanguineum has a waisted appearance ( Brooks, 2007).
Sympetrum sanguineum is most common around shallow lakes, ponds, ditches and canals, that are well vegetated. Although it is found throughout Ireland, in the United Kingdom it is mostly found in the south of England, with migrants from Continental Europe boosting the population. It flies from late June to October. The larvae feed on the roots of plants, including bulrushes Typha spp. and horsetails Equisetum spp., usually completing their lifecycle in one year ( Brooks, 2007).
The flight of S. sanguineum has been investigated extensively ( Wakeling & Ellington, 1997a; Wakeling & Ellington, 1997b; Wakeling & Ellington, 1997c), and some of the information gained has been used to help with the development of types of miniature flying robots known as flapping-wing micro aerial vehicles ( Szabo & D’Eleuterio, 2018).
We present a chromosomally complete genome sequence for Sympetrum sanguineum based on one adult male specimen collected at Wytham Woods, Oxfordshire, UK as part of the Darwin Tree of Life Project. This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland ( Blaxter et al., 2022).
Genome sequence report
The genome of an adult male Sympetrum sanguineum ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 25.47 Gb (gigabases) from 2.11 million reads, providing approximately 23-fold coverage. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 109.20 Gb from 723.20 million reads. Specimen and sequencing details are provided in Table 1.
Photograph of the Sympetrum sanguineum (ioSymSang1) specimen used for genome sequencing.
Table 1.: Specimen and sequencing data for Sympetrum sanguineum.
Haplotype 1 and haplotype 2 were combined for manual curation. Assembly errors, including 716 missing joins or mis-joins and 5 haplotypic duplications were corrected during curation. The result was to increase the assembly length by 1.68% and the scaffold number by 0.9%, and to increase the scaffold N50 by 2.01%.
The final haplotype 1 assembly has a total length of 1,500.53 Mb in 1,387 sequence scaffolds, with 628 gaps, and a scaffold N50 of 96.0 Mb ( Table 2). The snail plot in Figure 2 provides a summary of the assembly statistics for haplotype 1, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.
Table 2.: Genome assembly data for Sympetrum sanguineum, both haplotypes.
Genome assembly of Sympetrum sanguineum, ioSymSang1.hap1.1: metrics.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 insecta_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Sympetrum_sanguineum/dataset/GCA_964198025.1/snail.
Genome assembly of Sympetrum sanguineum, ioSymSang1.hap1.1: BlobToolKit GC-coverage plot.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/Sympetrum_sanguineum/dataset/GCA_964198025.1/blob.
Genome assembly of Sympetrum sanguineum ioSymSang1.hap1.1: 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/Sympetrum_sanguineum/dataset/GCA_964198025.1/cumulative.
For haplotype 1, most (83.39%) of the assembly sequence was assigned to 13 chromosomal-level scaffolds, representing 12 autosomes and the X sex chromosome. The specimen is an XO male. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 3). During manual curation, haplotypic inversions were identified on Chromosome 2 from 113–128.1 Mb, Chromosome 3 from 95–108.5 Mb, Chromosome 6 from 55.7–63 Mb, Chromosome 8 from 9.8–60.7 Mb and Chromosome 11 from 16.60–38.26 Mb. The order and orientation may not be certain for highly repetitive regions identified on Chromosome 1 from 0 to 14 Mb and Chromosome 7 from 86.5 to 95.9 Mb. The mitochondrial genome was also assembled and is included both as a contig within the multifasta file of the genome submission and as a standalone record in GenBank.
Genome assembly of Sympetrum sanguineum ioSymSang1.hap1.1: Hi-C contact map of the ioSymSang1.hap1.1 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=KYHOGRoOSn2EckXqEOT1-Q.
Table 3.: Chromosomal pseudomolecules in the genome assembly of Sympetrum sanguineum, ioSymSang1.
The estimated Quality Value (QV) of the haplotype 1 assembly is 62.7 with k-mer completeness of 80.59%, and the assembly has a BUSCO v5.4.3 completeness of 95.9% (single = 94.3%, duplicated = 1.6%), using the insecta_odb10 reference set ( n = 1,367).
For haplotype 2, the estimated Quality Value (QV) of the final assembly is 63.5 with k-mer completeness of 75.16%. The assembly has a BUSCO v5.4.3 completeness of 91.6% (single = 90.3%, duplicated = 1.3%), using the insecta_odb10 reference set ( n = 1,367). The k-mer completeness of the combined assemblies is 98.66%.
Metadata for specimens, BOLD barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/197176.
Methods
Sample acquisition and DNA barcoding
An adult male Sympetrum sanguineum (specimen ID Ox001974, ToLID ioSymSang1) was collected from Wytham Woods, Berkshire, United Kingdom (latitude 51.76, longitude –1.34) on 2021-10-10 by netting. The specimen was collected and identified by Liam Crowley (University of Oxford) 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 specimens and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI). 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).
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 core procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io ( Denton et al., 2023b). The ioSymSang1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023), and tissue from the thorax 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 preparation
Tissue from the thorax of the ioSymSang1 sample was processed at the WSI Scientific Operations core, using the Arima-HiC v2 kit. Frozen tissue (stored at –80 °C) was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde. After crosslinking, the tissue was homogenised using the Diagnocine Power Masher-II and BioMasher-II tubes and pestles. Following the kit manufacturer's instructions, crosslinked DNA was digested using a restriction enzyme master mix. The 5’-overhangs were then 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.
Library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Pacific Biosciences HiFi circular consensus DNA sequencing libraries were prepared using the PacBio Express Template Preparation Kit v2.0 (Pacific Biosciences, California, USA) as per the manufacturer’s instructions. The kit includes the reagents required for removal of single-strand overhangs, DNA damage repair, end repair/A-tailing, adapter ligation, and nuclease treatment. Library preparation also included a library purification step using AMPure PB beads (Pacific Biosciences, California, USA) and size selection step to remove templates shorter than 3 kb using AMPure PB modified SPRI. DNA concentration was quantified using the Qubit Fluorometer v2.0 and Qubit HS Assay Kit and the final library fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument and 165kb gDNA and 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 between 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.
For Hi-C library preparation, DNA was fragmented to a size of 400 to 600 bp using a Covaris E220 sonicator. The DNA was then enriched, barcoded, and amplified using the NEBNext Ultra II DNA Library Prep Kit following manufacturers’ instructions. 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 **
The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021; Cheng et al., 2022) in Hi-C phasing mode, resulting in a pair of haplotype-resolved assemblies. 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. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).
** Evaluation of the final assembly **
The final assembly was post-processed and evaluated using the three Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines: sanger-tol/readmapping ( Surana et al., 2023a), sanger-tol/genomenote ( Surana et al., 2023b), and sanger-tol/blobtoolkit ( Muffato et al., 2024). The readmapping pipeline aligns the Hi-C reads using bwa-mem2 ( Vasimuddin et al., 2019) and combines the alignment files with SAMtools ( Danecek et al., 2021). The genomenote pipeline converts the Hi-C alignments 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). This pipeline computes k-mer completeness and QV consensus quality values with FastK and MERQURY.FK, and runs BUSCO ( Manni et al., 2021) to assess completeness.
The blobtoolkit pipeline is a Nextflow 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 ( Buchfink et al., 2021) blastp. The genome is also split 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 with DIAMOND blastx. Genome sequences without a hit are chunked with 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 genome evaluation pipelines were 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.
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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- 2Allio R Schomaker-Bastos A Romiguier J : Mito Finder: efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 32243090 PMC 7497042 · doi ↗ · pubmed ↗
- 3Altschul SF Gish W Miller W : Basic local alignment search tool. J Mol Biol. 1990;215(3):403–410. 10.1016/S 0022-2836(05)80360-2 2231712 · doi ↗ · pubmed ↗
- 4Bateman A Martin MJ Orchard S : Uni Prot: the universal protein knowledgebase in 2023. Nucleic Acids Res. 2023;51(D 1):D 523–D 531. 10.1093/nar/gkac 1052 36408920 PMC 9825514 · doi ↗ · pubmed ↗
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- 8Brooks S : Field guide to the dragonflies and damselflies of Great Britain and Ireland. Gillingham: British Wildlife Publishing.2007.