The genome sequence of the Locust Fly, Stomorhina lunata (Fabricius, 1805)

We present a genome assembly from an individual female Stomorhina lunata (the Locust Fly; Arthropoda; Insecta; Diptera; Rhiniidae). The genome sequence is 728.1 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 16.49 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,358 protein coding genes.


Background
Stomorhina lunata (Fabricius, 1805) is a species of fly from the family Calliphoridae (blow flies), subfamily Rhiniinae.Rhiniinae used to be considered as a separate family (Kutty et al., 2010), however, recently it has been reclassified as a subfamily and placed within Calliphoridae (Yan et al., 2021).This classification has been accepted in Britain (Chandler, 2023).
Stomorhina lunata can be easily identified in the field due to its unique appearance.The thorax has three black undusted stripes, and the abdomen has yellow (male) or greyish-yellow (female) markings.The lower part of the face is shining black and strongly protruding as in some species of Syrphidae.Parts of the gena, postgena, occiput, anepisternum and katepisternum are yellow or white, with dense pale hairs.The live flies have stripy eyes, a feature that disappears in dead specimens (Draber-Mońko, 2004;Rognes, 1991;Sivell, 2021).Body length 5-9 mm (Rognes, 1991).
Stomorhina lunata is widely distributed in the Palaearctic, Afrotropical and Oriental Regions, and in Bermuda in the Nearctic Region (Rognes, 1991;Thomas-Cabianca et al., 2023).In Britain it is frequently seen during summer and early autumn.It is widely distributed in southern and central Britain, with a few recent records from Scotland (Rhodes & MacDonald, 2018;Sivell, 2021;Wilkinson, 2022).The flight season is from July to October, with occasional records from June, November, and December (Calliphoridae and Polleniidae Recording Scheme, 2023;Sivell, 2021).This species used to be considered an occasional vagrant in Britain (Colyer, 1947;Dear, 1981;D'Assis-Fonseca, 1947;Van Emden, 1954;Wainwright, 1949), however in the past twenty years it has become more common and widely distributed and is now likely breeding locally (Calliphoridae and Polleniidae Recording Scheme, 2023;Sivell, 2021;Wilkinson, 2022).
The high-quality genome of Stomorhina lunata 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.Here we present a chromosomally complete genome sequence for Stomorhina lunata, based on a female from Hartslock Nature Reserve, England.

Genome sequence report
The genome was sequenced from one female Stomorhina lunata (Figure 1) collected from Hartslock Reserve, Oxfordshire, UK (51.51,.A total of 42-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 155 missing joins or mis-joins and removed 24 haplotypic duplications, reducing the assembly length by 0.84% and the scaffold number by 38.51%, and increasing the scaffold N50 by 0.69%. The final assembly has a total length of 728.1 Mb in 107 sequence scaffolds with a scaffold N50 of 129.6 Mb (Table 1).Most (99.51%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 5 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).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/1606781.

Sample acquisition and nucleic acid extraction
A female Stomorhina lunata (idStoLuna1) was collected from Hartslock Reserve, Oxfordshire, UK (latitude 51.51, longitude -1.11) on 2020-08-20.The specimen was collected by Ryan Mitchell (Natural History Museum) using an aerial net.The specimen was identified by the collector and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idStoLuna1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Thorax tissue 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 abdomen tissue of idStoLuna1 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 head tissue of idStoLuna1 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 (Zhou et al., 2023).The assembly was checked for contamination and corrected using the gEVAL system   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.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016)

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  The assembled genome and annotations serve as invaluable sources for advancing our further understanding of this species and its ecological role.A minor point of contention pertains to the lack of a detailed explanation regarding identifying the X sex chromosome.

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? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect 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.

Figure 2 .
Figure 2. Genome assembly of Stomorhina lunata, idStoLuna1.1: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 728,100,380 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 (186,698,790 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (129,649,560 and 119,364,945 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/idStoLuna1.1/dataset/CAKOFX01/snail.

Figure 5 .
Figure 5. Genome assembly of Stomorhina lunata, idStoLuna1.1:Hi-C contact map of the idStoLuna1.1 alternate haplotype 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=LbSE7mKdTZaujqShK85QNA.

©
2023 Kang L. 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.Le KangInstitute of Zoology, Chinese Academy of Sciences, Beijing, 100101, ChinaThe paper showcases the genome assembly and annotation of Stomorhina lunata, commonly known as the Locust Fly.The genome spans 728.1 megabases and is organized into chromosomal pseudomolecules, encompassing the X sex chromosome.The mitochondrial genome, covering 16.49 kilobases, has also been successfully assembled.Ensembl-based gene annotation unveiled a total of 18,358 protein-coding genes.The sequencing employed Pacific Biosciences single-molecule HiFi long reads, followed by assembly refinement through curation efforts.The genome annotation process involved aligning transcriptomic data and specific UniProt proteins to the genome.This study significantly contributes to the Darwin Tree of Life Project, which aims to sequence all eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

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.