The genome sequence of a tachinid fly, Cistogaster globosa (Fabricius, 1775)

We present a genome assembly from an individual male Cistogaster globosa (a tachinid fly; Arthropoda; Insecta; Diptera; Tachinidae). The genome sequence is 837.8 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 16.97 kilobases in length. Gene annotation of this assembly on Ensembl identified 29,591 protein coding genes.

Cistogaster globosa larvae are parasites of shieldbugs of the genus Aelia (Raper & Smith, 2002).Eggs are laid on the dorsal surface of the host's abdomen, after which the larva leave the host to pupate on the ground (Khitsova & Golub, 2014).In Britain, C. globosa has only been reared from A. acuminata but is known to target various Aelia species in other habitat domains (Raper & Smith, 2002).The fly can often be observed settled on low growing umbels in dry grasslands, the same habitat in which its host can be found (Howe & Woodman, 2001;Raper & Smith, 2002).C. globosa was previously awarded a national RDB1 status in 1987 but subsequently saw rapid expansion across the UK (Falk, 1991;Gibbs, 2007;Shirt, 1987).This is thought to be in response to a warming climate, and has led to a proposed downgrade to RDB2 status (Raper & Smith, 2002).
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 and ecology of C. globosa.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 male Cistogaster globosa (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 25-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 63 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the assembly length by 0.23% and the scaffold number by 3.1%, and increasing the scaffold N50 by 0.34%. The final assembly has a total length of 837.8 Mb in 1033 sequence scaffolds with a scaffold N50 of 122.9 Mb (Table 1).Most (83.68%) 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).In chromosome 3, the order and orientation of scaffolds is uncertain in the region 26.94 to 30.60 Mb.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/1918221

Sample acquisition and nucleic acid extraction
The specimen used for genome sequencing was a male C. globosa (specimen ID Ox000765, individual idCisGlob1) was collected from Wytham Woods, Oxfordshire (biological vice-country Berkshire), UK (latitude 51.77, longitude -1.33) on 2020-08-04 by netting.The specimen used for Hi-C data was a male C. globosa (specimen ID Ox001751, individual idCisGlob2), netted in the same location on 2021-07-17.Both specimens were collected and identified by Steven Falk (University of Oxford), and were preserved on dry ice.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.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on the Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from whole organism tissue of idCisGlob2 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
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Cistogaster globosa assembly (GCA_937654795.1) in Ensembl Rapid Release.

Charles Plessy
Okinawa Institute of Science and Technology, Okinawa, Japan This data note follows the standard structure of the Darwin Tree of Life Consortium and is easy to follow.The primary assembly ticks most of the checkboxes of quality except perhaps for the number of scaffolds, which is above a thousand.I am concerned by the alternate haplotype assembly (GCA_937654215.1),which is longer of ~300 Mb.This does not make sense to me as 1) these haplotypes were sequenced from a single individual and 2) the primary assembly contains both the X and the Y chromosomes.Therefore if there were any significant difference in length, I would expect the alternate assembly to be shorter.Can the authors explain the difference or double-check the alternate haplotype assembly for duplications or contaminations?
Is the rationale for creating the dataset(s) clearly described?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: 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, however I have significant reservations, as outlined above.

Arun Arumugaperumal
Department of Biotechnology, Rajalakshmi Engineering College, Chennai, Tamilnadu, India Several tachinid fly genomes were sequenced and reported as part of Darwin Tree of Life project.This one is Cistogaster globosa (Fabricius, 1775).A google scholar search with keywords " Cistogaster globosa genome" did not yield any other report other than this.So this should be the first whole genome sequence of the fly.The data article reports an assembly of length 837.8 Mb covering 7 chromosomes.Also a mitogenome of 16.97 kb was reported.The authors annotated a total of 29,591 genes in the genome.
The photograph of the insect presented was not focussed well.The tube was focused and the insect was out of focus.
Long read technology was used for the sequencing experiment.The authors have used PacBio Sequel II platform with 25X coverage.The contig N50 value was 4.1 Mb which is very good for annotation.The authors have used Ensembl to automatically annotate their genome data.The genome assembly completeness was checked using BUSCO analysis.The analysis results show that the assembly at hand is about 98.7 % complete.Taken all together, the data reported herewith is of huge importance when it comes to designing molecular biology experiments on this organism.
Since several of the tachinid fly genomes are available, the authors could have made a comparison or could have estimated the pan-genome.

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

Jaakko Pohjoismäki
Department of Environmental and Biological Sciences, University of Eastern Finland, Joensuu, Finland This genome note presents another nice addition to the growing list of reference genomes from the DToL.Unlike many other bug-killing flies (Phasiinae), Cistogaster globosa is relatively easy to identify, even from the rather small image in the figure 1.As the species is sexually dimorphic (females are almost entirely shining black), it is possible to confirm the sex of the specimen to be male, as stated.This notable sexual dimorphism might be actually noteworthy to mention in the background?
I am surprised to read that the species has been having some endangered status.To my experience it is not very particular with its habitat demand as Aelia are all around where there's a bit of low growth, dryish grasslands, such as roadsides, suburban wastelands etc.
I had a hard time in finding out what RDB 1 and 2 mean, this could have been made more clear.As googling was futile, I asked ChatGTP, who considered RBD 1 comparable to CR and RBD 2 to VU in the IUCN terminology.I hope she got it right.
For readers outside of the UK, these details would be interesting to know.
The genome report looks good.Waiting to get to browse through the genes online.
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: Diptera, biodiversity, 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 Cistogaster globosa, idCisGlob1.1: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 837,775,133 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 (175,753,085 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (122,899,074 and 327,718 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/idCisGlob1.1/dataset/CALMVC01/ snail.

Figure 5 .
Figure 5. Genome assembly of Cistogaster globosa, idCisGlob1.1:Hi-C contact map of the idCisGlob1.1 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=I5N2JMIbSj2thdM1paISdg.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Cistogaster globosa, idCisGlob1. INSDC accession Chromosome Length (Mb) GC%
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', 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:

Table 3 . Software tools: versions and sources.
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.

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.
https://doi.org/10.21956/wellcomeopenres.22063.r68728© 2023 Pohjoismäki J.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.