The genome sequence of a satellite fly, Leucophora obtusa (Zetterstedt, 1837)

We present a genome assembly from an individual female Leucophora obtusa (a satellite fly; Arthropoda; Insecta; Diptera; Anthomyiidae). The genome sequence is 1,289.8 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 18.72 kilobases in length.


Background
Leucophora is a genus of root-maggot flies in the family Anthomyiidae, which has over 60 described species, with Leucophora obtusa being recorded most often.Leucophora obtusa is found across Japan, Europe and North America (GBIF Secretariat, 2022;Suwa, 1974).L. obtusa parasitise the larvae of Andrena bees, and can often be found near their nests (Huckett, 1940;Polidori et al., 2005).The common name "satellite fly" comes from the observed behaviour of the female fly hovering or "orbiting" around bee nests.Females are seen to shadow the host bee back to its burrow where she will oviposit in the tumulus of the nest entrance, and L. obtusa larvae are parasitic on the host's brood (Michener & Rettenmeyer, 1956;Polidori et al., 2005).
This species is notoriously difficult to distinguish from other flies in the Leucophora genus and has often been misidentified throughout the literature (Collin, 2009;Huckett, 1940).The genus itself can be distinguished by broad parafacials at the base of the antennae and are yellow basally and dark brownish apically (Suwa, 1974).L. obtusa is seen as a particularly hairy species of this genus, with a hairy thorax, abdomen, and legs (Huckett, 1940).It possesses long, erect hairs on the abdominal sternites and lateral margins of the scutellum, and the mid tibia and hind femur also have long fine ventral setae (Suwa, 1974).
We hope that this novel, chromosomally complete genome sequence, developed as part of the Darwin Tree of Life Project, can be of benefit to continued understanding of the biology of L. obtusa.This project is 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 female Leucophora obtusa (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76,.A total of 33-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 72 missing joins or mis-joins and removed 3 haplotypic duplications, reducing the scaffold number by 17.21%, and increasing the scaffold N50 by 1.22%. The final assembly has a total length of 1,289.8Mb in 201 sequence scaffolds with a scaffold N50 of 230.2 Mb (Table 1).Most (99.17%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The exact order and orientation of the scaffolds in the repetitive centromeres is unknown.Since idLeuObtu2 is a female XX sample lacking sequence data from the heterogametic species, the X chromosome remains unidentified.This is because, in Diptera, homology is unreliable for sex chromosome identification due to frequent turnover of these chromosomes (Vicoso & Bachtrog, 2015).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/2588525.

Sample acquisition and nucleic acid extraction
Leucophora obtusa specimens were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.76, longitude -1.34) on 2021-04-23 by netting.The specimens were collected and identified by Steven Falk (independent researcher), and were then preserved on dry ice.The specimen used for genome sequencing and Hi-C data was a female with specimen ID Ox001285 (ToLID idLeuObtu2), while the specimen used for RNA sequencing has specimen ID Ox001305 (ToLID idLeuObtu3).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idLeuObtu2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.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 whole organism tissue of idLeuObtu3 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.were also generated from remaining tissue of idLeuObtu2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.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.

Genome assembly, curation and evaluation
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019)

Software tool Version
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 This study conduct genome sequencing using HGS approach.Most of the resulting data can be scaffolded into 6 chromosomal pseudomolecules.In addition, a 18.72 kilobases mitogenome was reconstructed from the genome sequencing data.Many new sequencing assembling software were employed in this study.The overall writing of the article is good.Although the raw data of the genome sequencing is already included in PRJEB57107, more detailed annotations regarding the genome (including the mitochondrial genome) should be publicly disclosed for future research.
I suggested this article to be indexed.

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

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: Insect systematics, molecular biology 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 Leucophora obtusa, idLeuObtu2.1: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 1,289,857,006 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 (373,316,775 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (230,154,751 and 193,475,527 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/idLeuObtu2.1/dataset/CATLKA01/snail.

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

Figure 3 .
Figure 3. Genome assembly of Leucophora obtusa, idLeuObtu2.1: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/idLeuObtu2.1/dataset/CATLKA01/blob.

Figure 4 .
Figure 4. Genome assembly of Leucophora obtusa, idLeuObtu2.1: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/idLeuObtu2.1/dataset/CATLKA01/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Leucophora obtusa, idLeuObtu2.1:Hi-C contact map of the idLeuObtu2.1 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=DbS5c8XOSS6dl4NdSeCc7A.

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

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.
Reviewer Report 23 October 2023 https://doi.org/10.21956/wellcomeopenres.22059.r66575©2023Xi Y.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.Yuqiang XiState Key Laboratory of Wheat and Maize Crop Science/Henan International Laboratory for Green Pest Control, Henan Agricultural University, Zhengzhou, China