The genome sequence of the common grass-veneer, Agriphila tristella (Denis & Schiffermüller, 1775)

We present a genome assembly from an individual male Agriphila tristella (the common grass-veneer; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 802 megabases in span. Most of the assembly (99.83%) is scaffolded into 23 chromosomal pseudomolecules with the Z sex chromosome assembled. The mitochondrial genome was also assembled and is 15.3 kilobases in length.


Background
The common grass-veneer Agriphila tristella (Denis & Schiffermüller, 1775) is a micro-moth of the Crambinae subfamily.It can usually be recognised by its yellow median streak on the forewing which branches into four 'fingers' towards the apex of the wing.However, the species can be quite variable and difficult to separate from Agriphila selasella.In these cases, the prominent facial cone and differences in the genitalia can be used to identify A. tristella reliably (Lewis, 2012).The species is common in grassland and rough meadows throughout the British Isles, where the eggs are laid on various grasses.
A. tristella larvae can be found from September to June, feeding in a vertical silken gallery along the lower part of a grass stem.Pupae can then be found in June and July within oval frass-covered silken cocoons in loose soil amongst the grass roots.The adults typically fly between late June to mid-September, with a peak in August.During this time, they can be readily disturbed by day or attracted to light at night (Langmaid et al., 2018).
The genome of the common grass-veneer was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all the named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from a single male A. tristella (Figure 1), collected in Wytham Woods, Oxford, Berkshire, UK.A total of 29-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 56-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 86 missing/misjoins and removed 40 haplotypic duplications, reducing the assembly size by 3.91% and the scaffold number by 39.22%, and increasing the scaffold N50 by 32.77%.
The final assembly has a total length of 802 Mb in 62 sequence scaffolds with a scaffold N50 of 51.7 Mb (Table 1).Most of the assembly sequence (99.83%) was assigned to 23 chromosomal-level scaffolds, representing 22 autosomes (numbered by sequence length) and the Z sex chromosome (Figure 2-Figure 5; Table 2).Heterozygous inversion was   observed on chromosome 1 (19.75-29.97Mb).A large size differential between haplotypes on several chromosomes was observed, with additional sequence not aligning to comparators.Since difficulty was experienced in reconciling the chromosome 13 longer haplotype with the Hi-C map, 3.3 Mb of the chromosome was left in an alternate assembly.The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.0% (single 97.4%, duplicated 0.6%) using the lepidoptera_odb10 reference set (n = 5,286).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A single male A. tristella specimen (ilAgrTris1) was collected in Wytham Woods, Oxford, Berkshire, UK (latitude 51.772, longitude -1.338) by Douglas Boyes (University of Oxford),  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.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 Chromium read cloud sequencing libraries were constructed according to the manufacturers' instructions.
Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were generated in the Tree of Life laboratory from head tissue of ilAgrTris1 using the Arima v2 kit and sequenced on a NovaSeq 6000 instrument.
The genome was analysed and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020).Table 3   I note two omissions in this report compared to an earlier report that I reviewed: (1) Genome annotation-is there a reason why this assembly hasn't been run through the Ensembl rapid annotation pathway?(2) A link to the metadata, such as kmer spectra-which can be a really useful resource.
The level of heterozygosity in the genome individual seems quite marked and, from the haplotype size differences recorded, it would appear that structural variation may be an important feature of genome biology in this species.Whilst to explore this in detail is probably beyond the scope of these reports, it would have been nice to have these "quirks" also presented as tables/figures.As the reports of the primary assemblies become increasingly standardized (and these short reports are models in the field), as a collective I would love to see the DToL and its partners explore ways to effectively disseminate learnings from unexpected and challenging findings.

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.

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: Phylogenetic analysis of lepidopteran moths using complete mitochondrial genome sequence 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 A. tristella specimen taken prior to preservation and processing.

Figure 2 .
Figure 2. Genome assembly of A. tristella, ilAgrTris1.1: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 801,775,791 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 (62,134,667 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (51,702,748 and 19,521,324 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/ilAgrTris1.1/dataset/CAKMRQ01/snail.

Figure 3 .
Figure 3. Genome assembly of A. tristella, ilAgrTris1.1: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/ilAgrTris1.1/dataset/CAKMRQ01/blob.

INSDC accession Chromosome Size (Mb) GC%
contains a list of all software tool versions used, where appropriate.Ethics/compliance issuesThe 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 A. tristella 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.Members of theTree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.
https://doi.org/10.21956/wellcomeopenres.20589.r70181© 2024 Whibley A. 1 The University of Auckland, Auckland, Auckland, New Zealand 2 Bragato Research Institute, Blenheim, New Zealand Review of the genome assembly of Agriphila tristella by Douglas Boyes and colleagues at the University of Oxford and Wellcome Trust Sanger Centre.The authors have assembled another extremely high-quality genome and provided comprehensive and clearly structured information to support this effort.Yet again, the well-informed natural history detail that is included provides very helpful framing.Methods, analyses and reporting are all exemplary.