The genome sequence of the variegated flesh fly, Sarcophaga variegata (Scopoli, 1763)

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.


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%.
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: 39.1-41.2Mb.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.
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.

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  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  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.
Table 3. Software tools: versions and sources.

Software tool Version
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

Alex Makunin
Wellcome Sanger Institute, Hinxton, England, UK The data note by Falk et al. describes the genome assembly for Sarcophaga variegata, which seems to be among the six chromosomal assemblies for Sarcophaga species available at the time of writing.The data is presented in a standardised way and the only issue I can see is that the sources of transcriptomic data for gene annotation were not indicated (I assume those were some public datasets).
Similarly to the previous reviewer, I would appreciate if the chromosome names and grid were displayed in the Hi-C plot at Fig 5.
It might be also useful to indicate that the blob plot at Fig 3 was generated from a subset of 10x data alignment to the reference genome.On the similar line, inclusion of mitochondrion in the blob plot makes it hard to see sex chromosomes below the autosomes -it seems there is at least one sex-linked scaffold that is similar in size to Y.
It is also worth noting that the reported chromosome number agrees with the typical 2n=12 karyotype for Sarcophaga.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.This report presents the first complete genome sequence of the flesh fly Sarcophaga variegata, a close relative of S. carnaria.As the Sarcophaga genus is extremely speciose, obtaining genomes from key species and eventually whole sub-genera will be useful the examine the drivers of speciation in this taxa.

Reviewer
The report follows the standard structure established by the tree of life project, with the important tables and figures that describe the quality of the genome.As far as I can tell the data has been acquired using state of the art methods.
One thing I noticed however is how unorganized the Hi-C contact map appears to be (Fig 5).I am more familiar with data generated in lepidoptera where cis interactions are dominant, resulting in clearly distinguishable chromosomal scaffolds on the contact map.Looking around other full genomes in the Wellcome Open website, I noticed that other dipteran genomes suffer from the same high frequency of trans interactions.I would like to authors to add a line or two of explanation about this phenomenon.I also think it would be helpful to add grey lines to delimit chromosomes on that figure (e.g.see ref [1])

Figure 2 .
Figure 2. 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.

Figure 3 .
Figure 3. 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.

Figure 4 .
Figure 4. 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.

Figure 5 .
Figure 5. 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.
Expertise: comparative genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Reviewer Report 29 February 2024 https://doi.org/10.21956/wellcomeopenres.21581.r74945© 2024 Doucet D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Daniel DoucetNatural Resources Canada, Sault Ste.Marie, Ontario, Canada

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.