The genome sequence of the Light Brocade, Lacanobia w-latinum (Hufnagel, 1766)

We present a genome assembly from an individual male Lacanobia w-latinum (the Light Brocade; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 903.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.38 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,592 protein coding genes.


Background
Climate change can have a range of impacts on insect populations including effects on distribution, dispersal, abundance, and life cycle parameters.These may be induced directly, through insects responding to changes in temperature or precipitation, or indirectly through responses to changes in plant communities, competitors, predators, parasites or pathogens (Cohen et al., 2018;McCarty et al., 2017).A progressive shift of flight season to earlier in the year has been noted for many, though not all, species of Lepidoptera in Britain and Ireland, in parallel to climate change (Randle et al., 2019).The Light Brocade, Lacanobia w-latinum, is a species for which the flight period has clearly advanced in southern Britain over the past 50 years, with records from 1970-1979 peaking in mid-June compared to late May for 2000-2016(Randle et al., 2019)).
Lacanobia w-latinum is a striking member of the family Noctuidae.The forewings of the adult moth are neatly patterned with russet-brown and white markings, including a broad silvery cross-band following the termen of the wing and pronounced 'dog-tooth' terminal markings typical of this genus.The moth is distributed widely across Europe with many records from the UK, Austria, Switzerland, Netherlands, France, Denmark and southern regions of Sweden.There are also scattered records further east from Ukraine, Russia, Iran, Kazakhstan and Uzbekistan (GBIF Secretariat, 2022).In Britain, the moth is found predominantly south of a line running from the Bristol Channel to the Wash in calcareous grasslands, downland and heathland habitats, although it can also be found in open woodland and gardens (Randle et al., 2019).It has not been recorded from Ireland.The larvae are nocturnal and feed in late summer on the leaves of common broom (Cytisus scoparius), dyer's greenweed (Genista tinctoria), knotgrass (Polygonum aviculare) and some other low growing plants; the pupal stage overwinters (Bretherton et al., 1983).
A complete genome sequence of Lacanobia w-latinum may facilitate studies into adaptations to calcareous habitats and genetic responses to climate change, and will add to the growing set of genomic resources for studying lepidopteran evolution.

Genome sequence report
The genome was sequenced from one male Lacanobia w-latinum (Figure 1) collected from Wytham Woods,Oxfordshire (51.77,.A total of 48-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 105 missing joins or mis-joins and removed 21 haplotypic duplications, reducing the assembly length by 0.47% and the scaffold number by 83.35%, and increasing the scaffold N50 by 6.94%. The final assembly has a total length of 903.9 Mb in 145 sequence scaffolds with a scaffold N50 of 29.5 Mb (Table 1).Most (99 %) 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/987426.

Sample acquisition and nucleic acid extraction
The specimen used for genome sequencing was a male Lacanobia w-latinum (specimen ID Ox001930, ilLacWlai2), collected   DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilLacWlai2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.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 tissue of ilLacWlai2 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 Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus 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 was 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 tissue of ilLacWlai2 that has 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).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  (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant 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 Lacanobia w-latinum assembly (GCA_947578705.1). in Ensembl Rapid Release.Table 3. Software tools: versions and sources.

Manuela Lopez Villavicencio
Institut de Systématique, Evolution, Biodiversité, Muséum National d'Histoire Naturelle, Paris, France This article reports the genome assembly of a male individual of the European moth Lacanobia wlatinum.The assembly included the autosomes, the Z sexual chromosome and the mitochondrial genome.I found really interesting the authors mention that some features of the species, as the flight period, seem to be changing due to climate change.
The article is clearly written and pleasant to read.It shows convincing evidences for a high-quality assembly based on BUSCO scores.The methods for genome assembly, quality test and Hi-C scaffolding are relevant and up-to-date.There are sufficient details of methods and materials provided to allow replication by others.
The assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes plus the sexual Z chromosome.
As I am interested in butterfly genomes, I would have loved to have other interesting information as genome-wide level of heterozygosity (estimated with jellyfish and genomescope).I also think it is important to provide BUSCO quality assessment for the annotation because I usually find overestimation of gene numbers and high levels of duplication when using BRAKER and this could be caused by predicted isoforms of the same gene.
Overall, I think the release of this well-assembled and annotated genome is a very useful contribution and I recommend the indexing of this article.
Is the rationale for creating the dataset(s) clearly described?

Yue Yu
Tufts University, Medford, Massachusetts, USA The article is a clear and concise report on a genome assembly of the Light Brocade moth, Lacanobia w-latinum.The species is documented to have advanced spring phenology associated with climate change, and the authors point out that the genome sequence would be useful for studying the species' response to climate change as well as adaptation to calcareous habitats.Cutting-edge sequencing technologies are employed to assemble long scaffolds (using PacBio) with chromosomal/interaction information (using Hi-C).The resulting assembly appears to be of high quality (e.g.BUSCO 99%) and provides a solid reference genome for future studies.
While the report is overall succinct, a few places would benefit from more details for reproducibility, completeness, and interpretation of data.For example: Methods mentioned tissue set aside for Hi-C sequencing.What was the tissue (e.g.head)?
Similarly, what was the tissue used for RNA extraction? 1.
What parameters were used in the software tools?Even if all were at default values, explicitly stating this might be helpful for the readers.

2.
How was the RNA-Seq data analysed?Additionally, it was unclear whether the RNA-Seq data was used for genome annotation.I could be wrong but as I understand it, BRAKER2 in the default protein mode is for when there is no transcriptomic data available.

3.
While quality metrics for the final assembly were provided, I would be interested to see a summary of the sequencing and mapping quality.I could find some, but not all, of this information in the link provided.

4.
Minor comment: Typo/missing word in Abstract: "chromosomal, " Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Partly
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Population genomics, evolutionary genetics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Lacanobia w-latinum, ilLacWlai2.1: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 903,922,692 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 (48,999,040 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (29,461,643 and 20,845,940 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/Lacanobia wlatinum/dataset/CANPUP01/snail.

Figure 3 .
Figure 3. Genome assembly of Lacanobia w-latinum, ilLacWlai2.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Lacanobiawlatinum/dataset/CANPUP01/blob.

Figure 4 .
Figure 4. Genome assembly of Lacanobia w-latinum, ilLacWlai2.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Lacanobiawlatinum/dataset/CANPUP01/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Lacanobia w-latinum, ilLacWlai2.1:Hi-C contact map of the ilLacWlai2.1 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=GCV-ScdiSVyHt69lEDLeMQ.

the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
Yes, but please include BUSCO scores for annotation Are the datasets clearly presented in a useable and accessible format?No competing interests were disclosed.
○YesAre sufficient details of methods and materials provided to allow replication by others?○ ○ Yes Is

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 31 July 2023 https://doi.org/10.21956/wellcomeopenres.21770.r63431© 2023 Yu Y.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.