key: cord-0809733-6yw27w6k
authors: Sivell, Olga; Sivell, Duncan
title: The genome sequence of the St Mark’s fly, Bibio marci (Linnaeus, 1758)
date: 2021-10-22
journal: Wellcome Open Res
DOI: 10.12688/wellcomeopenres.17265.1
sha: cd380c4bc0a70592fb9a6363d44110155d4168e7
doc_id: 809733
cord_uid: 6yw27w6k

We present a genome assembly from an individual male Bibio marci (the St Mark’s fly; Arthropoda; Insecta; Diptera; Bibionidae). The genome sequence is 340 megabases in span. The complete assembly is scaffolded into six chromosomal pseudomolecules, with the X sex chromosome assembled.

Bibio marci (Diptera, Bibionidae), known as the St Mark's fly, is a common and widely distributed species in Britain and Ireland. It can be found in grassland and at woodland edges, with a preference for lowland sites. The single flight period occurs in spring, from April to June in Britain and from May to June in Ireland (Chandler, 2017; D'Arcy-Burt & Chandler, 1987; Freeman & Lane, 1985) .

The males of B. marci swarm around hedges and high around trees, while females and mating pairs can be seen on vegetation (D'Arcy-Burt & Chandler, 1987; Freeman & Lane, 1985) . A gravid female digs up a cell in the soil, into which all of her eggs are oviposited in a single clutch. The female dies soon afterwards. The adult lifespan is short, likely less than a week (Skartveit, 1997) . The eggs hatch after approximately one month (Freeman & Lane, 1985) . The larvae of B. marci require humid conditions, are gregarious and can be found in the upper layers of soil, leaf litter, manure and in vegetable matter. They feed on decaying vegetation and the subterranean parts of living plants (Freeman & Lane, 1985; Skartveit, 1997) . Pupation occurs in cells dug out by larvae in the soil or rotten wood, with one pupa per cell (Allen, 1975; Skartveit, 1997) . This stage lasts about three weeks, after which the adult flies emerge (Freeman & Lane, 1985) .

Larvae of B. marci are occasional pests of grass, cereals, and other crops (D'arcy-Burt & Blackshaw, 1991) . They have been reported causing damage to celery, asparagus, rose, lettuce, grass (lawn), Saxifraga and Polyanthus in Britain (Anderson, 1919; Edwards, 1925; Freeman & Lane, 1985) , and potato tubers in Ireland (Carpenter, 1920) . B. marci larvae are an important food source for birds, particularly game birds (Freeman & Lane, 1985; Parmenter, 1941) .

The high-quality genome sequence described here is the first to be reported for Bibio marci and has been generated as part of the Darwin Tree of Life project. The sequence will aid understanding of the biology, physiology and ecology of the species.

The genome was sequenced from a single male B. marci collected from Wigmore Park, Wigmore, Luton, England (latitude 51.88378, longitude -0.36861422). A total of 53-fold coverage in Pacific Biosciences single-molecule long reads and 127-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 14 missing/misjoins, reducing the scaffold number by 57.14%.

The final assembly has a total length of 340 Mb in six sequence scaffolds with a scaffold N50 of 54.6 Mb ( Table 1 ). The complete assembly sequence was assigned to chromosomallevel scaffolds, representing five autosomes (numbered by sequence length) and the X sex chromosome (Figure 1-Figure 4 ; Table 2 ). B. marci has an unknown sex chromosome system and no Y chromosome was recovered, despite the fact that the specimen was identified as male. However, there is no strong evidence to indicate that X and Y have been incorrectly merged together: the X chromosome has good contiguity and there is no evidence of misassembly. There is also good Hi-C linking across scaffold gaps. The mitochondrial genome was also assembled and is 13.2 kb in length. was provided by Duncan Sivell, Natural History Museum, London, based on (Freeman & Lane, 1985) . The sample was snap-frozen using dry ice and stored in a CoolRack. The sample was collected during a Covid-19 lockdown and owing to logistical issues, no image of the sample was taken.

DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilGlaAlex1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Tissue from the abdomen was disrupted to a fine powder using a powermasher. Fragment size analysis of 0.01-0.5 ng of DNA was then performed using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200-ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size between 12-20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solidphase 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. Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were generated from abdomen tissue in the WSI Tree of Life Laboratory using the Arima v2.0 kit and sequenced in the Scientific Operations core on an Illumina NovaSeq 6000 instrument.

Assembly was carried out with Hifiasm (Cheng et al., 2021); haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012) . The assembly was then scaffolded with Hi-C data (Rao et al., 2014 ) using SALSA2 (Ghurye et al., 2019 . The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021) . Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021) . The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020) . Table 3 contains a list of all software tool versions used, where appropriate. The genome sequence is released openly for reuse. The B. marci genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 .

The voucher specimen has been accessioned at the Natural History Museum, London, under accession number NHMUK014111013.

and Y chromosome contigs should be mentioned in the methods (if nothing else, this confirms the fly as possessing only one X chromosome).

I appreciate that no photo of the sample is available, but I think for context it would be really nice to include a photo of this species, albeit not the actual sample sequenced. I would also like to see a one sentence description (or even a photo!) of the collection habitat.

There is no mention of RNAseq, although I believe this is standard for DToL genomes. Is RNAseq data from this sample available? PRJEB45104 suggests that it is, but it is not mentioned here.

6.

Bibionid and Scatopsid Flies. Diptera: Bibionidae & Scatopsidae. Bibionid and Scatopsid Flies. Diptera: Bibionidae & Scatopsidae

Identifying and Removing Haplotypic Duplication in Primary Genome Assemblies

PubMed Abstract | Publisher Full Text | Free Full Text Parmenter L: Bibio Marci L. Captured by Garden Warbler. Entomologist's Monthly Magazine

A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping

BUSCO: Assessing Genome Assembly and Annotation Completeness with Single-Copy Orthologs

marcelauliano/MitoHiFi: mitohifi_v2.0. 2021. Publisher Full Text

Utilization of Dietary Protein in the Litter-Dwelling Larva of Bibio marci (Diptera: Bibionidae)

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

Current Peer Review Status: Version 1 Reviewer Report 19 January 2022 https://doi.org/10. 21956/wellcomeopenres.19083.r48111 © 2022 Obbard D. 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.

Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK This data note reports the sequencing and assembly of the genome of Bibio marci as part of the "Darwin Tree of Life" programme. In common with other data notes from this research effort, the reporting is standardised and quite brief. As such, I have very few comments to make.The approach is state-of-the-art, the raw data appear to be of a suitably high quality, and the assembly methods are appropriate. The provision of a voucher specimen is excellent. The public availability of raw data and genome assembly is excellent. The resulting genome is likely to be of very high quality, and I have no doubt that it will be of great value to any researchers working on this group of lies, or on the comparative or evolutionary genomics of insects more generally. The writing is clear, concise, and very easy to read. The authors have also managed to include sufficient allusion to the biology and research context of this fly to position the data note within the larger research literature.Below, I note a few inconsistencies or points that could usefully be expanded upon:It would be good to refer to recent research literature to provide more biological context (and useful cross links within the literature). I wonder whether an additional sentence in the introduction outlining the work by Jan Frouz and colleagues (Frouz et al. ( 2019 1 ), Frouz et al.( 2015 2 )) would be appropriate?

Latitude and longitude are given to five and eight decimal places, respectively. This is an implied precision of approximately 1.1m and 0.7mm. Such precision seems unrealistic for a netted fly, where four decimal places (nearest 10m) seems more likely.

I know it is standard DToL methodology, but I would like to see "Manual assembly curation corrected …" expanded into 2-3 sentences, explaining how curation was done.3.The absence of an assembled Y chromosome is extremely interesting, and probably warrants a mention in the abstract. I assume the X chromosome is the 'blob' that has half the coverage of the rest of the genome? I think the use of coverage to identify potential X 4.

Are the datasets clearly presented in a useable and accessible format? YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Evolutionary genomics of Drosophila and their pathogens.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.