The genome sequence of a stiletto fly, Thereva unica (Harris, 1780)

We present a genome assembly from an individual female Thereva unica (a stiletto fly; Arthropoda; Insecta; Diptera; Therevidae). The genome sequence is 910.1 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 17.66 kilobases in length.


Background
Thereva unica (Harris, 1780) is a member of the Therevidae (Diptera), known as stiletto flies on account of their conical abdomen.Short pubescence covers most of the body and in some species is brilliant silver but in T. unica is drab brown.This species was long known in Britain as T. bipunctata Meigen, 1820 as there was uncertainty over the identity of Harris's species (Chandler, 1998).His painting is unhelpful, but his description of the female's frons with its two shining black spots is accurate (Harris, 1780) and is similar only in occasional specimens of two rare British species and of the common T. nobilitata (Fabricius).On balance it seems likely that Harris's specimen was the same as Meigen's T. bipunctata.Species of Thereva can be difficult to name accurately, so the genome will help clarify the taxonomy.Larvae of Thereva cannot be identified using morphology so genomic identification will aid ecological research of this life stage.
Thereva unica is found on fixed dunes on the coast from Cumbria to Yorkshire, with disjunct populations in Scotland, including the Outer Hebrides.Inland it is found in dry sandy heaths in the Surrey area, the Breckland of East Anglia and isolated heaths elsewhere in England.
Therevid larvae are long, thin, and fairly featureless, but are remarkable for possessing a smooth dry cuticle which aids their 'swimming' through particulate substrates, rather like a stiff eel.They are hunting predators, detecting their prey by its vibrations and subduing it rapidly with a venom (Smith, 1989;Stubbs & Drake, 2014).They are probably unspecific in their choice of prey, taking any other arthropods and worms although Irwin and Lyneborg (1981) state that beetle larvae are preferred.In related species living in dry sand, prey captured on the surface is dragged back into the sand (MD, pers.obs.).The feeding behaviour of adults is unclear but males of some species of Thereva swarm, so an energy intake would be necessary.

Genome sequence report
The genome was sequenced from one female Thereva unica (Figure 1) collected from Loe Valley (River Cober), England, UK (50.09, -5.29).A total of 36-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 249 missing joins or mis-joins and removed 48 haplotypic duplications, reducing the assembly length by 2.35% and the scaffold number by 18.25%, and increasing the scaffold N50 by 70.63%.
The final assembly has a total length of 910.1 Mb in 940 sequence scaffolds with a scaffold N50 of 167.4 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (92.52%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 6 autosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The exact order and orientation of the scaffolds in the repetitive centromeres is unknown.As it is a female XX sample without a comparator species, the X chromosome is unidentified.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.

Sample acquisition and nucleic acid extraction
Thereva unica specimens was collected using an aerial net from Loe Valley (River Cober), England, UK (latitude 50.09, longitude -5.29) on 2021-06-29.The specimens were collected   by Martin Drake and identified by Chris Spilling (both of the Dipterists Forum) and preserved by dry freezing at -80°C.The sample with specimen ID NHMUK014537453 (ToLID idThe-Unic2), a female, was used for DNA sequencing was, and the sample with specimen ID NHMUK014537421 (ToLID idTheUnic1), a male, was used for Hi-C sequencing.SEQUEL II instrument.Hi-C data were also generated from head and thorax tissue of idTheUnic1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (  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.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome 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 • Legality 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,

Alex Makunin
Wellcome Sanger Institute, Hinxton, England, UK The data note by Drake et al. presents the first reference genome assembly for the representative species stiletto fly family, Thereva unica.The report follows the standard structure for DToL project genome notes and it looks comprehensive and cohesive.
Interesting Hi-C patterns resembling inversions can be seen in chromosome 3 (FIgure 5).

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Although the sex chromosome was not identified at this time, the Thereva nobilitata genome (GCA_963855945.1,released by DToL a few months later) suggested X chromosome is the smallest chromosome based on synteny with idTolCing1 (Tolmerus cingulatus) representing the same subfamily Asiloidea.Synteny between the two Thereva species genomes indicate chromosome 6 of Thereva unica as X chromosome.Interestingly, this chromosome also has slightly elevated coverage and GC content (Figure 3).

Benoit Nabholz
Universite de Montpellier, Montpellier, Occitanie, France The manuscript presents the genome of a Therevidae fly, Thereva unica, produced by the Darwin Tree of Life project (DToL).Once again, I applaud the project for rapidly releasing all the information and data transparently.This project is a model of open science and should set the standard for consortium genome projects.This is the first genome published for the family (as opposed to the >500 assemblies currently available for Drosophila).
I have very few comments.I have check all the links and all the data is available.The genome is chromosome scale.
Here are my minor comments : The coverage of long reads is somewhat lower compared to other genomes I have reviewed for the project, mostly butterflies.Here, the coverage is 36X, whereas it was above 45X for most of the other genome assemblies I have checked (except Hydrotaea diabolus, which is another fly).For instance, the coverage of the other unpublished Thereva genome exceeds 99X, as noted on the NCBI website : https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_963855945.1/.I wonder if this lower coverage will eventually lead to assembly errors, and I'm a bit concerned about that.Maybe it is enough using HiFi technology?

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The coverage information for Hi-C data should be provided.The authors provided a clear research motivation when preparing the dataset, thoroughly explaining the ecological and taxonomic significance of sequencing the genome of Thereva unica.
The experimental protocols are appropriate and technically sound, including methods for sample collection, DNA extraction, and sequencing, which ensure the rigor and reliability of the research.However, in terms of the presentation and accessibility of the dataset, although the data is publicly available, the completeness of chromosome mapping is slightly lacking, which might affect the usability of specific analyses.From the standpoint of a simple data note, the overall quality of the genome assembly is satisfactory, but the genome annotation results are not accessible via the provided links.Overall, this article performs well in many critical aspects, but there is still room for improvement in the clarity and ease of use of the data presentation.
The data is accessible, but the completeness of chromosome mapping(92%) is slightly below the standard(95%), the genome annotation results are not accessible via the provided links.which could affect usability for certain types of analyses.

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?Partly Competing Interests: No competing interests were disclosed.

Figure 2 .
Figure 2. Genome assembly of Thereva unica, idTheUnic2.1: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 910,162,612 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 (258,850,354 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (167,368,244 and 26,588,518 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/idTheUnic2_1/dataset/idTheUnic2_1/snail.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.The sample was prepared for DNA extraction in the Tree of Life core laboratory.The idTheUnic2 sample was weighed and

Figure 3 .
Figure 3. Genome assembly of Thereva unica, idTheUnic2.1: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/idTheUnic2_1/dataset/idTheUnic2_1/blob.

Figure 4 .
Figure 4. Genome assembly of Thereva unica, idTheUnic2.1: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/idTheUnic2_1/dataset/idTheUnic2_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Thereva unica, idTheUnic2.1:Hi-C contact map of the idTheUnic2.1 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=IU_qBrpWToWNrAiItglZPA.
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.

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Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.ReviewerExpertise: 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 16 May 2024 https://doi.org/10.21956/wellcomeopenres.23047.r78805© 2024 Nabholz B. 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.

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
○IsCompeting 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.
https://doi.org/10.21956/wellcomeopenres.23047.r82503© 2024 Du J. Juan Du Department of Entomology and MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China