Abstract
We present a genome assembly from an individual male Xestia xanthographa (the square-spot rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 934 megabases in span. The majority of the assembly (99.94%) is scaffolded into 31 chromosomal pseudomolecules, with the Z sex chromosome assembled.
Keywords: Xestia xanthographa, square-spot rustic, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Noctuoidea; Noctuidae; Noctuinae; Noctuini; Xestiai; Xestia xanthographa (Schiffermuller, 1775) (NCBI:txid988049).
Background
Xestia xanthographa (square-spot rustic) is a widespread noctuid moth found across much of the Palearctic, Europe, North Africa and North America; its larvae are nocturnal feeders on various grasses. In the UK, adults are abundant in late summer from August to September and the species overwinters as a larva. Xestia xanthographa was a key species in a recent study revealing the detrimental effects of street-lighting on caterpillar abundance in the UK ( Boyes et al., 2021). The species has also been recorded as a common prey species for autumn-flying bats ( Razgour et al., 2011), and, as an adaptation to facilitate bat avoidance, the auditory sensitivity of X. xanthographa is broadly tuned with an optimal frequency of 30 kHz ( Norman & Jones, 2008).
The genome of X. xanthographa, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all of the named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for X. xanthographa, based on one male specimen from Wytham Woods, Oxfordshire, UK.
Genome sequence report
The genome was sequenced from a single male X. xanthographa collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude -1.337) ( Figure 1). A total of 27-fold coverage in Pacific Biosciences single-molecule long reads (N50 12 kb) and 40-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 17 haplotypic duplications, reducing the assembly size by 1.41% and scaffold number by 44.86%, and increasing the scaffold N50 by 1.61%.
Figure 1. Image of the Xestia xanthographa specimens taken prior to preservation and processing.
Above, ilXesXant, used for genome and Hi-C sequencing; below, ilXesXant2, used for RNA-Seq.
The final assembly has a total length of 934 Mb in 59 sequence scaffolds with a scaffold N50 of 31 Mb ( Table 1). Of the assembly sequence, 99.94% 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 ( Simão et al., 2015) completeness of 98.7% 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.
Figure 2. Genome assembly of Xestia xanthographa, ilXesXant1.2: 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 933,882,218 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 (42,568,189 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (31,247,186 and 23,512,362 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/ilXesXant1.2/dataset/CAJHXD02/snail.
Figure 5. Genome assembly of Xestia xanthographa, ilXesXant1.2: Hi-C contact map.
Hi-C contact map of the ilXesXant1.2 assembly, visualised in HiGlass. Chromosomes are given in size order from left to right and top to bottom.
Table 1. Genome data for Xestia xanthographa, ilXesXant1.2.
| Project accession data | |
|---|---|
| Assembly identifier | ilXesXant1.2 |
| Species | Xestia xanthographa |
| Specimen | ilXesXant1 |
| NCBI taxonomy ID | NCBI:txid988049 |
| BioProject | PRJEB42066 |
| BioSample ID | SAMEA7520195 |
| Isolate information | Male, head/abdomen/thorax |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR6635594 |
| 10X Genomics Illumina | ERR6002560-ERR6002563 |
| Hi-C Illumina | ERR6002564 |
| Illumina polyA RNA-seq | ERR6002565, ERR6787402 |
| Genome assembly | |
| Assembly accession | GCA_905147715.2 |
| Accession of alternate haplotype | GCA_905147755.2 |
| Span (Mb) | 934 |
| Number of contigs | 301 |
| Contig N50 length (Mb) | 9.1 |
| Number of scaffolds | 59 |
| Scaffold N50 length (Mb) | 31.2 |
| Longest scaffold (Mb) | 35.5 |
| BUSCO * genome score | C:98.7%[S:97.9%,D:0.8%],
F:0.2%,M:1.1%,n:5286 |
*BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.1.2. C= complete [S= single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/ilXesXant1.2/dataset/CAJHXD02/busco.
Figure 3. Genome assembly of Xestia xanthographa, ilXesXant1.2: 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/ilXesXant1.2/dataset/CAJHXD02/blob.
Figure 4. Genome assembly of Xestia xanthographa, ilXesXant1.2: cumulative sequence.
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/ilXesXant1.2/dataset/CAJHXD02/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Xestia xanthographa, ilXesXant1.2.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| LR990642.1 | 1 | 35.50 | 38.7 |
| LR990643.1 | 2 | 34.37 | 38.5 |
| LR990644.1 | 3 | 34.35 | 38.8 |
| LR990645.1 | 4 | 33.93 | 38.7 |
| LR990646.1 | 5 | 32.94 | 38.3 |
| LR990647.1 | 6 | 32.23 | 38.5 |
| LR990648.1 | 7 | 32.15 | 38.6 |
| LR990649.1 | 8 | 32.03 | 38.9 |
| LR990650.1 | 9 | 31.69 | 38.7 |
| LR990651.1 | 10 | 31.50 | 38.9 |
| LR990652.1 | 11 | 31.49 | 38.8 |
| LR990653.1 | 12 | 31.35 | 38.3 |
| LR990654.1 | 13 | 31.25 | 39 |
| LR990655.1 | 14 | 31.05 | 38.6 |
| LR990656.1 | 15 | 30.95 | 38.5 |
| LR990657.1 | 16 | 30.88 | 38.5 |
| LR990658.1 | 17 | 30.80 | 38.7 |
| LR990659.1 | 18 | 30.66 | 38.4 |
| LR990660.1 | 19 | 30.04 | 38.7 |
| LR990661.1 | 20 | 29.75 | 38.6 |
| LR990662.1 | 21 | 29.67 | 38.3 |
| LR990663.1 | 22 | 29.39 | 38.7 |
| LR990664.1 | 23 | 27.72 | 38.8 |
| LR990665.1 | 24 | 27.14 | 38.6 |
| LR990666.1 | 25 | 24.91 | 39.1 |
| LR990667.1 | 26 | 23.51 | 39 |
| LR990668.1 | 27 | 21.65 | 38.9 |
| LR990669.1 | 28 | 21.50 | 38.7 |
| LR990670.1 | 29 | 20.05 | 39.4 |
| LR990671.1 | 30 | 19.43 | 40.1 |
| LR990641.1 | Z | 42.57 | 38.1 |
| LR990672.1 | MT | 0.02 | 19.2 |
| - | - | 7.42 | 41.1 |
Methods
Sample acquisition and nucleic acid extraction
A male X. xanthographa (ilXesXant1) and a second specimen of unknown sex (ilXesXant2) were collected from Wytham Woods, Oxfordshire, UK (latitude 51.772, longitude -1.337) by Douglas Boyes, University of Oxford, using a light trap. The specimens were identified by the same individual and snap-frozen on dry ice.
DNA was extracted from whole organism tissue of ilXesXant1 at the Wellcome Sanger Institute (WSI) Scientific Operations core from the whole organism using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions. RNA was extracted from thorax/abdomen tissue of ilXesXant2 in the Tree of Life Laboratory at the WSI using TRIzol (Invitrogen), 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 and 10X Genomics Chromium read cloud 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. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq X (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were generated from abdomen tissue of ilXesXant1 using the Arima v1.0 kit and sequenced on HiSeq X.
Genome assembly
Assembly was carried out with Hifiasm ( Cheng et al., 2021). Haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes ( Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using SALSA2 ( Ghurye et al., 2019). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performed annotation using MitoFinder ( Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.
Table 3. Software tools used.
| Software tool | Version | Source |
|---|---|---|
| HiCanu | 1.0 | Cheng et al., 2021 |
| purge_dups | 1.2.3 | Guan et al., 2020 |
| SALSA2 | 2.2 | Ghurye et al., 2019 |
| longranger align | 2.2.2 |
https://support.10xgenomics.com/
genome-exome/software/pipelines/latest/ advanced/other-pipelines |
| freebayes | 1.3.1-17-gaa2ace8 | Garrison & Marth, 2012 |
| MitoHiFi | 1.0 | Uliano-Silva et al., 2021 |
| gEVAL | N/A | Chow et al., 2016 |
| PretextView | 0.1.x | https://github.com/wtsi-hpag/PretextView |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| BlobToolKit | 2.6.4 | Challis et al., 2020 |
Data availability
European Nucleotide Archive: Xestia xanthographa (square-spot rustic). Accession number PRJEB42066; https://identifiers.org/ena.embl/PRJEB42066.
The genome sequence is released openly for reuse. The X. xanthographa 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 Table 1.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.5746938.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.5744972.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.5744840.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.5746904.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5743293.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.5638618.
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