The genome sequence of the peppered moth, Biston betularia Linnaeus, 1758

We present a genome assembly from an individual male Biston betularia (the peppered moth; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 405 megabases in span. The majority of the assembly (99.99%) is scaffolded into 31 chromosomal pseudomolecules, with the Z sex chromosome assembled.Gene annotation of this assembly on Ensembl has identified 12,251 protein coding genes.


Background
The peppered moth, Biston betularia, is widely distributed throughout Europe, Asia and North America. The species has one generation per year, with adults flying between May and August in England. Larvae mimic twigs in their form, and can even change colour to match their surroundings (Eacock et al., 2019;Eacock et al., 2017). Larvae feed on a wide variety of deciduous trees and bushes, including birch, blackthorn and roses. Individuals overwinter underground as pupae. A pale typica form is white, peppered with black on wings and body while a melanic, carbonaria form with white spots is associated with areas with higher atmospheric pollution levels. The two forms can interbreed resulting in intermediate forms. Genetically distinct are insularia, with a range of intermediate colour patterns. Industrial melanism in the peppered moth is a classic example of rapid adaptive response to environmental change Cook, 2003). High levels of coal pollution during the industrial revolution led to a rise in the frequency of the carbonaria form in urban areas due to selective predation. In recent decades, the frequency of the melanic form has decreased, in line with reduced pollution levels. The genetic basis of industrial melanism has been attributed to the insertion of a large transposable element into the first intron of the gene cortex ( Van't Hof et al., 2016). This event occurred in Britain in approximately 1819, in line with the historical record ( Van't Hof et al., 2016). Interestingly, cortex has been repeatedly associated with colour pattern variation in diverse lepidopteran species, including in Heliconius butterflies where it is a major determinant of scale cell identity (Livraghi et al., 2021;Nadeau et al., 2016;Van Belleghem et al., 2017). Biston betularia has a karyotype of 31 chromosomes ( Van't Hof et al., 2013).

Genome sequence report
The genome was sequenced from one male B. betularia ( Figure 1) collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.772, longitude -1.338). A total of 27-fold coverage in Pacific Biosciences single-molecule long reads and 91-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 8 missing/misjoins, reducing the scaffold number by 15.79% and increasing the scaffold N50 by 2.39%.
The final assembly has a total length of 405 Mb in 32 sequence scaffolds with a scaffold N50 of 14.7 Mb ( Table 1). The majority of the assembly sequence (99.99%) was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length, and the Z chromosome   Table 2). The assembly has a BUSCO v5.1.2 (Manni et al., 2021) completeness of 98.7% (single 98.3%, duplicated 0.4%) using the lepidoptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Genome annotation report
The ilBisBetu1.1 genome was annotated using the Ensembl rapid annotation pipeline (Table 1; https://rapid.ensembl.org/Bis-ton_betularia_GCA_905404145.1/). The resulting annotation includes 19,758 transcribed mRNAs from 12,251 protein-coding and 2.985 non-coding genes. There are 1.61 coding transcripts per gene and 7.88 exons per transcript.

Sample acquisition and DNA extraction
A single male B. betularia (ilBisBetu1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.772, longitude -1.338) by Douglas Boyes, UKCEH, using a light trap. The sample was identified by the same individual, and preserved on dry ice.  DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The ilBisBetu1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax tissue 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 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 X (10X) instruments. Hi-C data were generated from head tissue using the Arima Hi-C+ kit and sequenced on HiSeq X.

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  Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Biston betularia assembly (GCA_905404145.1). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Ethics/compliance issues
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner. The submission of materials by a Darwin Tree of Life Partner is subject to the Darwin Tree of Life Project Sampling Code of Practice. By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for,