Version Changes
Revised. Amendments from Version 1
In Version 2 of this data note we have updated the chromosome map in Figure 5 to a labelled version. We have updated the assembly quality metrics using the current version of Merqury.FK (1.1.2), and have included the Earth Biogenome Project reference standard.
Abstract
We present a genome assembly from an individual male Ceraclea dissimilis (cased caddisfly; Arthropoda; Insecta; Trichoptera; Leptoceridae). The genome sequence is 452.7 megabases in span. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules, including the Z sex chromosome. The assembly achieves the Earth Biogenome Project reference standard of 6.C.Q65. The mitochondrial genome has also been assembled and is 15.87 kilobases in length.
Keywords: Ceraclea dissimilis, cased caddisfly, genome sequence, chromosomal, Trichoptera
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Trichoptera; Integripalpia; Brevitentoria; Leptoceroidea; Leptoceridae; Leptocerinae; Athripsodini; Ceraclea; Ceraclea dissimilis (Stephens, 1836) (NCBI:txid446427).
Background
Ceraclea dissimilis is a cased caddis in the family Leptoceridae, which are easily recognised by their very long antennae. The adult has a pale spot on the trailing edge of a plain brown wing ( Barnard & Ross, 2012). The larval case is made of sand grains and strongly curved with the top edge overhanging the bottom edge ( Wallace et al., 2003). Most species within the genus Ceraclea feed on freshwater sponges and often incorporate them into their cases. Other species within the genus are obligate feeders on sponges but not dissimili ( Biggs, 2023). The larvae occur in both still and flowing water with the adults flying from June to September ( Barnard & Ross, 2012). It is the most common and smallest Ceraclea and is found throughout Britain ( Barnard & Ross, 2012). It is widespread in Europe ( O’Connor, 2015).
The genome of Ceraclea dissimilis 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 chromosomal-level whole genome sequence for Ceraclea dissimilis, based on one male specimen from Great Yarmouth, England.
Genome sequence report
The genome was sequenced from a specimen of Ceraclea dissimilis ( Figure 1) collected from the River Yare, Great Yarmouth, England, UK (52.61, –1.39). A total of 54-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 3 missing joins or mis-joins and removed one haplotypic duplication, reducing the assembly length by 0.36% and increasing the scaffold number by 2.94%.
Figure 1. Photograph of the Ceraclea dissimilis (iiCerDiss1) specimen used for genome sequencing.
The final assembly has a total length of 452.7 Mb in 34 sequence scaffolds with a scaffold N50 of 19.2 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, 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. Most (99.95%) of the assembly sequence was assigned to 25 chromosomal-level scaffolds, representing 24 autosomes and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 2). Chromosome Z was assigned based on synteny to Athripsodes cinereus (GCA_947579605.1) ( Wallace et al., 2023). 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.
Figure 2. Genome assembly of Ceraclea dissimilis, iiCerDiss1.1: metrics.
The BlobToolKit snail plot 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 452,751,329 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 (24,671,832 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,172,844 and 13,701,724 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/Ceraclea_dissimilis/dataset/GCA_963576895.1/snail.
Figure 3. Genome assembly of Ceraclea dissimilis, iiCerDiss1.1: BlobToolKit GC-coverage plot.
Sequences are coloured by phylum. Circles are sized in proportion to sequence length. Histograms show the distribution of sequence length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Ceraclea_dissimilis/dataset/GCA_963576895.1/blob.
Figure 4. Genome assembly of Ceraclea dissimilis, iiCerDiss1.1: BlobToolKit cumulative sequence plot.
The grey line shows cumulative length for all sequences. Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Ceraclea_dissimilis/dataset/GCA_963576895.1/cumulative.
Figure 5. Genome assembly of Ceraclea dissimilis, iiCerDiss1.1: Hi-C contact map of the iiCerDiss1.1 assembly, visualised using PretextView and PretextSnapshot.
Chromosomes are shown in order of size from left to right and top to bottom. An interactive version of this figure may be viewed in HiGlass at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=R0UhMd8dQp6r7SfOSiyUqw.
Table 1. Genome data for Ceraclea dissimilis, iiCerDiss1.1.
| Project accession data | ||
|---|---|---|
| Assembly identifier | iiCerDiss1.1 | |
| Species | Ceraclea dissimilis | |
| Specimen | iiCerDiss1 | |
| NCBI taxonomy ID | 446427 | |
| BioProject | PRJEB66416 | |
| BioSample ID | SAMEA112963029 | |
| Isolate information | iiCerDiss1, male: whole organism (PacBio DNA and Illumina
Hi-C sequencing) |
|
| Assembly metrics * | Benchmark | |
| Consensus quality (QV) | Primary: 65.4; alternate: 65.7; combined: 65.6 | ≥ 40 |
| k-mer completeness | Primary: 74.47%; alternate: 74.24%; combined: 98.86% | ≥ 95% |
| BUSCO ** | C:95.9%[S:95.1%,D:0.8%],
F:2.5%,M:1.6%,n:2,124 |
S > 90%; D < 5% |
| Percentage of assembly mapped
to chromosomes |
99.95% | ≥ 95% |
| Sex chromosomes | Z | localised homologous pairs |
| Organelles | Mitochondrial genome: 15.87 kb | complete single alleles |
| Raw data accessions | ||
| PacificBiosciences Sequel IIe | ERR12085120 | |
| Hi-C Illumina | ERR12102407 | |
| Genome assembly | ||
| Assembly accession | GCA_963576895.1 | |
| Accession of alternate haplotype | GCA_963576855.1 | |
| Span (Mb) | 452.7 | |
| Number of contigs | 91 | |
| Contig N50 length (Mb) | 9.6 | |
| Number of scaffolds | 34 | |
| Scaffold N50 length (Mb) | 19.2 | |
| Longest scaffold (Mb) | 24.67 | |
* Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from Rhie et al. (2021).
** BUSCO scores based on the endopterygota_odb10 BUSCO set using version v5.4.3. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/Ceraclea_dissimilis/dataset/GCA_963576895.1/busco.
Table 2. Chromosomal pseudomolecules in the genome assembly of Ceraclea dissimilis, iiCerDiss1.
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| OY756358.1 | 1 | 24.67 | 34.5 |
| OY756359.1 | 2 | 24.34 | 34.0 |
| OY756360.1 | 3 | 24.15 | 34.5 |
| OY756361.1 | 4 | 22.85 | 34.0 |
| OY756362.1 | 5 | 21.42 | 34.0 |
| OY756363.1 | 6 | 20.11 | 34.0 |
| OY756364.1 | 7 | 20.03 | 34.0 |
| OY756365.1 | 8 | 19.88 | 34.5 |
| OY756366.1 | 9 | 19.63 | 34.5 |
| OY756367.1 | 10 | 19.31 | 34.5 |
| OY756369.1 | 11 | 18.98 | 34.5 |
| OY756370.1 | 12 | 18.44 | 34.5 |
| OY756371.1 | 13 | 17.84 | 34.5 |
| OY756372.1 | 14 | 17.12 | 34.5 |
| OY756373.1 | 15 | 16.88 | 35.0 |
| OY756374.1 | 16 | 16.85 | 35.0 |
| OY756375.1 | 17 | 16.77 | 35.0 |
| OY756376.1 | 18 | 16.72 | 35.0 |
| OY756377.1 | 19 | 16.21 | 35.0 |
| OY756378.1 | 20 | 13.87 | 36.0 |
| OY756379.1 | 21 | 13.7 | 36.0 |
| OY756380.1 | 22 | 12.61 | 36.0 |
| OY756381.1 | 23 | 10.85 | 36.5 |
| OY756382.1 | 24 | 10.06 | 37.0 |
| OY756368.1 | Z | 19.17 | 34.0 |
| OY756383.1 | MT | 0.02 | 18.5 |
Table 1 also lists the assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The combined primary and alternate assemblies achieve an estimated QV of 65.6. The k-mer completeness is 74.47% for the primary assembly, 74.24% for the alternate haplotype, and 98.86% for the combined assemblies. The primary assembly has a BUSCO v5.4.3 completeness of 95.9% (single = 95.1%, duplicated = 0.8%), using the endopterygota_odb10 reference set ( n = 2,124). The EBP metric for the primary assembly is 6.C.Q65, meeting the recommended reference standard.
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/446427.
Methods
Sample acquisition and nucleic acid extraction
The male Ceraclea dissimilis (specimen ID NHMUK015059552, ToLID iiCerDiss1) that was used for genome sequencing was collected from River Yare, Great Yarmouth, England, UK (latitude 52.61, longitude –1.39) on 2022-07-06. The specimens were collected and identified by Derek Coleman (Dipterists Forum). Ten specimens with the initial identification of Ceraclea dissimilis were taken at actinic light. Five of the ten specimens were examined to confirm the initial identification and the remaining five were sent to the Darwin Tree of Life project at the Natural History Museum. Species identification was confirmed by DNA barcoding. To maintain the “field to lab” cold chain, the specimens were stored, handled and delivered on dry ice until reaching the whole genome sequencing lab at the Wellcome Sanger Institute (WSI).
The workflow for high molecular weight (HMW) DNA extraction at the WSI Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. The iiCerDiss1 sample was prepared for DNA extraction at the WSI Tree of Life Core Laboratory: it was weighed and dissected on dry ice ( Jay et al., 2023), and 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 ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. 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.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).
Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe instrument. Hi-C data were also generated from remaining tissue of iiCerDiss1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Genome assembly and curation
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.
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 then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected using the TreeVal pipeline ( Pointon et al., 2023). Manual curation was performed using JBrowse2 ( Diesh et al., 2023), HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) to select the final mitochondrial contig and to ensure the general quality of the sequence.
Final assembly evaluation
The Hi-C reads were aligned with bwa-mem2 ( Vasimuddin et al., 2019) and alignment files were combined with SAMtools ( Danecek et al., 2021). These alignments were transformed into a contact map with BEDTools ( Quinlan & Hall, 2010) and the Cooler tool suite ( Abdennur & Mirny, 2020), which is then visualised with HiGlass ( Kerpedjiev et al., 2018).
The Merqury.FK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for the primary and alternate haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics. The NCBI datasets API ( Sayers et al., 2024) was used to compile the assembly statistics table.
The sanger-tol/blobtoolkit pipeline ( Muffato et al., 2024) is a Nextflow port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads with 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 lineage, 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 taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx. Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). All those outputs are combined with the blobtools suite into a blobdir for visualisation.
The pipeline was developed using nf-core tooling ( Ewels et al., 2020). It uses MultiQC ( Ewels et al., 2016), the Biocontainers infrastructure ( da Veiga Leprevost et al., 2017), and the Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017) containerisation solutions.
Table 3 contains a list of relevant software tool versions and sources.
Table 3. Software tools: 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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>] and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 2; peer review: 2 approved, 1 approved with reservations]
Data availability
European Nucleotide Archive: Ceraclea dissimilis. Accession number PRJEB66416; https://identifiers.org/ena.embl/PRJEB66416 ( Wellcome Sanger Institute, 2023). The genome sequence is released openly for reuse. The Ceraclea dissimilis 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. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.
Author information
Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.7139035.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.
Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.10066175.
Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.10043364.
Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.10066637.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.
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