The genome sequence of the Squinting Bush Brown, Bicyclus anynana (Butler, 1879)

We present a genome assembly from an individual female Bicyclus anynana (the Squinting Bush Brown; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 457.2 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.1 kilobases in length.


Background
Bicyclus anynana, commonly known as the Squinting Bush Brown, is a widely distributed, medium-sized, Afrotropical satyrid butterfly, inhabiting grasslands and forest edges. Its seasonally plastic phenotype and modular pattern of wing eye spots (Figure 1), combined with ease of rearing in captivity, have made it a model system for evo-devo and life-history research (Brakefield et al., 2009). In nature, the primary larval host plant is Oplismenus compositus, and the adults feed on rotting fruit; the laboratory stocks have been domesticated onto maize and wheatgrass.
The standard karyotype consists of WZ females and ZZ males (n = 28), but the W chromosome is exceptionally small and remains to be assembled (Beldade et al., 2009;Van't Hof et al., 2008). The present genome is the third independent assembly (Murugesan et al., 2022;Nowell et al., 2017), which will support ongoing research on a wide range of fundamental questions. These include the genetics and development of wing colour pattern (Prakash et al., 2022), regulation of phenotypic plasticity (Tian & Monteiro, 2022), responses to climate change (Oostra et al., 2018), genetics of inbreeding depression (Saccheri et al., 2020), and sex-determination mechanisms.

Genome sequence report
The genome was sequenced from one female Bicyclus anynana from a collection at the University of Liverpool. A total of 20-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 117 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the scaffold number by 32.52%.
The final assembly has a total length of 457.2 Mb in 83 sequence scaffolds with a scaffold N50 of 17.4 Mb (Table 1). Most (99.51%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 autosomes and the Z 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/110368.

Sample acquisition and nucleic acid extraction
Three Bicyclus anynana (ilBicAnyn1, ilBicAnyn2 and ilBicAnyn3) were collected from laboratory stock at the University of Liverpool on 15 August 2015. The specimens were killed and stored in a -80°C freezer.
The samples were prepared and DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilBicAnyn1 sample was weighed and dissected on dry ice. 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. 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 whole organism tissue of ilBicAnyn3 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   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) instruments. Hi-C data were also generated from whole organism tissue of ilBicAnyn2 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). 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.
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

Ethics and compliance issues
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner. The submission   The genome sequence is released openly for reuse. The Bicyclus anynana 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 using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.