The genome sequence of the St Mark’s fly, Bibio marci (Linnaeus, 1758)

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.


Introduction
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).
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.

Genome sequence report
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.

5.
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.