The genome sequence of the Marbled White Spot, Protodeltote pygarga (Hufnagel, 1766)

We present a genome assembly from an individual male Protodeltote pygarga (the Marbled White Spot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 421.1 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 15.48 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,784 protein coding genes.


Background
The Marbled White Spot (Protodeltote pygarga) is a small noctuid moth.This species has undergone a marked range expansion in Britain and Ireland in recent decades.It now occurs widely in grassland, heathland, woodland and moorland habitats, but is absent from Scotland and northern England (Randle et al., 2019).Its distribution extends across much of Europe to western and central Asia, and it is also recorded from Japan, Korea and China (GBIF Secretariat, 2023).
The larval host plants are grasses.This species has a single annual generation and overwinters as a pupa underground (Waring et al., 2017).
The genome of the marbled white spot, Protodeltote pygarga, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomally complete genome sequence for Protodeltote pygarga, based on one male specimen from Wytham Woods, Oxfordshire.

Genome sequence report
The genome was sequenced from one male Protodeltote pygarga (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 92-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 7 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the assembly length by 0.45%. The final assembly has a total length of 421.1 Mb in 33 sequence scaffolds with a scaffold N50 of 14.9 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of reads on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.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 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.

Sample acquisition and nucleic acid extraction
A male Protodeltote pygarga (specimen ID Ox000464, ToLID ilProPyga1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, 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.
High molecular weight (HMW) DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI), following a sequence of core procedures: sample preparation; sample homogenisation; HMW DNA extraction; DNA fragmentation; and DNA clean-up.In sample preparation, the ilProPyga1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the abdomen was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA was sheared  * Assembly metric benchmarks are adapted from column VGP-2020 of "Table 1: Proposed standards and metrics for defining genome assembly quality" from (Rhie et al., 2021).
into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.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.
Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus 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 Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from thorax tissue of ilProPyga1 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 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    Table 3. Software tools: versions and sources.

Tyler Scott Alioto
Fundacion Centro Nacional de Analisi Genomico, Barcelona, Catalonia, Spain The genome note submitted by Boyes et al. reports the genome sequence of the Marbled White Spot, Proto Deltore pygarga.P. pygarga is a small moth that has increased its range across southern Britain and Ireland.It is distributed widely in Europe and Asia as well.It has a single annual generation, pupating underground over winter and the larval host plants are grasses.The male specimen sequenced was collected from Wytham Woods.92-fold coverage of HiFi reads were generated and assembled with Hifiasm, run through purge_dups, scaffolded with 141 Gb (335x) of Arima2 Hi-C data using YaHS, and manually curated with few interventions necessary.
The assembly is chromosome-scale and of high quality, meeting the minimum standards recommended by the Earth Biogenome Project.All protocols are appropriate and welldocumented.All data conforms with FAIR principles, with read data and assemblies being available in the ENA.Blob toolkit figures are interactive.Additional QC data are provided at https://tolqc.cog.sanger.ac.uk/, supplementing the figures published in the data note.
No major comments.
Minor comments: A ZZ-male was collected and sequenced.I believe it is usually standard practice to try to sequence the heterogametic sex.The authors should provide an explanation for why they chose a ZZ male.Were there difficulties in collecting females?Are ZW females too hard to assemble?It would help the rest of the reference genome sequencing community to know the rationale for choices made or be made aware of difficulties encountered.

1.
A general comment on the genome-note engine.I would like to see the Table 1 comparison to the now-old VGP standards "Assembly metric benchmarks are adapted from column VGP-2020 of "Table 1: Proposed standards and metrics for defining genome assembly quality" from (Rhie et al., 2021)."changed to a comparison to the latest EBP metric standards.Especially for non-vertebrate genomes.

2.
Is there a way to indicate what protein set was used to annotate the genome with BRAKER2? 3.
In order to reproduce the annotation, this information would seem necessary.Please find a way to include such info (from Ensembl rapid) in the genome note template.

Maria Nilsson
Senckenberg Biodiversity and Climate Research Centre, Frankfurt am Main, Germany The genome of the Marbled White Spot (Protodeltote pygarga) has been sequenced from a single male individual captured using a light trap.The Marbled White Spot is a commonly occurring moth that is found throughout Europe and has seen a range expansion in Britain.The genome assembly is 421 Mb large and is scaffolded to 33 scaffolds.The genome could not be fully phased.The QV value is 69.4 and the BUSCO gene completeness at 99.0, which indicate a very complete assembly.The contamination screen found minimal contamination and gene annotation identified 17,784 genes.
Minor comments: 1) The mitochondrial genome has been assembled, but seems to not be extracted/submitted separately.It would be helpful to have the mt genome submitted separate.It would be beneficial for those that are interested in the mitochondrial genome.2) Please clarify for what analyzes that the abdomen DNA was used for.The material and methods describe DNA isolation from the abdomen, while all of a sudden thorax DNA is used for HiC.This contradiction should be clarified.
3)The data set used for the blob plot in Figure 2, ERR9248441_cov, is not listed anywhere in table 1. Reviewer Expertise: Genomics, Conservation genomics, Biogeography 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, however I have significant reservations, as outlined above.

Figure 2 .
Figure 2. Genome assembly of Protodeltote pygarga, ilProPyga1.2: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 421,153,487 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 (21,803,012 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (14,901,961 and 10,302,340 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/Protodeltote%20pygarga/dataset/CAMPPH01/snail.

Figure 3 .
Figure 3. Genome assembly of Protodeltote pygarga, ilProPyga1.2:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to sequence 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/Protodeltote%20pygarga/dataset/CAMPPH01/blob.

Figure 5 .
Figure 5. Genome assembly of Protodeltote pygarga, ilProPyga1.2:Hi-C contact map of the ilProPyga1.2assembly, 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=NRR27qs_Twah2X0btFEQTQ.
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.Reviewer Expertise: genomics, genome assembly, gene prediction, annotation 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.Reviewer Report 11 June 2024 https://doi.org/10.21956/wellcomeopenres.22770.r78595© 2024 Nilsson M. 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.
: the high quality genome assembly of the common Marbled White Spot offers possibilities to study the range expansion of evolutionary properties of the moth family Noctuidae.Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: phylogenomics, transposable elements, mitogenomics 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, however I have significant reservations, as outlined above.Reviewer Report 07 May 2024 https://doi.org/10.21956/wellcomeopenres.22770.r78587© 2024 Makunin A. 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.Alex Makunin Wellcome Sanger Institute, Hinxton, England, UK The manuscript by Boyes et al present the chromosomal genome assembly for Protodeltote pygarga moth species.This study is seemingly filling a gap in taxonomic sampling by providing the first reference genome assembly for Eustrotiinae subfamily of Noctuidae family.Overall, the genome note is cohesive and well structured.I can see two minor issues: -DNA extraction is described in detail only for abdomen, which seemingly was used for PacBio sequencing, but not for thorax which was used for HI-C.-The reasoning for Z chromosome assignment in a homogametic sample is not provided -was it based on size (largest chromosome)?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?Yes Are the datasets clearly presented in a useable and accessible format?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.

Table 3
contains a list of relevant software tool versions and sources.Genome annotationThe BRAKER2 pipeline(Brůna et al., 2021)was used in the default protein mode to generate annotation for the Protodeltote pygarga assembly (GCA_936450705.2) in Ensembl Rapid Release.mission 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