The genome sequence of the Orange-tailed Clearwing, Synanthedon andrenaeformis (Laspeyres, 1801)

We present a genome assembly from an individual male Synanthedon andrenaeformis (the Orange-tailed Clearwing; Arthropoda; Insecta; Lepidoptera; Sesiidae). The genome sequence is 348.4 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 16.65 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,867 protein coding genes.


Background
The Orange-tailed Clearwing Synanthedon andrenaeformis is a diurnal moth in the family Sesiidae found across the western Palaearctic region, particularly in south, central and eastern Europe (GBIF Secretariat, 2023).The adult moth has a shiny black body marked with delicate yellow bands, and narrow black-edged, otherwise transparent, wings.The common name derives from a fan-shaped 'anal tuft' of yellow hair-scales at the terminus of the abdomen.
Like many of the Clearwing moths, the geographical distribution of the species was poorly known until the development of specific pheromone lures, such as the VES lure that attracts males of S. andrenaeformis and S. vespiformis (Priesner et al., 1986;Sims et al., 2020;Taylor, 2021).It is now clear that in Britain, S. andrenaeformis has a distribution that traces a Z-shape across the south of England: essentially a line running from the Bristol Channel to East Anglia, then southwest to Dorset, and east to Kent.Most of this distribution follows a discontinuous band of chalk and associated scrub and chalk downland habitat (GBIF Secretariat, 2023;NBN Atlas Partnership, 2023).The wood-boring larvae of S. andrenaeformis feed in galleries formed inside the branches of the Wayfaring tree Viburnum lantana and the Guelder-rose Viburnum opulus (Rothschild, 1906;South, 1939); both primary food plants are associated with calcareous soils.It has been proposed that dogwood Cornus sanguinea may be an alternative food plant (Sims et al., 2020).Larvae pass through two winters before the adult moths emerge in May to July; a characteristic 3 mm disc-shaped bark cap may be found over the future emergence hole (Rothschild, 1906).
Here we report a complete genome sequence for the Orange-tailed Clearwing Synanthedon andrenaeformis determined as part of the Darwin Tree of Life project.The genome sequence of S. andrenaeformis will facilitate research into host-plant specificity and the biology of wood-boring insects, and contribute to the growing set of resources for studying the evolution of Lepidoptera.

Genome sequence report
The genome was sequenced from a male Synanthedon andrenaeformis (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 106-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 8 missing joins or mis-joins, reducing the scaffold number by 2.78%, and increasing the scaffold N50 by 0.55%. The final assembly has a total length of 348.4 Mb in 34 sequence scaffolds with a scaffold N50 of 12.4 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds 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 (99.96%) 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
Two male Synanthedon andrenaeformis specimens were collected from Wytham Woods, Oxfordshire (biological vicecounty Berkshire), UK (latitude 51.77, longitude -1.34) on   In sample preparation, the ilSynAndr1 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 Manual MagAttract v1 protocol (Strickland et al., 2023b).DNA was sheared 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., 2023a): 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.
RNA was extracted from abdomen tissue of ilSynAndr2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

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 head and thorax tissue of ilSynAndr1 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 and corrected as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio 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.
Table 3 contains a list of relevant software tool versions and sources.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 In this Data Note, Boyes, Holland and colleagues present a reference genome assembly and gene annotation of the Orange-tailed Clearwing (Synanthedon andrenaeformis).The reference is highquality, using appropriate tools and with comprehensive reporting of the sample collection, data generation and analysis and all associated metadata.Links to data accessions are functional.

Minor comments:
Please preface geographical co-ordinates with latitude and longitude.I will continue to query whether this templated detail is correct: "in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA."This ratio of beads is not size-selective, I believe this should be 0.6x, as in the protocols.ioGuidelines.

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 Competing Interests: No competing interests were disclosed.

Gareth Weedall
Liverpool John Moores University, Liverpool, England, UK The data note paper reports the whole genome sequencing, assembly and annotation of the orange-tailed clearwing (Synanthedon andrenaeformis), a diurnal moth with wood-boring larvae.
The rationale for generating the genome assembly, including to provide a platform for further research on host-plant specificity, is clearly presented.The methodology is, as far as I can judge it, scientifically sound and are clearly described.The data (project accession PRJEB51451) are publicly accessible.
The data note paper reports the whole genome sequencing, assembly and annotation of the orange-tailed clearwing (Synanthedon andrenaeformis), a diurnal moth with wood-boring larvae.
The rationale for generating the genome assembly, including to provide a platform for further research on host-plant specificity, is clearly presented.The methodology is, as far as I can judge it, scientifically sound and are clearly described.The data (project accession PRJEB51451) are publicly accessible.
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 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 2 .
Figure 2. Genome assembly of Synanthedon andrenaeformis, ilSynAndr1.2:metrics.The BlobToolKit snail plot 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 348,388,281 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 (17,284,882 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (12,440,847 and 8,142,002 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/CAKZFN02/dataset/CAKZFN02/snail.

Figure 3 .
Figure 3. Genome assembly of Synanthedon andrenaeformis, ilSynAndr1.2:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKZFN02/dataset/CAKZFN02/blob.

Figure 4 .
Figure 4. Genome assembly of Synanthedon andrenaeformis, ilSynAndr1.2:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKZFN02/dataset/CAKZFN02/cumulative.

Figure 5 .
Figure 5. Genome assembly of Synanthedon andrenaeformis, ilSynAndr1.2:Hi-C contact map of the ilSynAndr1.2assembly, 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=bvCHwvS8TbCvV2D-CzlYDw.

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.