The genome sequence of the Ragwort Fly, Sphenella marginata (Fallén, 1814)

We present a genome assembly from an individual female Sphenella marginata (the Ragwort Fly; Arthropoda; Insecta; Diptera; Tephritidae). The genome sequence is 595.2 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.82 kilobases in length.


Background
Sphenella marginata (Fallén, 1814) is a species of fly in the family Tephritidae.This species is found throughout the Palearctic region.Sphenella marginata likely has the widest natural distribution of any Tephritid (White, 1988).In Britain, it is found towards the south of England and Wales with only rare occurrence further north.In Ireland, it has been officially recorded in Northern Ireland only, but it is likely found further south, as S. marginata distribution follows the distribution of ragworts and groundsels.No IUCN Global Red List category has been published as of the date of this publication.
Colloquially S. marginata is known as the Ragwort Fly.This is due to the larvae being known to feed within the capitulum of numerous Senecio plants (Schmidl, 1972), otherwise known as ragworts and groundsels.The activity of S.marginata larvae causes galls, where flower heads swell at the base and bracts enlarge.Usually the flower head will contain only one white-coloured larvae or brown puparium.The species is diurnal.
Adults have long geniculate mouthparts, indicating the possibility of nectar feeding (White, 1988).The frons and legs are yellow-orange, the thorax has an earthy brown colour, whilst the abdomen has a dark brown colour.The eyes of adult flies are verdant green.The costal margins of the forewings are spotted brown, with a full dark brown bar across the apex and where the abdomen begins, whilst the hindwings are clear.
The genome of the Ragwort Fly, Sphenella marginata, 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.

Genome sequence report
The genome was sequenced from one Sphenella marginata (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.33).A total of 31-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 318 missing joins or mis-joins and removed 22 haplotypic duplications, reducing the assembly length by 0.53% and the scaffold number by 62.38%, and increasing the scaffold N50 by 46.76%.
The final assembly has a total length of 595.2 Mb in 119 sequence scaffolds with a scaffold N50 of 109.6 Mb (Table 1).Most (99.36%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds.Chromosomescale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The sole sex chromosome in this assembly is chromosome 1, which has approximately two-thirds coverage compared to the autosomes.There is no evidence of a Y chromosome, thus the data indicates that this species has homomorphic sex chromosomes.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/594017.

Sample acquisition and nucleic acid extraction
The Sphenella marginata specimen (specimen ID Ox000842, individual idSphMarg1) used for sequencing was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.33) on 2020-08-04 by netting.The specimen was collected and identified by Steven Falk (independent researcher).The specimen used to generate Hi-C data and for RNA sequencing (specimen ID Ox001853, individual idSphMarg2) was collected by Ryan Mitchell (independent researcher) from the same location on 2021-08-21.Both specimens were preserved on dry ice.DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idSphMarg1 sample was weighed and dissected on dry ice.Tissue from the whole organism was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.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.
RNA was extracted from tissue of idSphMarg2 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from tissue of idSphMarg2 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).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS  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     Overall, the data note is sufficient for its purposes, though there are a few suggestions that would improve its reproducibility and utility as a resource.
The data note explains the source of collection of the specimens but does not explicitly mention identifying marks that distinguish this species from other similar flies, though this information can presumably be found in other sources, which the authors could reference.

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Though RNA was sequenced, the annotation is not included, hindering the utility of this resource.I would prefer annotations to be published alongside the genome assemblies, but I understand the benefit of publishing the genome assembly sooner.

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The method section contains references and links to pipelines, packages, and versions, but does not include all details to make this fully replicable (on parameters used, etc.).

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

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Lab and bioinformatics methods and analyses are state of the art.The writing is straightforward and clear.
○ My only substantial comment revolves around the sex of the specimens and the nature of the sex chromosome: I could not find any indication with the sex of the sequenced specimens, and it is not clear how the authors determined that chromosome 1 is actually the sex chromosome.I would urge the authors to add this information.
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.
Reviewer Expertise: population genetics, population genomics, bioinformatcs 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.

Figure 2 .
Figure 2. Genome assembly of Sphenella marginata, idSphMarg1.1: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 595,252,386 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 (129,039,367 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (109,573,589 and 66,161,324 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/idSphMarg1.1/dataset/CATOCK01/snail.

Figure 3 .
Figure 3. Genome assembly of Sphenella marginata, idSphMarg1.1: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/idSphMarg1.1/dataset/CATOCK01/blob.

Figure 4 .
Figure 4. Genome assembly of Sphenella marginata, idSphMarg1.1: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/idSphMarg1.1/dataset/CATOCK01/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Sphenella marginata, idSphMarg1.1:Hi-C contact map of the idSphMarg1.1 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=GGd34c4iSHOvGtSr3Yyt6Q.

Table 3
Wellcome Sanger Institute -Legal and GovernanceThe 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',

Table 2 . Chromosomal pseudomolecules in the genome assembly of Sphenella marginata, idSphMarg1. INSDC accession Chromosome Length (Mb) GC%
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: