The genome sequence of the Sycamore Piercer, Pammene aurita (Razowski, 1991)

TL;DR

This paper presents the genome sequence of the Sycamore Piercer moth, including its chromosomes and mitochondrial DNA.

Contribution

The novel contribution is the first genome assembly of Pammene aurita, including chromosomal scaffolding and mitochondrial genome.

Findings

The genome assembly spans 1,041.8 megabases and is scaffolded into 28 chromosomal pseudomolecules.

The mitochondrial genome is 16.7 kilobases long and has been fully assembled.

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.

Figures

Photograph of the Pammene aurita (ilPamAuri1) specimen used for genome sequencing.

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.

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.

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.

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

Tables

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

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

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

Full text

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%.

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.

Table 1.: Genome data for Pammene aurita, ilPamAuri1.1.

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.

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.

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.

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 2.: Chromosomal pseudomolecules in the genome assembly of Pammene aurita, ilPamAuri1.

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.

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.