The genome sequence of a soldier fly, Nemotelus pantherinus (Linnaeus, 1758)
Anthony Irwin, Ryan Mitchell, Olga Sivell, Komal Kumar Raja, Henrique Antoniolli
TL;DR
This paper presents the genome sequence of the soldier fly Nemotelus pantherinus, including its chromosomal structure and gene annotations.
Contribution
The study provides a high-quality genome assembly and gene annotation for the soldier fly Nemotelus pantherinus.
Findings
The genome assembly is 804.69 megabases long, with 95.93% scaffolded into 6 chromosomal pseudomolecules.
The mitochondrial genome is 19.1 kilobases long and has been assembled.
Ensembl identified 27,389 protein-coding genes in the assembly.
Abstract
We present a genome assembly from a male specimen of Nemotelus pantherinus (soldierfly; Arthropoda; Insecta; Diptera; Stratiomyidae). The genome sequence has a total length of 804.69 megabases. Most of the assembly (95.93%) is scaffolded into 6 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled, with a length of 19.1 kilobases. Gene annotation of this assembly on Ensembl identified 27,389 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
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Figure 5| Project information | |||
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| Nemotelus pantherinus | ||
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| PRJEB73393 | ||
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| SAMEA112964080 | ||
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| 2867254 | ||
| Specimen information | |||
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| idNemPant2 | SAMEA112975209 | Whole organism |
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| idNemPant1 | SAMEA11025283 | whole organism |
| Sequencing information | |||
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| ERR12723456 | 9.23e+08 | 139.3 |
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| ERR12721045 | 2.33e+06 | 23.98 |
| Genome assembly | ||
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| Assembly name | idNemPant2.1 | |
| Assembly accession | GCA_963978885.1 | |
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| Assembly level for primary assembly | chromosome | |
| Span (Mb) | 804.69 | |
| Number of contigs | 685 | |
| Number of scaffolds | 149 | |
| Longest scaffold (Mb) | 269.57 | |
| Assembly metric | Measure |
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| Contig N50 length | 7.1 Mb |
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| Scaffold N50 length | 241.31 Mb |
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| Consensus quality (QV) | Primary: 56.9; alternate: 57.3; combined: 57.2 |
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| Primary: 65.34%; alternate: 63.18%; combined: 98.91% |
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| BUSCO
| C:93.9%[S:93.0%,D:0.9%], F:1.8%,M:4.4%,n:3,285 |
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| Percentage of assembly mapped to
| 95.93% |
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| Sex chromosomes | X and Y |
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| Organelles | Mitochondrial genome: 19.1 kb |
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| INSDC accession | Name | Length (Mb) | GC% |
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| 1 | 241.31 | 36.5 | |
| 2 | 269.57 | 37 | |
| 3 | 147.08 | 36.5 | |
| 4 | 99.03 | 36.5 | |
| X | 12.82 | 36.5 | |
| Y | 2.13 | 49.5 | |
| MT | 0.02 | 22 |
| Software tool | Version | Source |
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| BLAST | 2.14.0 |
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| BlobToolKit | 4.3.9 |
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| BUSCO | 5.5.0 |
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| bwa-mem2 | 2.2.1 |
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| DIAMOND | 2.1.8 |
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| fasta_windows | 0.2.4 |
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| FastK | 666652151335353eef2fcd58880bcef5bc2928e1 |
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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 | 44086069ee7d4d3f6f3f0012569789ec138f42b84aa44357826c0
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| MerquryFK | d00d98157618f4e8d1a9190026b19b471055b22e |
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| Minimap2 | 2.24-r1122 |
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| MitoHiFi | 3 |
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| MultiQC | 1.14, 1.17, and 1.18 |
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| Nextflow | 23.04.1 |
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| PretextView | 0.2.5 |
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| PretextSnapshot | - |
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| purge_dups | 1.2.5 |
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| samtools | 1.19.2 |
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| sanger-tol/ascc | 0.1.0 |
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| sanger-tol/
| 0.4.0 |
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| Seqtk | 1.3 |
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| Singularity | 3.9.0 |
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| TreeVal | 1.2.0 |
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| YaHS | 1.2a.2 |
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- —Wellcome Trust
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Taxonomy
TopicsInsect symbiosis and bacterial influences · Genomics and Phylogenetic Studies · Environmental DNA in Biodiversity Studies
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Stratiomyomorpha; Stratiomyidae; Nemotelinae; Nemotelus; Nemotelus pantherinus (Linnaeus, 1758) (NCBI:txid2867254)
Background
Nemotelus pantherinus (Linnaeus, 1758) is a small black and white fly from the family Stratiomyidae (Diptera) commonly called soldierflies. The body length of 4.25–5.5 mm. It is a characteristic species that can be identified in the field upon closer examination. The similar looking N. notatus Zetterstedt, 1842 and N. uliginosus (Linnaeus, 1767) both have white stripes on the sides of the thorax, but these are absent in N. pantherinus. The species exhibits sexual dimorphism with females lacking white markings on the face but have white triangular marks on the tergites, while males have more extensive white markings on the abdomen. This is the only British Nemotelus in which the underside of the abdomen is mostly white ( Stubbs & Drake, 2014; Zeegers & Schulten, 2022). There are two forms of this species, one with long face, and the other with short face. The latter was described as a separate species fraternus Loew. It is now considered to be a synonym of N. pantherinus ( Woodley, 2001).
Nemotelus pantherinus is a Palaearctic species, recorded from Albania, Armenia, Austria, Azerbaijan, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, England, Estonia, France, Germany, Greece, Hungary, Ireland, Israel, Italy, Latvia, Morocco, Netherlands, Norway, Poland, Roumania, Russia, Slovakia, Spain, Sweden, Switzerland, Tajikistan, Turkey, Wales and Yugoslavia ( Woodley, 2001). It is common in southern England and Wales, uncommon in northern England and absent in Scotland, with scattered records from Ireland ( Drake, 1991; Stubbs & Drake, 2014). The fly occurs in fens and damp meadows, often with seepages. The flight period is from June to August ( Stubbs & Drake, 2014).
The larvae have been found in fens and seepages in moss covered by a layer of flowing water. They are yellow to pale brown with a dark median stripe and irregular dark oblique stripes on either side of it ( Brindle, 1964; Rozkošný, 1982; Stubbs & Drake, 2014).
The high-quality genome of Nemotelus pantherinus 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. The genome is based on a specimen from Marston Marsh, Norwich, England. It will aid research on this species as well as on phylogeny of Stratiomyidae.
Genome sequence report
Sequencing data
The genome of a specimen of Nemotelus pantherinus ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 23.98 Gb (gigabases) from 2.33 million reads, which were used to assemble the genome. GenomeScope analysis estimated the haploid genome size at 762.10 Mb, with a heterozygosity of 2.95% and repeat content of 39.46%. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 30 coverage. Hi-C sequencing produced 139.30 Gb from 922.54 million reads, used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
Photograph of the Nemotelus pantherinus (idNemPant2) specimen used for genome sequencing.
Table 1.: Specimen and sequencing data for Nemotelus pantherinus.
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The assembly was improved by manual curation, which corrected 200 misjoins or missing joins and removed 22 haplotypic duplications. These interventions reduced the total assembly length by 1.5%, decreased the scaffold count by 34.21%, also decreasing the scaffold N50 by 50.93%. The final assembly has a total length of 804.69 Mb in 149 scaffolds, with 536 gaps, and a scaffold N50 of 241.31 Mb ( Table 2).
Table 2.: Genome assembly data for Nemotelus pantherinus.
The snail plot in Figure 2 provides a summary of the assembly statistics, indicating the distribution of scaffold lengths and other assembly metrics. Figure 3 shows the distribution of scaffolds by GC proportion and coverage. Figure 4 presents a cumulative assembly plot, with separate curves representing different scaffold subsets assigned to various phyla, illustrating the completeness of the assembly.
Genome assembly of Nemotelus pantherinus, idNemPant2.1: 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 diptera_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963978885.1/dataset/GCA_963978885.1/snail.
Genome assembly of Nemotelus pantherinus, idNemPant2.1: 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/GCA_963978885.1/dataset/GCA_963978885.1/blob.
Genome assembly of Nemotelus pantherinus, idNemPant2.1: 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/GCA_963978885.1/dataset/GCA_963978885.1/cumulative.
Most of the assembly sequence (95.93%) was assigned to 6 chromosomal-level scaffolds, representing 4 autosomes and the X and Y sex chromosomes. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 5; Table 3). During curation, the X and Y chromosomes were identified based on PacBio read coverage. The Y chromosome is unscaffolded, as the Hi-C data were from a female sample.
Genome assembly of Nemotelus pantherinus: Hi-C contact map of the idNemPant2.1 assembly, generated using PretextSnapshot.
Table 3.: Chromosomal pseudomolecules in the genome assembly of Nemotelus pantherinus, idNemPant2.
The mitochondrial genome was also assembled. This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
Assembly quality metrics
The estimated Quality Value (QV) and k-mer completeness metrics, along with BUSCO completeness scores, were calculated for each haplotype and the combined assembly. The QV reflects the base-level accuracy of the assembly, while k-mer completeness indicates the proportion of expected k-mers identified in the assembly. BUSCO scores provide a measure of completeness based on benchmarking universal single-copy orthologues.
The combined primary and alternate assemblies achieve an estimated QV of 57.2. The k-mer completeness is 65.34% for the primary haplotype and 63.18% for the alternate haplotype; and 98.91% for the combined primary and alternate assemblies. BUSCO v.5.5.0 analysis using the diptera_odb10 reference set ( n = 3,285) identified 93.9% of the expected gene set (single = 93.0%, duplicated = 0.9%).
Table 2 provides assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The primary assembly achieves the EBP reference standard of 6.C.Q56.
Genome annotation report
The Nemotelus pantherinus genome assembly (GCA_963978885.1) was annotated externally by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 27,882 transcribed mRNAs from 27,389 protein-coding genes. The average transcript length is 4,536.92 bp, with 3.19 exons per transcript. For further information about the annotation, please refer to https://beta.ensembl.org/species/887196f1-a3f3-4bb4-8488-66cca063470c.
Methods
Sample acquisition and DNA barcoding
The specimen used for genome sequencing was an adult male Nemotelus pantherinus (specimen ID NHMUK015059309, ToLID idNemPant2), collected from Marston Marsh, Norwich, England, United Kingdom (latitude 52.6, longitude 1.27) on 2022-07-04 by handpicking. The specimen was collected and identified by Anthony Irwin (Castle Museum Study Centre) and preserved by dry freezing (–80 °C).
A second specimen was used for Hi-C sequencing (specimen ID NHMUK014036862, ToLID idNemPant1). It was an adult specimen collected from Parsonage Moor, Abington, England, United Kingdom (latitude 51.6942, longitude –1.3344) on 2021-06-19 by aerial net. The specimen was collected by Ryan Mitchell and Olga Sivell (Natural History Museum), identified by Ryan Mitchell and preserved by liquid nitrogen.
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 each specimen and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI) ( Pereira et al., 2022). 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).
Metadata collection for samples adhered to the Darwin Tree of Life project standards described by Lawniczak et al. (2022).
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 procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification ( Howard et al., 2025). Detailed protocols are available on protocols.io ( Denton et al., 2023b). The idNemPant2 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( Jay et al., 2023). Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted using the Automated MagAttract v2 protocol ( Oatley et al., 2023a). 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 ( Oatley et al., 2023b). 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. For this sample, the extracted DNA had a Qubit concentration of 6.3 ng/μL and a yield of 819.00 ng.
Hi-C sample preparation and crosslinking
Hi-C data were generated from the idNemPant1 sample using the Arima-HiC v2 kit (Arima Genomics) with 20–50 mg of frozen tissue (stored at –80 °C). As per manufacturer’s instructions, tissue was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde concentration, and a final formaldehyde concentration of 2%. The tissue was then homogenised using the Diagnocine Power Masher-II. The crosslinked DNA was digested using a restriction enzyme master mix, then biotinylated and ligated. A clean up was performed with SPRIselect beads prior to library preparation. DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and Qubit HS Assay Kit, and sample biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
Library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core.
** PacBio HiFi **
At a minimum, samples were required to have an average fragment size exceeding 8 kb and a total mass over 400 ng to proceed to the low-input SMRTbell Prep Kit 3.0 protocol (Pacific Biosciences), depending on genome size and sequencing depth required. Libraries were prepared using the SMRTbell Prep Kit 3.0 as per the manufacturer's instructions. The kit includes the reagents required for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead cleanup, and nuclease treatment. Size-selection and clean-up were carried out using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified using the Qubit Fluorometer v4.0 (ThermoFisher Scientific) with Qubit 1X dsDNA HS assay kit and the final library fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) and the gDNA 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 in the range 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.
** Hi-C **
For Hi-C library preparation, the biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size-selected to 400–600 bp using SPRISelect beads. DNA was then enriched using Arima-HiC v2 Enrichment beads. The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) was used for end repair, A-tailing, and adapter ligation, following a modified protocol in which library preparation is carried out while the DNA remains bound to the enrichment beads. PCR amplification was performed using KAPA HiFi HotStart mix and custom dual-indexed adapters (Integrated DNA Technologies) in a 96-well plate format. Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, samples were amplified for 10–16 PCR cycles. Post-PCR clean-up was carried out using SPRISelect beads. The libraries were quantified using the Accuclear Ultra High Sensitivity dsDNA Standards Assay kit (Biotium) and normalised to 10 ng/μL before sequencing. Hi-C sequencing was performed on the Illumina NovaSeq 6000 instrument using 150 bp paired-end reads.
Genome assembly, curation and evaluation
** Assembly **
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.
The HiFi reads were first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded 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. Flat files and maps used in curation were generated via the TreeVal pipeline ( Pointon et al., 2023). Manual curation was conducted primarily in PretextView ( Harry, 2022) and HiGlass ( Kerpedjiev et al., 2018), with additional insights provided by JBrowse2 ( Diesh et al., 2023). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were amended, and duplicate sequences were tagged and removed. Sex chromosomes were identified by read coverage analysis. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation.
** Assembly quality assessment **
The Merqury.FK tool ( Rhie et al., 2020), run in a Singularity container ( Kurtzer et al., 2017), was used 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 genome was analysed in the blobtoolkit pipeline, a Nextflow ( Di Tommaso et al., 2017) port of the previous Snakemake Blobtoolkit pipeline ( Challis et al., 2020). It aligns the PacBio reads in SAMtools ( Danecek et al., 2021) 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 blastp ( Buchfink et al., 2021). The genome is also divided 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 using DIAMOND blastx. Genome sequences without a hit are chunked using 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 blobtoolkit pipeline was 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.
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