The genome sequence of the wall brown, Lasiommata megera (Linnaeus, 1767) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Lasiommata megera (the wall brown; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 488 megabases in span. The majority of the assembly (99.97%) is scaffolded into 30 chromosomal pseudomolecules with the W and Z sex chromosomes assembled. The complete mitochondrial genome was also assembled and is 15.3 kilobases in length.


Background
The wall brown, Lasiommata megera (Linnaeus 1767), is a widely distributed butterfly found across the Palearctic. This species inhabits open sunny places such as grasslands and sand dunes, and is known for basking on bare surfaces such as walls and rocks. Larvae feed on various grasses including false broom (B. sylvaticum), tor-grass (Brachypodium pinnatum) and bents (Agrostis spp.). Forewings possess a single large eyespot, and hindwings contain four smaller eyespots, set against orange and brown markings. This species is generally bivoltine; adults can be found on the wing from May to October.
In the British Isles, this butterfly is widespread but scarce, with a higher density towards the coast. Since the 1970s, the wall brown has experienced a major decline in both abundance and occurrence in the British Isles, with an 87% decrease in abundance in southern Britain due to loss of colonies (Fox et al., 2015). The Wall has also experienced a significant decline in abundance across Europe based on the European Grassland Butterfly Indicator (van Swaay et al., 2013). One potential explanation for this decline is that warmer conditions due to climate change, may be triggering a third generation, resulting in a high mortality rate in autumn (Van Dyck et al., 2015). Other changes in the environment, such as nitrogen deposition, have also been implicated in their decline (Klop et al., 2015). The Wall has an estimated genome size of 381 Mb based on flow cytometry (Mackintosh et al., 2019).

Genome sequence report
The genome was sequenced from a single female L. megera from Aberlady Bay, Scotland, UK (Figure 1). A total of 45-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 77-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 17 missing/misjoins and removed 1 haplotypic duplication, reducing the assembly size by 0.33% and the scaffold number by 24.56%, and increasing the scaffold N50 by 0.002%.
The final assembly has a total length of 488 Mb in 43 sequence scaffolds with a scaffold N50 of 17.8 Mb (Table 1). The majority, 99.97%, of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 28 autosomes (numbered by sequence length) and the W and Z sex chromosomes (Figure 2- Figure 5; Table 2). The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.6% (single 98.2%, duplicated 0.4%) using the lepidoptera_odb10 reference set (n=5,286). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Project accession data
Assembly identifier ilLasMege1.1

Raw data accessions
PacificBiosciences SEQUEL II ERR7224284

Genome assembly
Assembly accession GCA_928268935.  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 Chromium read cloud 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 (HiFi) and Illumina HiSeq (10X) instruments. Hi-C data were generated in the Tree of Life laboratory from whole organism tissue of ilLasMege3 using the Arima v2 kit and sequenced on a NovaSeq 6000 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). 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 (Howe et al., 2021) was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was    assembled using MitoHiFi (Uliano- Silva et al., 2021), which performs annotation using 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 L. megera 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.