The genome sequence of the Barred Red, Hylaea fasciaria (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Hylaea fasciaria (the Barred Red; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 327.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 17.18 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,216 protein coding genes.


Background
Hylaea fasciaria, commonly known as the Barred Red, is a moth species of the family Geometridae, subfamily Ennominae.It is a Palaearctic species, distributed across central and northern Europe, the Urals, Caucasus, Altai, and eastern Siberia (GBIF Secretariat, 2022).It is widespread and common in Britain and Ireland in regions of coniferous woodland (Kimber, 2023).
The Barred Red is a typical size for a Geometrid moth, with a wingspan of 27-40 mm.The Barred Red has two main forms: a rust red form (f. fasciaria) and a green form (f. prasinaria) (Sihvonen et al., 2014).The red form is found more commonly in Britain, while H. fasciaria prasinaria is found more frequently in Europe, with rare records from the south-east of England.In both forms, the front wings are slightly curved with thin transverse bands and a slightly dark closed midfield.These bands are pale whitish coloured in the rust-red form, while in the green form the colouring of the bands varies from pale whitish to beige to light brown.In addition to the two main forms, there are other colour variants with different shades of green or plain brown (Sihvonen et al., 2014).
The H. fasciaria caterpillar is greyish or greyish brown with some brownish smears and a light brown head, with series of small warts on the back.The caterpillar feeds on coniferous trees, including Pinus sylvestris and Picea abies.The larva grows slowly and enters hibernation in winter, usually roosting on green spruce twigs, and often stretching along a single needle.This behaviour has been observed to provide the larva with sufficient food while reducing the risk of predation and protecting the body of the caterpillar from sudden fluctuations in body temperature (Dvořáčková & Kulfan, 2009).
Pupation occurs in spring, lasting 4 to 6 weeks.On the continent, the adults fly in two generations from April to October, but the species is univoltine in Britain and northern Europe, where it flies from June to August (Waring et al., 2017).Adults are nocturnal, and come to light, sometimes in great numbers (Kimber, 2023).
The genome sequencing of Hylaea fasciaria, part of the Darwin Tree of Life project, is expected to broaden our ecological understanding of this moth.It will provide comprehensive DNA reference data for molecular surveillance of the species and offer valuable insights into its phylogenetic relationship with other similar species.

Genome sequence report
The genome was sequenced from one male Hylaea fasciaria (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76,.A total of 72-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 97-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 joins or misjoins and removed one haplotypic duplication, reducing the scaffold number by 17.5%, and increasing the scaffold N50 by 3.08%. The final assembly has a total length of 327.9 Mb in 33 sequence scaffolds with a scaffold N50 of 11.7 Mb (Table 1).Most (99.98%%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 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/722673.
The resulting annotation includes 17,406 transcribed mRNAs from 17,216 protein-coding genes.

Sample acquisition and nucleic acid extraction
A male Hylaea fasciaria (specimen ID Ox000468, individual ilHylFasc1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.76, longitude -1.34) on 2020-06-13 using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilHylFasc1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Head and thorax 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.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from head and thorax tissue of ilHylFasc1 that had been set aside, using the Arima2 kit and sequenced on the HiSeq X Ten 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 SALSA2 (Ghurye et al., 2019).The assembly was checked for contamination and corrected using the gEVAL system    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 work was done using Nextflow (Di Tommaso et al

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Hylaea fasciaria assembly (GCA_905147375.1) in Ensembl Rapid Release.

Wellcome Sanger Institute -Legal and Governance
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', which can be found in full on the Darwin Tree of Life website here.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, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Figure 2 .
Figure 2. Genome assembly of Hylaea fasciaria, ilHylFasc1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 327,936,668 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (17,069,495 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (11,686,647 and 7,753,621 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilHylFasc1.1/dataset/ CAJHVH01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Hylaea fasciaria, ilHylFasc1.1:Hi-C contact map of the ilHylFasc1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=Irlrd5-MSvOIilPqAGjoSQ.

Table 3
contains a list of relevant software tool versions and sources.
• Legality of collection, transfer and use (national and international) Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.