The genome sequence of a bluebottle, Calliphora vomitoria (Linnaeus, 1758)

We present a genome assembly from an individual male Calliphora vomitoria (a bluebottle; Arthropoda; Insecta; Diptera; Calliphoridae). The genome sequence is 708 megabases in span. Most of the assembly is scaffolded into six chromosomal pseudomolecules, including the assembled X sex chromosome. The mitochondrial genome has also been assembled and is 16.2 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,917 protein coding genes.


Background
Calliphora vomitoria belongs in the Diptera family Calliphoridae (blowflies).The species from the genus Calliphora are commonly called bluebottles due to their shiny metallic appearance.Calliphora vomitoria is easily identifiable due to the orange hairs on the postgena and lower part of gena (a 'ginger beard') that contrast with the black base colour (covered with grey dusting) on those parts of the head.The lower calypters are brown with a white rim and with dark brown hairs on the upper side.The anterior thoracic spiracle and basicosta are dark brown to black (Rognes, 1991;Sivell, 2021).This species is common and widely distributed in Britain (Davies, 1990;Davies, 1999;Davies & Laurence, 1992;MacLeod, 1943;MacLeod, 1963;MacLeod & Donnelly, 1956;Sivell, 2021); although less synanthropic than C. vicina (Draber-Mońko, 2004;Hwang & Turner, 2005).It prefers shady sites and is common in woodland (Davies, 1999;Green, 1951;MacLeod & Donnelly, 1956;van Emden, 1954) and uplands (Davies, 1990), up to 1070 m (Sivell, 2021), also in gardens and parks.The flight period is from March to October/November, although adults are occasionally seen during winter on warmer days (Sivell, 2021).The adults are attracted to carrion, faeces, stinkhorn fungus (Phallus impudicus Linnaeus, 1753), flowering plants and ripe fruit; they feed mainly on sugar.
Calliphora vomitoria is oviparous and anautogenous: females require a protein meal to reach maturity and produce eggs (Rivers & Dahlem, 2014).The larvae are saprophagous and feed on carrion.A female fly oviposits on the carcass, choosing shaded and moist locations (e.g. the mouth, ears, eyes, anus) to avoid desiccation of the eggs.This species has a strong preference for large carrion such as sheep (Davies, 1990, Davies, 1999) or human bodies, making it potentially useful in forensics.Its developmental rates have been researched using populations from North America (Greenberg & Tantawi, 1993;Kamal, 1958) and Europe (Marchenko, 2001;Niederegger et al., 2010;Niederegger et al., 2013) including Britain (Davies & Ratcliffe, 1994).Calliphora vomitoria can also cause secondary myiasis in living animals which have a primary infestation from another species.This is most frequent in sheep and is referred to as 'sheep-strike' (Haddow & Thomson, 1937;MacLeod, 1943;Morris & Titchener, 1997;Taylor et al., 2013).Calliphora vomitoria was also reported from a case of orbital myiasis in a human in USA (Wood & Slight, 1970).

Genome sequence report
The genome was sequenced from one male Calliphora vomitoria specimen (Figure 1) collected from Hever Castle, UK (51.188, 0.12).A total of 54-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 36 missing joins or mis-joins and removed five haplotypic duplications, reducing the assembly length by 0.53% and the scaffold number by 8.05%, and increasing the scaffold N50 by 1.38%.
The final assembly has a total length of 707.6 Mb in 137 sequence scaffolds with a scaffold N50 of 130.6 Mb (Table 1).Most (99.04%) of the assembly sequence was assigned to six chromosomal-level scaffolds, representing five 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).The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 99.1% using the OrthoDB v10 Diptera reference set (n = 3,285).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male Calliphora vomitoria (idCalVomi1) was collected from Hever Castle (latitude 51.188, longitude 0.12) on 27 August 2020.The specimen was caught by Olga Sivell (Natural History Museum) using an aerial net.The specimen was also identified by Olga Sivell and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The idCalVomi1 sample was weighed and  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 head tissue of idCalVomi1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Calliphora vomitoria assembly (GCA_942486065.1).Annotation was created primarily

Xin Liu
State Key Laboratory of Agricultural Genomics, BGI (Beijing Genomics Institute)-Shenzhen, Shenzhen, China Dr. Olga Sivell present a genome assembly from an individual male Calliphora vomitoria, which is an important genome data for this species study.There are some points should be revised.
The author has employed hic data to assemble the genome at the chromosome level and has assigned chromosome numbers based on their lengths, considering the shortest chromosome as the X chromosome.However, it is recommended to provide additional evidences, such as marker genes of X chromosome or collinearity with other model species, to support the claim that the shortest chromosome indeed corresponds to the X chromosome. 1.
The manuscript includes data from PacBio HiFi CCS, 10x genomics, and HiC libraries, but lacks an explanation of how the data from 10x genomics was utilized in the assembly method.It is advised to provide a detailed description, possibly in the form of a flow chart, that outlines the software, version, and parameters employed in each step of the assembly process.

2.
The YaHC tool, which the author used for assembling chromosomes, requires an update since the article has been published in Bioinformatics, while the author cited the reference from bioRxiv.Furthermore, the heat map in Figure 5 should clearly mark the boundary points of each chromosome as it is currently unclear.

3.
In the abstract, the author mentions the assembly of the mitochondria; however, there is a lack of description regarding the mitochondrial genome and gene annotation in the Results.
It is recommended to address this gap and provide the necessary details.

4.
The general genome annotation should include the annotation of repeat sequences.Therefore, it is suggested that the author supplements the annotation results and provides a description of the methodology employed for annotating repeat sequences.

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

Maxwell J Scott
North Carolina State University, Raleigh, USA The authors report a very high-quality assembly of the genome of the blow fly, Calliphora vomitoria .I have a few minor comments for the authors to consider: Since this is a genome paper, some information in the Background on the expected number of chromosomes and genome size if known (or genome sizes for other flies in the genus).Most blow flies have five autosomes and X and Y sex chromosomes.There may not be any cytogenetic studies published for this species but other Calliphora species have been studied.

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In the methods 10XGeneomics libraries were made in addition to the PacBio.However, it is not clear from the methods or results how the 10X reads contributed to the final assembly.

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How was the X chromosome scaffold identified?Since DNA was only sequenced from a single male, I assume this was done by comparison to other blow fly genomes.
○ Some additional labels on Figure 2 would help understand what is quite a complex figure.
○ I think the X chromosome scaffold is the small square at the bottom right of Figure 5.If so, a label and arrow pointing to this part of the plot would be helpful.Reviewer Expertise: Insect genetics 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
Figure 1.Calliphora vomitoria (Linnaeus, 1758) specimen NHMUK014444469.Photographs taken during sample preservation and processing.a) The specimen in dorsal view.b) The specimen in lateral view.Photographs by O. Sivell.© Trustees of the Natural History Museum, London.

Figure 2 .
Figure 2. Genome assembly of Calliphora vomitoria, idCalVomi1.2: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 706,678,423 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 (177,447,591 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 sequence lengths (131,076,759 and 116,750,479 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/idCalVomi1.1/dataset/CALNXL01/snail.

Figure 5 .
Figure 5. Genome assembly of Calliphora vomitoria, idCalVomi1.2:Hi-C contact map.Hi-C contact map of the idCalVomi1.2assembly, 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=P7Xf5smoSJG-hLdBKd_gtw.

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

Table 3 . Software tools and versions used. Software tool Version Source
Ethics/compliance issuesThe 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.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.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, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.
through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Peer Review Current Peer Review Status: Version 1
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.
Reviewer Report 19 June 2023 https://doi.org/10.21956/wellcomeopenres.20947.r58999© 2023 Scott M. 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.