The genome sequence of the hazel leaf-roller, Apoderus coryli (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Apoderus coryli (the hazel leaf roller; Arthropoda; Insecta; Coleoptera; Attelabidae). The genome sequence is 428 megabases in span. The majority (98.90%) of the assembly is scaffolded into 20 chromosomal pseudomolecules, with the X and Y sex chromosomes assembled.


Background
The hazel leaf-roller, Apoderus coryli, is a distinctive leafrolling weevil which is widespread and locally common across Europe and western Asia. In the UK, it is common in deciduous woodland across England and Wales, with a few records from southern Scotland. The main host plant is common hazel, Corylus avellana, although it is also known to infrequently feed on alder, birch, hornbeam and beech. It may be considered a minor pest of hazelnut cultivation (Gantner, 2000). Adults are 6-8 mm with red elytra, pronotum and bases of the femora. The head, underside and remainder of the legs are black. It has a distinctive elongate head and 'neck'. Adults are active from May, feeding on hazel leaves in which they leave characteristic round holes. Mating and oviposition occurs in early summer. Females lay a single bright orange egg around 1 mm in size on a leaf before cutting a line across the leaf with their mandibles and rolling it into a cylinder around the egg. The leaf rolling process may take 60-90 minutes and a single female may repeat this for up to 30 eggs across several days (Urban, 2014). Larvae develop and pupate within these leaf rolls. Second generation adults are active in late summer, the larvae of which overwinter within leaf rolls that drop to the ground and pupate in the spring. A previous study has shown that this species has 13 autosomal bivalents, that sex chromosomes exhibit a parachute association and that this species possesses a small amount of heterochromatin (Rozek et al., 2004).

Genome sequence report
The genome was sequenced from one male A. coryli (Figure 1) collected from Wytham Woods, Oxfordshire (biological vicecounty: Berkshire), UK (latitude 51.770, longitude -1.339). A total of 59-fold coverage in Pacific Biosciences single-molecule long reads and 93-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 57 missing/misjoins and removed 7 haplotypic duplications, reducing the assembly length by 0.41% and the scaffold number by 24.71%, and increasing the scaffold N50 by 24.75%.
The final assembly has a total length of 428 Mb in 128 sequence scaffolds with a scaffold N50 of 23.7 Mb ( Table 1). The majority, 98.90%, of the assembly sequence was assigned to 20 chromosomal-level scaffolds, representing 18 autosomes (numbered by sequence length), and the X and Y sex chromosome (Figure 2- Figure 5; Table 2). The assembly has a BUSCO v5.1.2 (Simão et al., 2015) completeness of 99.3% (single 97.7%, duplicated 1.6%) using the endopterygota_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and DNA extraction
A single male A. coryli was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.770, longitude -1.339) by Liam Crowley, University of Oxford, using a pooter. The sample was snap-frozen on dry ice and stored using a CoolRack.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The icApoCory1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Tissue from the whole organism was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. 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 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 and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were generated from further whole organism tissue using the Arima v2 Hi-C kit and sequenced on a HiSeq X instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021); haplotypic duplication was identified and removed with purge_ dups (Guan et al., 2020) with the -e flag. One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with  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) and annotated with MitoFinder (Allio et al., 2020). 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 A. coryli 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. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.