The genome sequence of the variegated flesh fly, Sarcophaga variegata (Scopoli, 1763)
Steven Falk, John F Mulley, Yuqiang Xi, Alex Makunin, Daniel Doucet
TL;DR
The study provides the genome sequence of the variegated flesh fly, including its chromosomes and mitochondrial DNA.
Contribution
The first genome assembly of Sarcophaga variegata, including sex chromosomes and mitochondrial DNA.
Findings
The genome assembly spans 718.5 megabases and includes 7 chromosomal pseudomolecules.
The mitochondrial genome is 18.7 kilobases long and fully assembled.
Gene annotation identified 16,660 protein-coding genes.
Abstract
We present a genome assembly from an individual male Sarcophaga variegata (the variegated flesh fly; Arthropoda; Insecta; Diptera; Sarcophagidae). The genome sequence is 718.5 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 18.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,660 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
Figure 2
Figure 3
Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | idSarVari1.1 | |
| Species |
| |
| Specimen | idSarVari1 | |
| NCBI taxonomy ID | 236851 | |
| BioProject | PRJEB48115 | |
| BioSample ID | SAMEA8603132 | |
| Isolate information | idSarVari1, male (genome sequencing and HiC) | |
| Assembly metrics
|
| |
| Consensus quality (QV) | 52.9 |
|
|
| 99.99% |
|
| BUSCO
| C:98.9%[S:98.4%,D:0.5%],
|
|
| Percentage of assembly mapped
| 98.85% |
|
| Sex chromosomes | X and Y chromosomes |
|
| Organelles | Mitochondrial genome assembled |
|
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR7123977, ERR7123978 | |
| 10X Genomics Illumina | ERR7113567–ERR7113570 | |
| Hi-C Illumina | ERR7113566 | |
| Genome assembly | ||
| Assembly accession | GCA_932273835.1 | |
|
| GCA_932276125.1 | |
| Span (Mb) | 718.5 | |
| Number of contigs | 602 | |
| Contig N50 length (Mb) | 7.4 | |
| Number of scaffolds | 119 | |
| Scaffold N50 length (Mb) | 130.2 | |
| Longest scaffold (Mb) | 167.1 | |
| Genome annotation | ||
| Number of protein-coding genes | 16,660 | |
| Number of non-coding genes | 11,439 | |
| Number of gene transcripts | 38,740 | |
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| 1 | 167.11 | 33.3 | |
| 2 | 141.46 | 33.1 | |
| 3 | 130.24 | 33.8 | |
| 4 | 129.56 | 33.9 | |
| 5 | 116.88 | 33.6 | |
| X | 10.72 | 33.6 | |
| Y | 3.57 | 32.8 | |
| MT | 0.02 | 21.7 | |
| - | unplaced | 18.92 | 31.7 |
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 3.4.0 |
|
| BUSCO | 5.3.2 |
|
| FreeBayes | 1.3.1-17-gaa2ace8 |
|
| Hifiasm | 0.15.3 |
|
| HiGlass | 1.11.6 |
|
| Long Ranger ALIGN | 2.2.2 |
|
| Merqury | MerquryFK |
|
| MitoHiFi | 2 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| SALSA | 2.2 |
|
| sanger-tol/genomenote | v1.0 |
|
| sanger-tol/readmapping | 1.1.0 |
|
- —Wellcome Trust
- —Wellcome Trust
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Taxonomy
TopicsForensic Entomology and Diptera Studies · Insect behavior and control techniques · Diptera species taxonomy and behavior
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha; Eremoneura; Cyclorrhapha; Schizophora; Calyptratae; Oestroidea; Sarcophagidae; Sarcophaginae; Sarcophaga; Sarcophaga variegata ( Scopoli, 1763) (NCBI:txid236851).
Background
The genus Sarcophaga comprises around 890 species within 169 subgenera ( Buenaventura & Pape, 2017), 36 of which have been recorded in Britain ( Whitmore et al., 2020). The variegated flesh fly Sarcophaga ( Sarcophaga) variegata (Diptera: Sarcophagidae) is a large (15–16 mm body length) flesh fly common across England and Wales, but rarer in Scotland ( NBN Atlas Partnership, 2021), with a wider Palaearctic distribution ( Pape, 1996). Adults have been recorded from April to September, reaching peak abundance in July and August in the UK. As is typical among flesh flies, adult S. variegata are black to grey overall, with longitudinal stripes on the thorax and a checked pattern on the abdomen, and so reliable identification of this species using morphological characters alone is challenging. Separation of this species from the other members of the so-called carnaria subgroup ( S. carnaria and S. subvicina) is especially difficult, requiring examination of male terminalia and, for females, DNA barcoding ( Jordaens et al., 2013; Schönberger et al., 2022). Cuticular hydrocarbons have also recently been proposed as a possible identification method ( Moore et al., 2021). S. variegata was described as Musca variegata by Giovanni Antonio Scopoli in 1763 in his Entomologia Carniolica ( Scopoli, 1763), and has in the past been regarded as a synonym of S. carnaria ( Van Emden, 1954).
Flesh flies are of forensic importantance ( Ren et al., 2018), including their role as vectors for the transfer of contaminating DNA ( Durdle, 2020), and S. variegata adults have been reported to be attracted to beef liver baits and pig carcasses ( Anton et al., 2011), and can be considered an indicators of summertime urban habitats ( Fremdt & Amendt, 2014). This S. variegata genome will be useful for the development of new molecular tools for species identification within this cryptic genus, for the investigation into the evolution of ovoviviparity; and as a resource for wider research into genome evolution in Diptera or insects more generally. Indeed, this genome sequence has already been used in an analysis of Hox cluster evolution in 243 insects ( Mulhair & Holland, 2022).
Genome sequence report
The genome was sequenced from one male Sarcophaga variegata specimen ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude – 1.33). A total of 35-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 71.0-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 410 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 75.21%, and increasing the scaffold N50 by 157.09%.
Photograph of the Sarcophaga variegata (idSarVari1) specimen used for genome sequencing.
The final assembly has a total length of 718.5 Mb in 119 sequence scaffolds with a scaffold N50 of 130.2 Mb ( Table 1). Most (98.85%) of the assembly sequence was assigned to 7 chromosomal-level scaffolds, representing 5 autosomes and the X and Y sex chromosomes. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 2– Figure 5; Table 2). The order and orientation of scaffolds is uncertain in the following regions: chromosome 4: 53.2–62.5 Mb, and chromosome 5: 39.1–41.2 Mb. 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 Sarcophaga variegata, idSarVari1.1.
Genome assembly of Sarcophaga variegata, idSarVari1.1: 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 718,474,625 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 (167,104,922 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (130,241,815 and 116,879,514 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 diptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idSarVari1.1/dataset/CAKNZP01/snail.
Genome assembly of Sarcophaga variegata, idSarVari1.1: 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/idSarVari1.1/dataset/CAKNZP01/blob.
Genome assembly of Sarcophaga variegata, idSarVari1.1: 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/idSarVari1.1/dataset/CAKNZP01/cumulative.
Genome assembly of Sarcophaga variegata, idSarVari1.1: Hi-C contact map of the idSarVari1.1 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=f8AEpuqjQR-5DIpdzaZLJA.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Sarcophaga variegata, idSarVari1.
The estimated Quality Value (QV) of the final assembly is 52.9 with k-mer completeness of 99.99%, and the assembly has a BUSCO v5.3.2 completeness of 98.9% (single = 98.4%, duplicated = 0.5%), using the diptera_odb10 reference set ( n = 3,285).
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/236851.
Genome annotation report
The S. variegata genome assembly (GCA_932273835.1) was annotated using the Ensembl rapid annotation pipeline ( Table 1; https://rapid.ensembl.org/Sarcophaga_variegata_GCA_932276125.1/Info/Index). The resulting annotation includes 38,740 transcribed mRNAs from 16,660 protein-coding and 11,439 non-coding genes.
Methods
Sample acquisition and nucleic acid extraction
A male Sarcophaga variegata (idSarVari1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude – 1.33) on 4 August 2020 by netting. The specimen was collected and identified by Steven Falk (independent researcher), and was then preserved on dry ice prior to processing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The idSarVari1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax 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 tissue of idSarVari1 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 SALSA2 ( Ghurye et al., 2019). 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 ( Simão et al., 2015; Manni et al., 2021) were calculated.
Table 3 contains a list of relevant software tool versions and sources.
Genome annotation
The Ensembl gene annotation system ( Aken et al., 2016) was used to generate annotation for the Sarcophaga variegata assembly (GCA_932276125.1). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt ( UniProt Consortium, 2019).
Legal and ethical review process for Darwin Tree of Life Partner submitted materials
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.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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