Abstract
We present a genome assembly from an individual male Pammene aurita (the Sycamore Piercer; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 1,041.8 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 16.7 kilobases in length.
Keywords: Pammene aurita, Sycamore Piercer, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Tortricoidea; Tortricidae; Olethreutinae; Grapholitini; Pammene; Pammene aurita (Razowski, 1991) (NCBI:txid1870148).
Background
The Sycamore Piercer Pammene aurita (Razowski, 1992) is a moth of the Tortricidae family. The adults exhibit attractive light orange markings on the forewings that fade to a dull brown towards the head. There is little variation in these markings ( Bradley et al., 1979). The English vernacular name of this species is derived from the feeding habits of the larvae, which feed internally in the seeds of Sycamore ( Acer pseudoplatanus), ‘piercing’ the seed and leaving a neat round hole when the larva exits the seed to pupate ( Bradley et al., 1979; Elliott et al., 2018; Hancock et al., 2015). Larvae feed between August and September, and the moth overwinters as a pupa, choosing dead wood or bark as a pupation site in captivity ( Bradley et al., 1979; Elliott et al., 2018; Hancock et al., 2015). Adults can be found between June and August, flying around Sycamores in the afternoon, and coming to light after dark ( Elliott et al., 2018).
Globally, P. aurita is confined to northern and central Europe ( GBIF Secretariat, 2022; Hancock et al., 2015). This species is a relatively recent addition to the British and Irish fauna, apparently colonising south-eastern England in the mid-20th century ( Bradley et al., 1979; Hancock et al., 2015). Since then, the moth has spread across England and Wales, and has been recorded in eastern Ireland ( Elliott et al., 2018).
The genome of Pammene aurita 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 Pammene aurita, based on one male specimen from Wytham Woods, Oxfordshire, UK.
Genome sequence report
The genome was sequenced from one male Pammene aurita specimen ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude –1.34). A total of 24-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 76 missing joins or mis-joins and removed 19 haplotypic duplications, reducing the assembly length by 1.13% and the scaffold number by 32.47%.
Figure 1. Photograph of the Pammene aurita (ilPamAuri1) specimen used for genome sequencing.
The final assembly has a total length of 1,041.8 Mb in 52 sequence scaffolds with a scaffold N50 of 37.5 Mb ( Table 1). Most (99.81%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 autosomes, and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 2– 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.
Figure 2. Genome assembly of Pammene aurita, ilPamAuri1.1: 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 1,041,812,917 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 (72,712,871 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (37,501,620 and 26,227,413 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/ilPamAuri1.1/dataset/CAMTYY01/snail.
Figure 5. Genome assembly of Pammene aurita, ilPamAuri1.1: Hi-C contact map.
Hi-C contact map of the ilPamAuri1.1 assembly, 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=XW4dzhIyThitRIyDG5Q5JQ
Table 1. Genome data for Pammene aurita, ilPamAuri1.1.
| Project accession data | ||
|---|---|---|
| Assembly identifier | ilPamAuri1.1 | |
| Species | Pammene aurita | |
| Specimen | ilPamAuri1 | |
| NCBI taxonomy ID | 1870148 | |
| BioProject | PRJEB55029 | |
| BioSample ID | SAMEA10979080 | |
| Isolate information | ilPamAuri1, male, whole organism (genome sequencing and Hi-C scaffolding) | |
| Assembly metrics * | Benchmark | |
| Consensus quality (QV) | 62.5 | ≥ 50 |
| k-mer completeness | 100% | ≥ 95% |
| BUSCO ** | C:98.1%[S:97.3%,D:0.8%],
F:0.5%,M:1.5%,n:5,286 |
C ≥ 95% |
| Percentage of assembly mapped to chromosomes | 99.81% | ≥ 95% |
| Sex chromosomes | Z chromosome | localised homologous pairs |
| Organelles | Mitochondrial genome assembled | complete single alleles |
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR10008907 | |
| Hi-C Illumina | ERR10015064 | |
| Genome assembly | ||
| Assembly accession | GCA_947086415.1 | |
| Accession of alternate haplotype | GCA_947086445.1 | |
| Span (Mb) | 1,041.8 | |
| Number of contigs | 245 | |
| Contig N50 length (Mb) | 9.2 | |
| Number of scaffolds | 51 | |
| Scaffold N50 length (Mb) | 37.5 | |
| Longest scaffold (Mb) | 72.7 | |
* 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).
** BUSCO scores based on the lepidoptera_odb10 BUSCO set using v5.3.2. 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/ilPamAuri1.1/dataset/CAMTYY01/busco.
Figure 3. Genome assembly of Pammene aurita, ilPamAuri1.1: 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/ilPamAuri1.1/dataset/CAMTYY01/blob.
Figure 4. Genome assembly of Pammene aurita, ilPamAuri1.1: 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/ilPamAuri1.1/dataset/CAMTYY01/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Pammene aurita, ilPamAuri1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| OX352290.1 | 1 | 55.38 | 37.2 |
| OX352291.1 | 2 | 49.73 | 37.3 |
| OX352292.1 | 3 | 47.15 | 37.4 |
| OX352296.1 | 4 | 41.96 | 37.4 |
| OX352293.1 | 5 | 44.29 | 37.5 |
| OX352299.1 | 6 | 37.83 | 37.4 |
| OX352297.1 | 7 | 40.1 | 37.4 |
| OX352298.1 | 8 | 39.56 | 37.3 |
| OX352294.1 | 9 | 43.25 | 37.5 |
| OX352300.1 | 10 | 37.5 | 37.5 |
| OX352314.1 | 11 | 25.18 | 37.2 |
| OX352301.1 | 12 | 36.88 | 37.3 |
| OX352303.1 | 13 | 35.41 | 37.5 |
| OX352295.1 | 14 | 42.62 | 37.6 |
| OX352304.1 | 15 | 34.59 | 37.3 |
| OX352305.1 | 16 | 34.48 | 37.4 |
| OX352306.1 | 17 | 34.37 | 37.5 |
| OX352308.1 | 18 | 32.99 | 37.5 |
| OX352302.1 | 19 | 35.48 | 37.4 |
| OX352307.1 | 20 | 33.65 | 37.3 |
| OX352309.1 | 21 | 29.8 | 37.3 |
| OX352310.1 | 22 | 28.75 | 37.9 |
| OX352315.1 | 23 | 24.24 | 37.6 |
| OX352313.1 | 24 | 25.35 | 38.9 |
| OX352312.1 | 25 | 26.23 | 38 |
| OX352316.1 | 26 | 23.78 | 37.8 |
| OX352311.1 | 27 | 26.6 | 39 |
| OX352289.1 | Z | 72.71 | 37.2 |
| OX352317.1 | MT | 0.02 | 18.3 |
The estimated Quality Value (QV) of the final assembly is 62.5 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 ( Manni et al., 2021) completeness of 98.1% (single 97.3%, duplicated 0.8%) using the lepidoptera_odb10 reference set ( n = 5,286).
Methods
Sample acquisition and nucleic acid extraction
A male Pammene aurita specimen (ilPamAuri1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude –1.34) on 24 July 2021. The specimen was taken from woodland habitat by Douglas Boyes (University of Oxford) using a light trap. The specimen was identified by the collector and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilPamAuri1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Whole organism tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. 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 sample. 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 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) instrument. Hi-C data were also generated from tissue of ilPamAuri1 using the Arima v2 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 as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and Pretext ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2022), which performed annotation using MitoFinder ( Allio et al., 2020). To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness ( Rhie et al., 2020). The genome was analysed, and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were generated within the BlobToolKit environment ( Challis et al., 2020). Table 3 contains a list of software tool versions and sources.
Table 3. Software tools: versions and sources.
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.0.7 | https://github.com/blobtoolkit/blobtoolkit |
| BUSCO | 5.3.2 | https://gitlab.com/ezlab/busco |
| Hifiasm | 0.16.1-r375 | https://github.com/chhylp123/hifiasm |
| HiGlass | 1.11.6 | https://github.com/higlass/higlass |
| Merqury | MerquryFK | https://github.com/thegenemyers/MERQURY.FK |
| MitoHiFi | 2 | https://github.com/marcelauliano/MitoHiFi |
| PretextView | 0.2 | https://github.com/wtsi-hpag/PretextView |
| purge_dups | 1.2.3 | https://github.com/dfguan/purge_dups |
| YaHS | yahs-1.1.91eebc2 | https://github.com/c-zhou/yahs |
Ethics and compliance issues
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. All efforts are undertaken to minimise the suffering of animals used for sequencing. 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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>) and the Darwin Tree of Life Discretionary Award (218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328</a>).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 3 approved]
Data availability
European Nucleotide Archive: Pammene aurita (sycamore piercer). Accession number PRJEB55029; https://identifiers.org/ena.embl/PRJEB55029. ( Wellcome Sanger Institute, 2022)
The genome sequence is released openly for reuse. The Pammene aurita 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 using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4789928.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.4783585.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.4790455.
Members of the Tree 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.
References
- Allio R, Schomaker-Bastos A, Romiguier J, et al. : MitoFinder: Efficient automated large‐scale extraction of mitogenomic data in target enrichment phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradley JD, Tremewan WG, Smith A: British Tortricoid Moths - Tortricidae: Olethreutinae.The Ray Society. 1979. [Google Scholar]
- Challis R, Richards E, Rajan J, et al. : BlobToolKit - interactive quality assessment of genome assemblies. G3 (Bethesda). 2020;10(4):1361–1374. 10.1534/g3.119.400908 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng H, Concepcion GT, Feng X, et al. : Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021;18(2):170–175. 10.1038/s41592-020-01056-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elliott B, et al. : Tortricidae.In: J.R. Langmaid, S.M. Palmer, and M.R. Young (eds) A Field Guide to the Smaller Moths of Great Britain and Ireland . The British Entomological and Natural History Society.2018;279. [Google Scholar]
- GBIF Secretariat: Pammene aurita Razowski, 1992, GBIF Backbone Taxonomy. Checklist dataset. 2022; (Accessed: 9 March 2023). 10.15468/39omei [DOI] [Google Scholar]
- Guan D, McCarthy SA, Wood J, et al. : Identifying and removing haplotypic duplication in primary genome assemblies. Bioinformatics. 2020;36(9):2896–2898. 10.1093/bioinformatics/btaa025 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hancock F, Bland KP, Razowski J: The Moths and Butterflies of Great Britain and Ireland.Leiden: BRILL,2015;5(2). [Google Scholar]
- Harry E: PretextView (Paired REad TEXTure Viewer): A desktop application for viewing pretext contact maps. 2022; (Accessed: 19 October 2022). Reference Source [Google Scholar]
- Howe K, Chow W, Collins J, et al. : Significantly improving the quality of genome assemblies through curation. GigaScience. Oxford University Press,2021;10(1):giaa153. 10.1093/gigascience/giaa153 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerpedjiev P, Abdennur N, Lekschas F, et al. : HiGlass: Web-based visual exploration and analysis of genome interaction maps. Genome Biol. 2018;19(1):125. 10.1186/s13059-018-1486-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manni M, Berkeley MR, Seppey M, et al. : BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Mol Biol Evol. 2021;38(10):4647–4654. 10.1093/molbev/msab199 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao SSP, Huntley MH, Durand NC, et al. : A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665–80. 10.1016/j.cell.2014.11.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhie A, McCarthy SA, Fedrigo O, et al. : Towards complete and error-free genome assemblies of all vertebrate species. Nature. 2021;592(7856):737–746. 10.1038/s41586-021-03451-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhie A, Walenz BP, Koren S, et al. : Merqury: Reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 2020;21(1):245. 10.1186/s13059-020-02134-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simão FA, Waterhouse RM, Ioannidis P, et al. : BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31(19):3210–2. 10.1093/bioinformatics/btv351 [DOI] [PubMed] [Google Scholar]
- Uliano-Silva M, Ferreira JGRN, Krasheninnikova K, et al. : MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio High Fidelity reads. bioRxiv. [Preprint],2022. 10.1101/2022.12.23.521667 [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wellcome Sanger Institute: The genome sequence of the Sycamore Piercer, Pammene aurita (Razowski, 1991). European Nucleotide Archive.[dataset], accession number PRJEB55029,2022.
- Zhou C, McCarthy SA, Durbin R: YaHS: yet another Hi-C scaffolding tool. Bioinformatics. Edited by C. Alkan,2023;39(1):btac808. 10.1093/bioinformatics/btac808 [DOI] [PMC free article] [PubMed] [Google Scholar]