The genome sequence of the flounced rustic, Luperina testacea (Denis & Schiffermüller, 1775)

We present a genome assembly from an individual male Luperina testacea (the flounced rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 601 megabases in span. The majority of the assembly (99.98%) is scaffolded into 31 chromosomal pseudomolecules, with the Z sex chromosome assembled. The mitochondrial genome was also assembled, and is 15.3 kilobases in length.


Background
Across much of north-west and central Europe, Luperina testacea, the flounced rustic, is a common moth of late summer and autumn.Widespread in Britain, Luperina testacea becomes much less frequent in Scotland.Eggs are laid at the bases of various grasses and the smooth, rather unpigmented larvae feed on the roots and lower stems, feeding slowly through the winter.Adults do not feed, are regular at light (predominantly males) and can be one of the most abundantly trapped moths from late July to early October, especially in open coastal areas or calcareous grasslands (Henwood et al., 2020;Waring et al., 2003).Abundance in southern Sweden is positively associated with soil disturbance (Tyler, 2020).
Adults are variable in appearance, particularly in the tone of the forewing.The hind wings are always strikingly pale, the fore wings usually have a distinct dark bar in the centre and a broad, paler subterminal area.

Genome sequence report
The genome was sequenced from one male L. testacea (Figure 1) collected from Hever Castle, England, UK (latitude 51.1884, longitude 0.1198).A total of 26-fold coverage in Pacific Biosciences single-molecule long reads and 76-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 24 missing/misjoins, reducing the assembly size by 0.01% and the scaffold number by 33.33%, and increasing the scaffold N50 by 4.57%.
The final assembly has a total length of 601 Mb in 34 sequence scaffolds with a scaffold N50 of 21.6 Mb (Table 1).The majority of the assembly sequence (99.98%) was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length), and the Z sex chromosome (Figure 2-Figure 5; Table 2).The assembly has a BUSCO v5.2.2 (Manni et al., 2021) completeness of 98.9% (single 98.6%, 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.

Sample acquisition and DNA extraction
A single male L. testacea (ilLupTest1) was collected from Hever Castle, England, UK (latitude 51.1884, longitude 0.1198) by Gavin Broad, Natural History Museum, using a light trap in   grassland near a lake.The sample was identified by the same individual, and preserved in liquid nitrogen.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute.The ilLupTest1 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  Illumina NovaSeq 6000 (10X) instruments.Hi-C data were generated from head tissue using the Arima Hi-C+ kit and sequenced on NovaSeq 6000.

Nicholas W VanKuren
Department of Ecology & Evolution, The University of Chicago, Chicago, Illinois, USA Broad and co-authors present a complete assembly of a common British moth, Luperina testacea.They use the standard pipeline for genome sequencing and assembly that have been applied to hundreds of TOL genomes over the past several years.
I sincerely appreciate the high quality and accessible data from all of these genome assembly projects, but I am frequently lost as to why we need to sequence to them all beyond "we can".I would love to see a very brief discussion of why this moth is particularly interesting, or at least into what context this genome sequence is being placed (does it fill some crucial phylogenetic gap, is it a pest, does it have a unique ecology?)It doesn't need to be regurgitated from any of the previous TOL/Sanger pubs, but just a sentence or two to give me a reason for why I should look at this genome would help a lot.
I have no issues with any of the methods, the excellent tools to access and analyze the data, or the sequence itself.

Susan McEvoy
1 Santa Barbara Botanic Garden, Mission Canyon, CA, USA 2 EEB, Meyer Lab, University of California Santa Cruz, Santa Cruz, CA, USA This article details the genome assembly of Luperina testacea, the flounced rustic moth of northwest and central Europe.Tissue from the thorax of a single male L. testacea was used for Pacific Biosciences HiFi and Chromium 10X sequencing, while the head was used to generate Arima Hi-C data.The HiFi reads were assembled with HiFiasm, polished with 10X reads in longranger, and scaffolded with Hi-C reads in SALSA2.The mitochondrial genome was assembled using MitoHiFi.The genome was manually curated using a visualization of the assembly in HiGlass.
The final reference was highly complete and contiguous relative to standard genome reference metrics, with 31 pseudomolecules and a sex chromosome, totaling 601 Mbp in length, and with 98.9% of BUSCO Lepidoptera genes found in a single copy.
Standard genome assembly protocols were used, and this article communicates details in a reproducible manner.The resulting datasets are also very clear.Any comments I can contribute are very minor and mostly just things I was curious about.
I marked "partial" regarding the rationale for creating the dataset just because it didn't seem explicit.My assumption is that it is because it is common across Europe as mentioned in the background.How does this genome fit into the current landscape of Lepidoptera genomics?Or maybe a similar statement?Also, as a plant specialist, I wasn't sure of the phrase "regular at light".If it is specific to entomology, perhaps it could be rewritten to be clear to a broader audience.
The protocols as described for DNA extraction, sequencing, assembly, and scaffolding are generally appropriate and commonly used elsewhere.I was a little puzzled at polishing the assembly with 75x of Chromium 10X instead of a whole-genome shotgun.10X is normally used for scaffolding, but perhaps it was not needed for this purpose as the Hi-C scaffolding was sufficient.I believe 10X would have a coverage bias that makes it less than ideal for polishing genome-wide, but maybe at 75x it is fine.I don't think this is a step I would recommend others replicate.
I was also wondering if there are any potential issues with separating thorax and head tissue and only using head for Hi-C.I'm just curious if there would be any issues with this.Maybe it is common with insects, I'm not sure.
Is the rationale for creating the dataset(s) clearly described?Partly

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
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 1 .
Figure 1.Image of the Luperina testacea (ilLupTest1) specimen taken prior to preservation and processing.

Figure 2 .
Figure 2. Genome assembly of Luperina testacea, ilLupTest1.1: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 601,512,407 bp assembly.The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (26,814,763 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (21,613,865 and 15,062,659 bp), respectively.The pale grey spiral shows the cumulative chromosome 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/ilLupTest1.1/dataset/CAKMJJ01/snail.

Figure 3 .
Figure 3. Genome assembly of Luperina testacea, ilLupTest1.1:GC coverage.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/ilLupTest1.1/dataset/CAKMJJ01/blob.

Figure 5 .
Figure 5. Genome assembly of Luperina testacea, ilLupTest1.1:Hi-C contact map.Hi-C contact map of the ilNotZicz1.1 assembly, visualised in HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this map is available here.

Table 3 . Software tools used.
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, and supplied to, the Darwin Tree of Life Project.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.The genome sequence is released openly for reuse.The L. testacea 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 Table1.

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for creating the dataset(s) clearly described? No Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 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? No Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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.