The genome sequence of the sycamore, Acronicta aceris (Linnaeus, 1758).

We present a genome assembly from an individual female Acronicta aceris (the sycamore; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 466 megabases in span. The complete assembly is scaffolded into 32 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Copyright:

Species taxonomy

Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Lepidoptera; Glossata; Ditrysia; Noctuoidea; Noctuidae; Acronictinae; Acronicta; Acronicta aceris (Linnaeus, 1758) (NCBI:txid987859).

Background

Acronicta aceris (sycamore moth) is a widely distributed noctuid moth found in Europe, Morocco and western regions of Asia. In the UK it is locally common in southeast and central England, with a flight season from June to August. Forewing colouration of the moth varies from silvery to dark grey, with variation in ground colour likely controlled by alleles at a single locus ( Majerus, 1986). The larvae of A. aceris are amongst the most colourful and flamboyant of all Lepidoptera caterpillars, bearing yellow and orange hairs arranged in striking ‘punk’ tufts along the body. As the common name suggests, the larvae feed on the leaves of sycamore ( Acer pseudoplatanus), other maples ( Acer sp.) and, particularly in urban and suburban areas, horse chestnut ( Aesculus hippocastanum). Larvae are active from July to September and overwintering occurs as a pupa in a double-layered cocoon in bark crevices or leaf litter. It is known to occasionally overwinter as a pupa through two winters before eclosing as an imago ( Waring ).

Genome sequence report

The genome was sequenced from one female A. berbera ( Figure 1) collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.772, longitude -1.338). A total of 39-fold coverage in Pacific Biosciences single-molecule long reads and 99-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 14 missing/misjoins, reducing the scaffold number by 20.00% and increasing the scaffold N50 by 4.33%.

Figure 1.

Image of the ilAcrAcer1 specimen captured prior to preservation and processing.

Specimen shown next to FluidX storage tube, 43.9 mm in length.

The final assembly has a total length of 466 Mb in 32 sequence scaffolds with a scaffold N50 of 16.1 Mb ( Table 1). The complete assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length), and the W and Z sex chromosome ( Figure 2– Figure 5; Table 2). The assembly has a BUSCO v5.1.2 ( Manni ) completeness of 99.0% (single 98.5%, duplicated 0.5%) 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.

Table 1.

Genome data for Acronicta aceris, ilAcrAcer1.1.

Project accession data
Assembly identifier	ilAcrAcer1.1
Species	Acronicta aceris
Specimen	ilAcrAcer1
NCBI taxonomy ID	NCBI:txid987859
BioProject	PRJEB45197
BioSample ID	SAMEA7701532
Isolate information	Female, abdomen (genome assembly), head/thorax (Hi-C)
Raw data accessions
PacificBiosciences SEQUEL II	ERR6406216
10X Genomics Illumina	ERR6054961-ERR6054964
Hi-C Illumina	ERR6054960
Genome assembly
Assembly accession	GCA_910591435.1
Accession of alternate haplotype	GCA_910591495.1
Span (Mb)	466
Number of contigs	45
Contig N50 length (Mb)	15.4
Number of scaffolds	32
Scaffold N50 length (Mb)	16.1
Longest scaffold (Mb)	19.0
BUSCO * genome score	C:99.0%[S:98.5%,D:0.5%],F:0.1%,M:0.9%,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/ilAcrAcer1.1/dataset/ilAcrAcer1_1/busco.

Figure 2.

Genome assembly of Acronicta aceris, ilAcrAcer1.1: 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 466,384,436 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 (20,910,575 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (16,061,144 and 10,539,460 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/ilAcrAcer1.1/dataset/ilAcrAcer1_1/snail.

Figure 5.

Genome assembly of Acronicta aceris, ilAcrAcer1.1: Hi-C contact map.

Hi-C contact map of the ilAcrAcer1.1 assembly, visualised in HiGlass.

Table 2.

Chromosomal pseudomolecules in the genome assembly of Acronicta aceris, ilAcrAcer1.1.

INSDC accession	Chromosome	Size (Mb)	GC%
OU342758.1	1	18.99	36.8
OU342759.1	2	18.58	37.1
OU342760.1	3	18.34	37.1
OU342761.1	4	18.02	36.9
OU342762.1	5	17.67	36.6
OU342763.1	6	17.51	36.3
OU342764.1	7	17.20	36.7
OU342765.1	8	16.99	37.2
OU342766.1	9	16.64	36.7
OU342767.1	10	16.27	36.5
OU342768.1	11	16.24	36.5
OU342769.1	12	16.06	37.1
OU342770.1	13	15.39	36.8
OU342771.1	14	15.23	36.8
OU342772.1	15	15.11	37.2
OU342773.1	16	14.79	36.7
OU342774.1	17	14.58	37.1
OU342775.1	18	14.39	37.4
OU342776.1	19	14.24	37.5
OU342777.1	20	14.04	37.1
OU342778.1	21	13.53	37.3
OU342779.1	22	11.82	37.8
OU342780.1	23	11.79	37.3
OU342781.1	24	11.31	37.8
OU342782.1	25	10.54	37.5
OU342783.1	26	9.86	37.7
OU342784.1	27	8.67	39.1
OU342785.1	28	7.85	38.6
OU342786.1	29	7.70	39.5
OU342787.1	30	6.64	38.9
OU342757.1	W	19.46	38.6
OU342756.1	Z	20.91	36.4
OU342788.1	MT	0.02	20.6

Genome assembly of Acronicta aceris, ilAcrAcer1.1: 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/ilAcrAcer1.1/dataset/ilAcrAcer1_1/blob.

Genome assembly of Acronicta aceris, ilAcrAcer1.1: 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/ilAcrAcer1.1/dataset/ilAcrAcer1_1/cumulative.

Methods

Sample acquisition and DNA extraction

A single female A. aceris (ilAcrAcer1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.772, longitude -1.338) by Douglas Boyes, UKCEH, using a light trap. The sample was identified by the same individual, and preserved on dry ice.

DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The ilAcrAcer1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Abdomen tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. Fragment size analysis of 0.01-0.5 ng of DNA was then performed using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200-ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size between 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 and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers’ instructions. Sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were generated from abdomen tissue using the Arima v2 Hi-C kit and sequenced on an Illumina NovaSeq 6000 X instrument.

Genome assembly

Assembly was carried out with Hifiasm ( Cheng ); haplotypic duplication was identified and removed with purge_dups ( Guan ). 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 ) using SALSA2 ( Ghurye ). The assembly was checked for contamination and corrected using the gEVAL system ( Chow ) as described previously ( Howe ). Manual curation ( Howe ) was performed using gEVAL, HiGlass ( Kerpedjiev ) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva ). The genome was analysed and BUSCO scores generated within the BlobToolKit environment ( Challis ). Table 3 contains a list of all software tool versions used, where appropriate.

Table 3.

Software tools used.

Software tool	Version	Source
Hifiasm	0.14	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	3.0	Uliano-Silva et al., 2021
gEVAL	N/A	Chow et al., 2016
HiGlass	1.11.6	Kerpedjiev et al., 2018
PretextView	0.2.x	https://github.com/wtsi-hpag/PretextView
BlobToolKit	2.6.2	Challis et al., 2020

Ethics/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. 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.

Data availability

European Nucleotide Archive: Acronicta aceris (the sycamore). Accession number PRJEB45197; https://identifiers.org/ena.embl/PRJEB45197.

The genome sequence is released openly for reuse. The A. aceris 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.

The Wellcome Open Research paper by Boyes et al. is a genome sequencing report of the sycamore, Acronicta aceris, found the Southeast and central parts of England.

The methods and strategies for sample collection (a female A. aceris), processing (dissection), preparation and quality control of DNA and sequencing are clearly described and appropriately referenced. Three complementary approaches including the PacificBiosciences SEQUEL II, the 10x genomics Illumina and Hi-C Illumina were used to generate high quality genome sequencing data. The data generated was further analysed very convincingly using the latest strategies for genome assembly, construction of 32 chromosomal pseudomolecules and assembly of sex chromosomes. The results are of excellent quality and add a lot more to the much-needed global genomic resources that will definitely support subsequent studies in population dynamics, distribution and the genomic changes that underpin adaptation to some environmental cues.

I fully support this publication but wish (in addition to the comments/inputs already provided by reviewer 1) to make the following suggestions for future consideration:

A stronger justification for sequencing this species and the added value to the already existing other genomic resources from closely related species.

Under the section on data availability, it would be beneficial to steer how the data will be annotated and if there will be need to generate RNAseq data for better annotation. Additionally, I could also be useful to mention if or how within the DToL initiative to there are plans to generate additional genomic resources to increase the usefulness of this dataset. For example, these genomic resources could be further analysed and discussed in the context of understanding the genomic differences amongst devastating pests (from outside the UK).

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Tropical livestock genomics and genetic improvement

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.

Boyes et al. have sequenced a genome of Acronicta aceris, a Lepidopteran from Noctuidae family. They have assembled the genome using PacBio HiFi long-read sequencing technology and scaffolded the genome using Illumina Novaseq based on short-read data obtained from chromosome conformation capture Hi-C technology. To polish the genome high-coverage 10X genomics short-read data was also generated. Based on the adopted methodologies for genome sequencing, assembly, and quality checks, I am very much convinced that the genome is of very high quality (Contig N50:15.4 Mb; Scaffold N50: 16.1 Mb; Longest contig: 19 Mb; BUSCO C: 99%) with 32 binned chromosomes including the sex chromosomes and a mitochondrial genome. However, I have some minor comments which might improve the quality of this genome note:

I think a few lines of the motivation behind the sequencing of this moth is missing in the background. Particularly, I would like to see why it is important to sequence the genome of this moth and what value it will bring to the overall Lepidopteran genomics.

Authors have performed one round of polishing using 10X short-read data, but they have not given any information about what proportion of variants they have observed before and after polishing. This will further add knowledge to the quality of the long-reads and to the overall assembly quality.

I think it would be useful to the readers if the authors add an estimate of the base-level accuracy (QV) for the assembly. QV values are useful for downstream applications like population or functional genetics. This stat could be easily calculated using Merqury (Rhie et al. Genome Biol  21, 245 (2020 ))

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Bioinformatics; Genome informatics; Integrative omics;

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.