The genome sequence of the Spruce-seed moth, Cydia strobilella (Linnaeus, 1758)

TL;DR

This paper provides the genome sequence of the spruce-seed moth, including detailed gene annotations and chromosomal scaffolding.

Contribution

The study presents a high-quality genome assembly and gene annotation for Cydia strobilella, including chromosomal pseudomolecules and mitochondrial DNA.

Findings

The genome assembly is 542.6 megabases in size and includes 28 chromosomal pseudomolecules.

Gene annotation identified 13,058 protein-coding genes.

The mitochondrial genome is 16.78 kilobases long and was fully assembled.

Abstract

We present a genome assembly from an individual male
Cydia strobilella (the spruce-seed moth; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 542.6 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.78 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,058 protein coding genes.

Figures

Photograph of the Cydia strobilella (ilCydStro3) specimen used for genome sequencing.

Genome assembly of Cydia strobilella, ilCydStro3.1: 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 542,605,525 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 (61,581,207 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,535,481 and 12,144,315 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/CANOQN01/dataset/CANOQN01/snail.

Genome assembly of Cydia strobilella, ilCydStro3.1: 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/CANOQN01/dataset/CANOQN01/blob.

Genome assembly of Cydia strobilella, ilCydStro3.1: 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/CANOQN01/dataset/CANOQN01/cumulative.

Genome assembly of Cydia strobilella, ilCydStro3.1: Hi-C contact map of the ilCydStro3.1 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=CM2o3MkBTOmtZxcqxZ1LZw.

Tables

Table 1.. Genome data for Cydia strobilella, ilCydStro3.1.

Project accession data
Assembly identifier	ilCydStro3.1
Species	Cydia strobilella
Specimen	ilCydStro3
NCBI taxonomy ID	1100964
BioProject	PRJEB57682
BioSample ID	SAMEA110451554
Isolate information	ilCydStro3
Assembly metrics *		Benchmark
Consensus quality (QV)	65.7	≥ 50
k-mer completeness	100.0%	≥ 95%
BUSCO **	C:98.2%[S:97.5%,D:0.7%], F:0.5%,M:1.3%,n:5,286	C ≥ 95%
Percentage of assembly mapped to chromosomes	99.99%	≥ 95%
Sex chromosomes	Z	localised homologous pairs
Organelles	Mitochondrial genome: 16.78 kb	complete single alleles
Raw data accessions
PacificBiosciences SEQUEL II	ERR10499363
Hi-C Illumina	ERR10501029
PolyA RNA-Seq Illumina	ERR10908614
Genome assembly
Assembly accession	GCA_947568885.1
Accession of alternate haplotype	GCA_947568895.1
Span (Mb)	542.6
Number of contigs	94
Contig N50 length (Mb)	10.6
Number of scaffolds	29
Scaffold N50 length (Mb)	19.5
Longest scaffold (Mb)	61.58
Genome annotation
Number of protein-coding genes	13,058
Number of non-coding genes	1,863
Number of gene transcripts	24,652

Table 2.. Chromosomal pseudomolecules in the genome assembly of Cydia strobilella, ilCydStro3.

INSDC accession	Chromosome	Length (Mb)	GC%
OX387676.1	1	36.76	37.5
OX387677.1	2	30.81	37.5
OX387678.1	3	24.0	37.5
OX387679.1	4	23.74	38.0
OX387680.1	5	23.35	38.0
OX387681.1	6	22.09	37.5
OX387682.1	7	20.71	37.5
OX387683.1	8	20.57	37.5
OX387684.1	9	19.54	38.0
OX387685.1	10	19.54	37.5
OX387686.1	11	18.92	38.0
OX387687.1	12	18.44	38.0
OX387688.1	13	17.49	38.0
OX387689.1	14	17.46	38.0
OX387690.1	15	17.22	38.0
OX387691.1	16	16.86	38.0
OX387692.1	17	16.83	38.0
OX387693.1	18	16.42	38.0
OX387694.1	19	14.57	38.0
OX387695.1	20	12.98	38.0
OX387696.1	21	12.79	38.5
OX387697.1	22	12.14	38.0
OX387698.1	23	11.04	37.5
OX387699.1	24	9.84	39.5
OX387700.1	25	9.5	38.0
OX387701.1	26	9.01	39.0
OX387702.1	27	8.4	38.0
OX387675.1	Z	61.58	38.0
OX387703.1	MT	0.02	19.5

Table 3.. Software tools: versions and sources.

Software tool	Version	Source
BlobToolKit	4.1.7	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
sanger-tol/ genomenote	v1.0	https://github.com/sanger-tol/genomenote
sanger-tol/ readmapping	1.1.0	https://github.com/sanger-tol/readmapping/tree/1.1.0
YaHS	1.2a	https://github.com/c-zhou/yahs

Full text

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Apoditrysia; Tortricoidea; Tortricidae; Olethreutinae; Grapholitini; Cydia; Cydia strobilella (Linnaeus, 1758) (NCBI:txid1100964).

Background

The genome of the spruce-seed moth, Cydia strobilella, 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 Cydia strobilella, based on one male specimen from Wytham Woods, Oxfordshire.

Genome sequence report

The genome was sequenced from one male Cydia strobilella ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76, –1.34). A total of 36-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 9 missing joins or mis-joins and removed two haplotypic duplications, reducing the scaffold number by 11.76%.

Photograph of the Cydia strobilella (ilCydStro3) specimen used for genome sequencing.

The final assembly has a total length of 542.6 Mb in 29 sequence scaffolds with a scaffold N50 of 19.5 Mb ( Table 1). The snailplot 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 (99.99%) 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 5; Table 2). The Z chromosome identified based on synteny with Cydia splendana (GCA_910591565.2) ( Boyes et al., 2023). 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.

Table 1.: Genome data for Cydia strobilella, ilCydStro3.1.

Genome assembly of Cydia strobilella, ilCydStro3.1: 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 542,605,525 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 (61,581,207 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,535,481 and 12,144,315 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/CANOQN01/dataset/CANOQN01/snail.

Genome assembly of Cydia strobilella, ilCydStro3.1: 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/CANOQN01/dataset/CANOQN01/blob.

Genome assembly of Cydia strobilella, ilCydStro3.1: 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/CANOQN01/dataset/CANOQN01/cumulative.

Genome assembly of Cydia strobilella, ilCydStro3.1: Hi-C contact map of the ilCydStro3.1 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=CM2o3MkBTOmtZxcqxZ1LZw.

Table 2.: Chromosomal pseudomolecules in the genome assembly of Cydia strobilella, ilCydStro3.

The estimated Quality Value (QV) of the final assembly is 65.7 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 98.2% (single = 97.5%, duplicated = 0.7%), using the lepidoptera_odb10 reference set ( n = 5,286).

Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/1100964.

Genome annotation report

The Cydia strobilella genome assembly (GCA_947568885.1) was annotated using the Ensembl rapid annotation pipeline at the European Bioinformatics Institute (EBI). The resulting annotation includes 24,652 transcribed mRNAs from 13,058 protein-coding and 1,863 non-coding genes ( Table 1; https://rapid.ensembl.org/Cydia_strobilella_GCA_947568895.1/Info/Index).

Methods

Sample acquisition and nucleic acid extraction

The Cydia strobilella specimens used for DNA and Hi-C sequencing (specimen ID Ox002025, ToLID ilCydStro3) and for RNA sequencing (specimen ID Ox002046, ToLID ilCydStro6) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.76, longitude –1.34) on 2022-03-21. The specimen was collected and identified by James Hammond (University of Oxford) and snap-frozen on dry ice.

Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io ( Denton et al., 2023b). The workflow for high molecular weight (HMW) DNA extraction at the WSI includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the ilCydStro3 sample was weighed and dissected on dry ice, setting aside tissue for Hi-C sequencing ( Jay et al., 2023). Tissue from the whole organism was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). HMW DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 31 ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): 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 whole organism tissue of ilCydStro6 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.

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 remaining tissue of ilCydStro3 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 Pretext ( 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.

A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.

Table 3 contains a list of relevant software tool versions and sources.

Genome annotation

The Ensembl Genebuild annotation system ( Aken et al., 2016) at the EBI was used to generate annotation for the Cydia strobilella assembly (GCA_947568885.1). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt ( UniProt Consortium, 2019).

Wellcome Sanger Institute – Legal and Governance

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’, 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.

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

•      Legality of collection, transfer and use (national and international)

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.