The genome sequence of the orange ladybird, Halyzia sedecimguttata (Linnaeus, 1758)

We present a genome assembly from an individual Halyzia sedecimguttata (the orange ladybird, Arthropoda; Insecta; Coleoptera, Coccinellidae). The genome sequence is 919.1 megabases in span. Most of the assembly is scaffolded into 10 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 21.0 kilobases in length. Gene annotation of this assembly on Ensembl identified 27,547 protein coding genes.


Background
The mildew-feeding orange ladybird, Halyzia sedecimguttata, was restricted to ancient woodland within the UK but in recent decades has been found on deciduous trees, including sycamore and ash, in many diverse habitats. Orange with sixteen white spots arranged in rows along the length, the adult beetle has distinct translucent edges. The larvae of the orange ladybird are almost fluorescent, with rows of black bristly tufts along the upper surface and black tips to the legs. The larvae are active much later than those of other ladybirds in the UK with the sculptured black pupae being seen into early winter. Some people may have been fortunate to observe tiny yellow fruiting bodies on the surface of ladybirds. This stunning fungal parasite of ladybirds was classified as one species -Hesperomyces virescens Thaxt., but recently this fungus has been revealed, through multi-locus phylogenetic analyses coupled with sequence-based methods, to be a complex of many species (Haelewaters et al., 2018). One isolate collected from Halyzia sedecimguttata has been named Hesperomyces halyziae (Haelewaters & Kesel, 2020) and is considered a sister to H. virescens s.l. from Harmonia axyridis, the harlequin ladybird. Harmonia axyridis is an invasive non-native species in many parts of the world (Roy et al., 2016) and appears to be causing distribution declines of Halyzia sedecimguttata and other ladybirds in the UK (Roy et al., 2012).

Genome sequence report
The genome was sequenced from one Halyzia sedecimguttata specimen ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -1.34). A total of 55-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 46-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 86 missing joins or mis-joins and removed 20 haplotypic duplications, reducing the assembly length by 1.38% and the scaffold number by 43.27%, and increasing the scaffold N50 by 0.16%] The final assembly has a total length of 919.1 Mb in 59 sequence scaffolds with a scaffold N50 of 111.0 Mb (Table 1). Most (99.91%) of the assembly sequence was assigned to 10 chromosomal-level scaffolds, representing 9 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). Heterozygous inversions were observed at ~110 Mb on chromosome 2, ~57 Mb on chromosome 5 and ~26 Mb on chromosome 8. The specimen is most likely male, as there is half coverage of the X chromosome ( Figure 5), and karyotyping data indicate that the Y chromosome is very small.
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/347359.

Sample acquisition and nucleic acid extraction
A Halyzia sedecimguttata specimen (icHalSede1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 29 January 2020. The specimen was found on a beech trunk by Liam Crowley (University of Oxford) and collected by potting. The specimen was identified by the collector and then snap-frozen on dry ice. This specimen was used for genome sequencing and Hi-C scaffolding.
A second Halyzia sedecimguttata specimen (icHalSede3) was collected from Parsons Green, London UK (latitude 51.48, longitude -0.18). The specimen was potted by Maxwell Barclay (Natural History Museum). The specimen was identified by the collector and then snap-frozen at -80°C. This specimen was used for RNA sequencing. DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The icHalSede1 sample was weighed and dissected on dry ice, setting aside tissue for Hi-C sequencing. Whole organism 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 icHalSede3 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions. RNA was 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

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) and Illumina NovaSeq 6000 (RNA-Seq and 10X) instruments. Hi-C data were also generated from tissue of icHalSede3 that had been set aside, using the Arima2 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 YaHS (Zhou et al., 2023). The assembly was checked for contamination 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. To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020). The genome was analysed within the BlobToolKit environment (Challis et al., 2020) andBUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated. Table 3 contains a list of software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Halyzia sedecimguttata assembly (GCA_937662695.1). in Ensembl Rapid Release.