The genome sequence of the narrow-cheeked clusterfly, Pollenia angustigena (Wainwright, 1940) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Pollenia angustigena (the narrow-cheeked clusterfly; Arthropoda; Insecta; Diptera; Polleniidae). The genome sequence is 1370.5 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 21.01 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,930 protein coding genes.


Background
Pollenia angustigena (Diptera, Polleniidae) is a medium-size (4.5-9.5 mm) black fly with a chequered dusting pattern on the abdomen and golden crinkly hairs on the thorax. Until recently it was placed within Calliphoridae, but the Polleninae subfamily was given family status following molecular research by Cerretti et al. (2019). The species was considered a synonym of Pollenia rudis (Fabricius, 1794), (e.g. in Van Emden (1954) it appears as P. rudis f. angustigena), but this was rectified by Rognes (1985).
This species is somewhat variable in colour and morphology (e.g. palpi are yellow to black, the basicosta is usually yellow but can be dark; the facial keel is usually sharp but is occasionally low and blunt). Pollenia angustigena can be confused with other Pollenia species, particularly P. rudis (Draber-Mońko, 2004;Rognes, 1987;Rognes, 1991;Sivell, 2021). The characteristic golden hairs on the posterior and posteroventral surface of the middle and hind femora can occasionally be found in P. rudis (particularly in specimens from central Europe), while the middle tibia, usually with a single anterodorsal (ad) bristle, can sometimes possess 2 to 3 ad, as in P. rudis (Rognes, 1987). Rarely the outer post-humeral bristle may be missing, as in P. griseotomentosa (Jacentkovský, 1944), however other characters should allow for a correct identification (Rognes, 1987). The males can be reliably separated based on the shape of the distiphallus and hairs on the anteroventral surface of the hind tibia, which are reclined in P. angustigena and erect and long in P. rudis (Rognes, 1991;Sivell, 2021). Also, the ground vestiture on the ventral side of the abdomen is reclined, and as dense as on the dorsal side, while in P. rudis the ventral vestiture is erect, and denser and finer than the dorsal vestiture. In females the shape of lateral sacs is distinctive (Rognes, 1991).
Pollenia are parasitoids and/or predators of earthworms. The eggs are laid in small batches on the ground and hatched larvae move through soil in search of a host (Rognes, 1991). The larvae (individually or in small groups) penetrate the worm, usually dorsally. They feed on it internally with their spiracles exposed to the outside. Third instar larva can also feed on the exterior of the worm and can move on to a different host if needed. The larvae pupate in the soil (Keilin, 1915;Rognes, 1987;Yahnke & George, 1972).
The first instar larva of P. angustigena was described by Szpila (2003), the puparium was described by Rognes (1987); the second and third instars remain unknown.
Pollenia angustigena is closely related to P. rudis and P. pediculata Macquart, 1834, as recently shown by molecular phylogenetic analysis using the three molecular markers, COI, Ef-1α and CAD (Szpila et al., 2023).
The high-quality genome of Pollenia angustigena was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for Pollenia angustigena, based on one specimen from Wytham Woods.

Genome sequence report
The genome was sequenced from one female Pollenia angustigena ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.33). A total of 29-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 28-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 56 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the assembly length by 0.35% and the scaffold number by 50.82%, and increasing the scaffold N50 by 17.67%. The final assembly has a total length of 1,370.5 Mb in 30 sequence scaffolds with a scaffold N50 of 255.4 Mb (Table 1). Most (99.96%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 5 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). 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/1266490.

Sample acquisition and nucleic acid extraction
A female Pollenia angustigena (idPolAngu1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.33) on 2020-08-04 by netting. The specimen was collected and identified by Steven Falk (independent researcher) and was then snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The idPolAngu1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax tissue 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. Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 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 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. RNA was extracted from abdomen tissue of idPolAngu1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions. RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit. Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions. Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. DNA and RNA sequencing were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina NovaSeq 6000 (RNA-Seq and 10X) instruments. Hi-C data were also generated from head tissue of idPolAngu1 using the Arimav2 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). One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger 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 as described previously (Howe et al., 2021). Manual curation was performed using HiGlass    Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020). This work was done using Nextflow (Di Tommaso et al., 2017) DSL2 pipelines "sanger-tol/readmapping" (Surana et al., 2023a) and "sanger-tol/genomenote" (Surana et al., 2023b). The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.  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 The genome sequence is released openly for reuse. The Pollenia angustigena 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.