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. 2026 Sep 11;11:628. [Version 1] doi: 10.12688/wellcomeopenres.27772.1

The genome sequence of a plutellid moth, Plutella hyperboreella Strand, 1902 (Lepidoptera: Plutellidae)

Mikko Vallinmäki 1, Marko Mutanen 2, Charlotte J Wright 3,a, Joana I Meier 3, Mark L Blaxter 3; Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team; Wellcome Sanger Institute Scientific Operations: Sequencing Operations; Wellcome Sanger Institute Tree of Life Core Informatics team; Tree of Life Core Informatics collective; Project Psyche Community
PMCID: PMC13613003  PMID: 42800963

Abstract

We present a genome assembly from a female specimen of Plutella hyperboreella (Arthropoda; Insecta; Lepidoptera; Plutellidae). The assembly contains two haplotypes with total lengths of 503.34 megabases and 426.42 megabases. Most of the haplotype 1 assembly (98.75%) was scaffolded into 31 chromosomal pseudomolecules, including the W and Z sex chromosomes. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 20.67 kilobases. Gene annotation of this assembly by Ensembl identified 12,347 protein-coding genes. This work is part of Project Psyche, a collaborative programme generating genomes for European butterflies and moths.

Keywords: Plutella hyperboreella, plutellid moth, genome sequence, chromosomal, Lepidoptera

Species taxonomy

Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Altocrustacea; Allotriocarida; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Eumetabola; Endopterygota; Aparaglossata; Panorpida; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Yponomeutoidea; Plutellidae; Plutella; Plutella hyperboreella Strand, 1902 (NCBI:txid1405359)

Background

The genus Plutella Schrank, 1802 (Yponomeutoidea: Plutellidae) includes the Diamondback moth, Plutella xylostella (Linnaeus, 1758), a widely distributed and infamous pest of cruciferous vegetables such as cabbage. It is less well known that the diamondback moth has many close relatives adapted to cold climatic conditions and therefore restricted to mountainous and Arctic regions. Recent studies have indicated that these cold-adapted Plutella are more diverse than previously thought (e.g. Baraniak, 2007; Søli et al., 2018). DNA barcodes suggest that more cold-adapted species remain to be described. Plutella hyperboreella Strand, 1902, is one of the arctic members of the genus. It is known from the entire Holarctic region, although recorded locations are few. Furthermore, given the taxonomic difficulties within the group, some populations may represent other closely related species. Consequently, the alpine P. geniatella Zeller, 1839 was long regarded as synonymous with P. hyperboreella. DNA barcodes support their status as distinct species and also confirm the status of P. hyperboreella as a Holarctic species, as the same DNA barcode BIN (BOLD:AAC3387) is known from Finland, Sweden, Russia and Canada. The monophyletic status of Plutella is highly questionable, as the genus Eidophasia is likely nested within Plutella. However, the monophyly of the family Plutellidae is strongly supported ( Sohn et al., 2013).

With a wingspan of 13–16 mm, Plutella hyperboreella (Figure 1) is a small moth, although it appears at least mid-sized among its congeners. Its wing pattern resembles that of most other Plutella, with the narrow forewings dorsally predominantly pale, forming a diamond-like pattern in resting position, while the forewings are otherwise mostly grey-brown except for the usually whitish costa, particularly terminally. The fringe is streaked white and brown. Sexual dimorphism is minor.

Figure 1. Voucher photograph of the Plutella hyperboreella (ilPluHype1) specimen used for genome sequencing.

Figure 1.

As in all Plutella, the diet consists of cruciferous plants (Brassicaceae). In its arctic habitats in Finland, the usual food plant is probably Arabis alpina from which the author MM has reared it, but it likely also feeds on, e.g. Draba spp. It occurs very locally in the alpine zone (tundra), typically on rocky slopes, often slate, and on cliffs. It is mostly day-active, like many arctic moths. In Finland, it is most often seen between midsummer and mid-July. It is very local and regarded as vulnerable in the country (VU).

The genome sequence of P. hyperboreella will aid in clarifying the phylogeny of Plutellidae, including the genus Plutella, which is probably not monophyletic. The genus would provide an interesting model for biogeographic and evolutionary studies. For example, why are so many Plutella species adapted to cold environments, while the diamondback moth has evolved to tolerate significantly different environmental conditions? Other life history differences between the two taxa include voltinism, ability to enter diapause, and migratory behaviours.

Methods

Sample acquisition

The specimen used for genome sequencing was an adult female Plutella hyperboreella (specimen ID OULUPSY000435, ToLID ilPluHype1; Figure 1), collected from South, Saana, Kilpisjärvi, Finland (latitude 69.0318, longitude 20.8467) on 2024-07-04. The specimen was collected and identified by Mikko Vallinmäki and Marko Mutanen (University of Oulu).

Nucleic acid extraction

Protocols for high molecular weight (HMW) DNA extraction developed at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory are available on protocols.io ( Howard et al., 2025). For HMW DNA extraction, tissue from the abdomen of ilPluHype1 was homogenised by powermashing using a PowerMasher II tissue disruptor.

HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the Femto Pulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 10.49 ng/μL and a yield of 493.03 ng, with a fragment size of 14.2 kb. The Genomic Quality Number (GQN) was 6.9.

PacBio HiFi library preparation and sequencing

Library preparation and sequencing took place in the WSI Scientific Operations core. Library PSYCHE15450949 was prepared with the SMRTbell Prep Kit 3.0 (Pacific Biosciences) according to the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified with a Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.

The library was sequenced on a Revio instrument (Pacific Biosciences, California, USA). The prepared libraries selected for multiplexing were pooled based on genome size, library molarity and the intended plex level. Primers were annealed and polymerases were bound to generate circularised complexes following the manufacturer’s instructions. Complexes were purified using SMRTbell beads, diluted to the Revio loading concentration, and spiked with a Revio sequencing internal control. The pooled libraries were sequenced on a Revio 25M Plex SMRT cell in a 2-plex run. SMRT Link software (Pacific Biosciences) was used to configure and monitor the run and to perform primary and secondary data analysis.

Hi-C

Sample preparation and crosslinking

Hi-C data were generated from the ilPluHype1 head sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.

Hi-C library preparation and sequencing

Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size-selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Libraries were amplified with KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).

Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again to create equimolar and/or weighted 2.8 nM pools. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Libraries were sequenced on the Illumina NovaSeq X, generating paired-end 150-bp reads.

Genome assembly

Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. The k-mer frequency distribution was analysed with GenomeScope (version 2.1.0) ( Ranallo-Benavidez et al., 2020), providing fitted point estimates of genome size, heterozygosity and repeat content.

The HiFi reads were assembled using Hifiasm in Hi-C phasing mode ( Cheng et al., 2021, 2022), producing two haplotypes. Hi-C reads ( Rao et al. , 2014) were mapped to contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al. , 2022), BUSCO ( Manni et al., 2021) and MerquryFK ( Rhie et al., 2020).

The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al. , 2023), which runs MitoFinder ( Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation

The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. The flat files and maps for curation were generated with TreeVal. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al. , 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). The two haplotype assemblies were combined for curation. Manual corrections included ten breaks and 45 joins. This reduced the scaffold count by 14.6%, increased the scaffold N50 by 6.5%, and increased the total assembly length by 1.2%. The curation process is described at sanger-tol/curation-resources. A Hi-C contact map of the haplotype 1 assembly was generated with PretextSnapshot.

Assembly quality assessment

The MerquryFK tool ( Rhie et al., 2020), run in a Singularity container ( Kurtzer et al., 2017), was used to evaluate k-mer completeness and assembly quality for both haplotypes using the k-mer databases ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.

The haplotype 1 assembly was assessed using BlobToolKit ( Challis et al., 2020). PacBio reads were mapped to the haplotype 1 assembly to derive sequence coverage, and BUSCO ( Manni et al., 2021) was used to assess gene-set completeness. DIAMOND BLASTp searches of extracted BUSCO proteins and DIAMOND BLASTx searches of assembly sequences against UniProt Reference Proteomes ( Buchfink et al., 2021), followed by BLASTn searches of sequences without DIAMOND BLASTx hits against the NCBI nt database ( Altschul et al., 1990), supported taxonomic assignment. Coverage, sequence composition, taxonomic assignment and BUSCO results were integrated into a BlobDir for interactive inspection and visualisation. The snail and blob plots were rendered locally from this hosted BlobDir using BlobTk v0.8.3. The scaffold-length radial scale function for the snail plot was set to linear ( Challis & Blaxter, 2026). The blob plot used circular markers with area proportional to scaffold length ( Challis et al., 2020).

We used busco-alg-painter v0.1.0, a Python reimplementation and extension of lep_busco_painter, to paint Merian elements along chromosomes. Merian elements represent the 32 ancestral linkage groups reconstructed for Lepidoptera ( Wright et al., 2024). The BUSCO-to-Merian reference assignments were derived from lep_busco_painter. Complete single-copy and duplicated BUSCO genes from the lepidoptera_odb10 results were assigned to Merian elements. Chromosome identifiers and lengths were obtained from NCBI Datasets, and BUSCO positions were plotted on chromosomes drawn to scale.

Genome sequence report

Sequence data

PacBio sequencing of the P. hyperboreella specimen generated 35.98 Gb (gigabases) from 3.96 million reads. We assembled the genome from these reads. GenomeScope analysis using a p = 2 model gave a k-mer-based haploid genome size estimate of 458.55 Mb, with heterozygosity of 1.02% and repeat content of 43.27%; the full-model fit was 96.47% (Figure 2). The GenomeScope estimates guided expectations for the assembly. The sequencing data provided approximately 76× coverage of the haplotype 1 assembly. Hi-C sequencing produced 151.95 Gb from 503.16 million reads, which were used to scaffold the haplotype 1 assembly. Table 1 summarises the specimen and sequencing details.

Figure 2. GenomeScope model fitted to the k-mer frequency distribution.

Figure 2.

The plot shows the observed and modelled k-mer spectra used to estimate genome size, heterozygosity and repeat content from unassembled sequencing reads.

Table 1. Specimen and sequencing data for P. hyperboreella (BioProject PRJEB92268).

Platform PacBio HiFi Hi-C
ToLID ilPluHype1 ilPluHype1
Specimen ID OULUPSY000435 OULUPSY000435
BioSample (source individual) SAMEA117574523 SAMEA117574523
BioSample (tissue) SAMEA117574591 SAMEA117574590
Tissue abdomen head
Instrument Revio (1 run) Illumina NovaSeq X
Library ID & run accession(s) PSYCHE15450949: ERR15282569 ERR15286553
Read count total 3.96 million reads 503.16 million read pairs
Base count total 35.98 Gb 151.95 Gb

Assembly statistics

The genome was assembled into two haplotypes using Hi-C phasing. The haplotype 1 assembly was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The haplotype 1 assembly, which is proposed as the reference assembly for this species, has a total length of 503.34 Mb in 163 scaffolds (excluding organelle sequences), with 68 gaps, and a scaffold N50 of 16.36 Mb (Table 2). The haplotype 1 assembly has a scaffold auN of 18.6 Mb.

Table 2. Genome assembly statistics for P. hyperboreella .

Genome assembly Haplotype 1 Haplotype 2
Assembly name ilPluHype1.hap1.1 ilPluHype1.hap2.1
Assembly accession GCA_966184305.1 GCA_966185215.1
Assembly level chromosome scaffold
Span (Mb) 503.34 426.42
Number of chromosomes 31 -
Number of contigs 231 195
Contig N50 (Mb) 13.89 8.14
Number of scaffolds (excluding organelle sequences) 163 141
Scaffold N50 (Mb) 16.36 15.71
Scaffold auN (Mb) 18.6 14.8
Longest scaffold length (Mb) 39.58 18.49
Sex chromosomes W and Z -
Organelles Mitochondrial genome: 20.67 kb -

Most of the haplotype 1 assembly sequence (98.75%) was assigned to 31 chromosome-level scaffolds, representing 29 autosomes and the W and Z sex chromosomes. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size (Figure 3; Table 3). Chromosome painting of the haplotype 1 assembly with Merian elements illustrates the distribution of orthologues along chromosomes and highlights patterns of chromosomal evolution relative to Lepidopteran ancestral linkage groups (Figure 4). The Z and W chromosomes were identified by copy number in the diploid assembly.

Figure 3. Hi-C contact map of the P. hyperboreella haplotype 1 assembly.

Figure 3.

Assembled chromosomes are shown in order of size and labelled along the axes. The plot was generated using PretextSnapshot.

Figure 4. Merian elements painted across chromosomes in the ilPluHype1.hap1.1 haplotype 1 assembly of P. hyperboreella.

Figure 4.

Chromosomes are drawn to scale, with the positions of orthologues shown as coloured bars. Each orthologue is coloured by the Merian element that it belongs to. All orthologues which could be assigned to Merian elements are shown.

The mitochondrial genome was also assembled (length 20.67 kb, OZ305772.1). This sequence is included as a contig in the multifasta file of the haplotype 1 genome submission and as a standalone record.

Table 3. Chromosomal pseudomolecules in the haplotype 1 genome assembly of P. hyperboreella ilPluHype1.

INSDC accession Molecule Length (Mb) GC% Assigned Merian elements
OZ305741.1 1 18.55 39 M17; M20
OZ305742.1 2 18.48 38.50 M2
OZ305743.1 3 18.02 38.50 M12
OZ305744.1 4 17.95 38 M9
OZ305745.1 5 17.84 38.50 M23
OZ305746.1 6 17.60 39 M1
OZ305747.1 7 17.45 39 M3
OZ305748.1 8 17.30 38.50 M5
OZ305749.1 9 16.62 38 M18
OZ305750.1 10 16.36 38.50 M8
OZ305751.1 11 15.94 38.50 M16
OZ305752.1 12 15.89 38.50 M7
OZ305753.1 13 15.35 38.50 M6
OZ305754.1 14 15.30 38.50 M4
OZ305755.1 15 15.03 39 M15
OZ305756.1 16 14.79 38.50 M21
OZ305757.1 17 14.74 39 M10
OZ305758.1 18 14.56 38.50 M22
OZ305759.1 19 14.45 39 M19
OZ305760.1 20 13.89 39 M11
OZ305761.1 21 12.72 39 M14
OZ305762.1 22 11.60 38.50 M24
OZ305763.1 23 11.57 39 M27
OZ305764.1 24 11.08 38.50 M26
OZ305765.1 25 10.24 39 M28
OZ305766.1 26 10.24 39.50 M31
OZ305767.1 27 9.53 39.50 M25
OZ305768.1 28 8.57 39.50 M29
OZ305769.1 29 8.10 41 M30
OZ305770.1 W 39.58 38.50 M13
OZ305771.1 Z 37.75 38 M13; MZ

Assembly quality metrics

For the haplotype 1 assembly, the estimated QV is 61.8, and for the haplotype 2 assembly, 61.9. When the two haplotypes are combined, the assembly achieves an estimated QV of 61.9. The k-mer completeness is 82.80% for the haplotype 1 assembly, 74.70% for the haplotype 2 assembly, and 99.41% for the combined haplotypes (Figure 5).

Figure 5. Evaluation of k-mer completeness using MerquryFK.

Figure 5.

This plot illustrates the recovery of k‐mers from the original read data in the haplotype 1 assembly, the haplotype 2 assembly and the combined haplotypes. The horizontal axis represents k‐mer multiplicity, and the vertical axis shows the number of k‐mers. The black curve represents k‐mers that appear in the reads but are not assembled. The green curve corresponds to k‐mers shared by both haplotypes, and the red and blue curves show k‐mers found only in one of the haplotypes.

BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5,286) identified 99.3% of the expected gene set (single = 97.5%, duplicated = 1.8%) in the haplotype 1 assembly. For the haplotype 2 assembly, BUSCO analysis identified 92.1% of the expected gene set (single = 91.4%, duplicated = 0.7%).

The snail plot in Figure 6 provides a summary of assembly contiguity, cumulative scaffold count, sequence composition and BUSCO completeness for the haplotype 1 assembly ( Challis & Blaxter, 2026). The relative sizes of the longest scaffold, scaffold N50 and N90, together with the dark-grey scaffold-length distribution, show how much of the assembly is contained in long scaffolds. The outer composition track shows variation in GC, AT and N content, while the central track shows the cumulative scaffold count and the BUSCO inset summarises gene-set completeness.

Figure 6. Assembly metrics for ilPluHype1.hap1.1, the haplotype 1 assembly.

Figure 6.

The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the haplotype 1 assembly, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. Scaffold length is shown on a linear radial scale, scaled to the longest scaffold. Red shading indicates the longest scaffold, and the deeper orange and pale orange shading represent the N50 and N90 lengths. The summary statistics include scaffold auN, the area under the scaffold Nx curve. The light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the lepidoptera_odb10 set is presented at the top right. The assembly can be explored interactively in the BlobToolKit viewer.

The blob plot in Figure 7 displays scaffolds from the haplotype 1 assembly by GC proportion and base coverage. Marker area is proportional to scaffold length, and colour indicates taxonomic assignment ( Challis et al., 2020).

Figure 7. BlobToolKit blob plot for ilPluHype1.hap1.1, the haplotype 1 assembly.

Figure 7.

The plot shows base coverage (vertical axis) and GC content (horizontal axis). Circles represent scaffolds; marker area is proportional to scaffold length, and colour indicates taxonomic assignment. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. The assembly can be explored interactively in the BlobToolKit viewer.

Table 4 lists the assembly metric benchmarks adapted from Rhie et al.. (2021) the Earth BioGenome Project (EBP) Report on Assembly Standards. The three-part EBP metric, calculated for the haplotype 1 assembly, is 7.C.Q61.

Table 4. Earth BioGenome Project summary metrics for the P. hyperboreella assembly.

Measure Value Benchmark
EBP summary (haplotype 1) 7.C.Q61 6.C.Q40
Contig N50 length 13.89 Mb > 1 Mb
Scaffold N50 length 16.36 Mb = chromosome N50
Consensus quality (QV) Haplotype 1: 61.8; haplotype 2: 61.9; combined: 61.9 ≥ 40
k-mer completeness Haplotype 1: 82.80%; Haplotype 2: 74.70%; combined: 99.41% > 90%
BUSCO Haplotype 1: C:99.3% [S:97.5%, D:1.8%], F:0.1%, M:0.6%, n:5,286; Haplotype 2: C:92.1% [S:91.4%, D:0.7%], F:0.1%, M:7.8%, n:5,286 S > 90%; D < 5%
Percentage of assembly assigned to chromosomes 98.75% ≥ 90%

Notes: The EBP summary uses log10(Contig N50); chromosome-level (C) or log10(Scaffold N50); Q (Merqury QV). BUSCO v6.0.0 using the lepidoptera_odb10 lineage dataset: C=complete; S=single-copy; D=duplicated; F=fragmented; M=missing; n=orthologues.

Genome annotation report

The P. hyperboreella genome assembly (GCA_966184305.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI). The annotation comprises 22,399 transcripts from 12,347 protein-coding genes and 2,229 non-coding genes. The average transcript length is 13,109.26 bp, with an average of 1.54 coding transcripts per gene and 6.46 exons per transcript. For further information, please refer to the Ensembl annotation page.

Data availability

European Nucleotide Archive: Plutella hyperboreella. Accession number: PRJEB92268; URL: https://identifiers.org/ena.embl/PRJEB92268. The genome sequence is released openly for reuse. The P. hyperboreella genome sequencing initiative is part of the Sanger Institute Tree of Life Programme ( PRJEB43745) and Project Psyche ( PRJEB71705). All raw sequence data and the haplotype assemblies have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.

Pipelines used for genome assembly at the WSI Tree of Life are available at https://pipelines.tol.sanger.ac.uk/pipelines. Table 5 lists software versions used in this study.

Table 5. Software versions and sources used for P. hyperboreella .

Software Version Source
BLAST 2.16.0+ https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/
BlobToolKit 4.4.6 https://github.com/genomehubs/blobtoolkit
BlobTk (hosted BlobDir) 0.5.1 https://github.com/genomehubs/blobtk
BlobTk (snail and blob plot renderer) 0.8.3 https://github.com/genomehubs/blobtk
BUSCO 6.0.0; 5.8.3 https://gitlab.com/ezlab/busco
busco-alg-painter 0.1.0 https://github.com/Karenvn/busco-alg-painter
bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2
DIAMOND 2.1.8 https://github.com/bbuchfink/diamond
fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows
FastK 1.2 https://github.com/thegenemyers/FASTK
GenomeScope 2.1.0 https://github.com/tbenavi1/genomescope2.0
Gfastats 1.3.6 https://github.com/vgl-hub/gfastats
Hifiasm 0.19.8-r603 https://github.com/chhylp123/hifiasm
HiGlass 1.13.4 https://github.com/higlass/higlass
MerquryFK 1.1.0-c1 https://github.com/thegenemyers/MERQURY.FK
Minimap2 2.28-r1209 https://github.com/lh3/minimap2
MitoHiFi 3.2.2 https://github.com/marcelauliano/MitoHiFi
MultiQC 1.14; 1.17 and 1.18 https://github.com/MultiQC/MultiQC
Nextflow 24.10.4 https://github.com/nextflow-io/nextflow
PretextSnapshot 0.0.6 https://github.com/sanger-tol/PretextSnapshot
PretextView 1.0.3 https://github.com/sanger-tol/PretextView
samtools 1.21 https://github.com/samtools/samtools
sanger-tol/ascc 0.1.0 https://github.com/sanger-tol/ascc
sanger-tol/blobtoolkit v0.9.0 https://github.com/sanger-tol/blobtoolkit
sanger-tol/curationpretext 1.4.2 https://github.com/sanger-tol/curationpretext
Seqtk 1.4-r122 https://github.com/lh3/seqtk
Singularity 3.9.0 https://github.com/sylabs/singularity
TreeVal v1.4.0 https://github.com/sanger-tol/treeval
YaHS 1.2.2 https://github.com/c-zhou/yahs

Author information

Contributors are listed at the following links:

Funding Statement

This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [220540]. The Oulu hub is supported by the Biodiverse Anthropocenes Research Profiling programme funded by the Research Council of Finland (decision #336449) and the University of Oulu.

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]

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Wellcome Open Res. 2026 Sep 24. doi: 10.21956/wellcomeopenres.30563.r169446

Reviewer response for version 1

Will Nash 1

The manuscript presents two haplotype assemblies of the moth Plutella hyperboreella, spanning 503.34 Mb & 426.42 Mb respectively. Hap 1 is assigned to 31 chromosome pseudomolecules. 12,347 protein coding genes are also annotated based on computational inference.

The background is well written and presents a strong rationale for studying the target species, based on cold adaptation, as well as links to pest species.

All methods are appropriate and current for generating a high-quality genome from a single small insect specimen. Wet lab methods are well documented through protocols.io, with specific modifications for the species also described. I am happy to see yields and fragment sizes included. Detailed library prep (PacBio and Hi-C) methodologies are included – this is great and extends reproducibility for small insect studies. Bioinformatic methods are very clear and well documented.

The sequencing report shows 3.96 million HiFi reads were used to assemble the genome. The k-mer spectrum shows a clear diploid profile, with a well-fitted GenomeScope model (96.47%), an estimated heterozygosity of 1.02%, and ample HiFi sequencing depth (~76×) for haplotype-resolved assembly. Assembly stats (98.75% chromosome-assigned, QV 61.8, BUSCO 99.3%) suggest a very strong assembly following scaffolding. The genome is computationally annotated, with 12,347 protein-coding genes, with an average transcript length of ~13 kb. It would be useful for the authors to state more explicitly what evidence sources were incorporated into the Ensembl annotation, particularly whether transcriptomic evidence was available. RNA-seq-supported annotation would provide additional confidence in gene models, although I recognise that obtaining suitable RNA material can be challenging for small insect specimens and may be beyond the scope of this Genome Note.

Overall, this is a well-produced and highly reproducible genome resource. I have only a small number of comments that I think would improve clarity:

  1. Difference in haplotype assembly span. Haplotype 1 is 503.34 Mb whereas haplotype 2 is 426.42 Mb. The W and Z chromosomes in haplotype 1 are 39.58 Mb and 37.75 Mb, respectively, together accounting for ~77.3 Mb, very close to the ~76.9 Mb difference between the two haplotype assemblies. This appears biologically consistent with the female WZ specimen and the representation of both sex chromosomes within haplotype 1. I suggest that the authors add a short explanation of the difference in assembly span, as it is otherwise quite striking when first encountering Table 2.

  2. Individual haplotype k-mer completeness. K-mer completeness is reported as 82.80% for haplotype 1 and 74.70% for haplotype 2, while the combined assembly reaches 99.41%. I think a sentence explaining the expected interpretation of the individual versus combined haplotype values would help readers, particularly given the phased nature of the assembly and the sex chromosomes.

  3. Sex chromosome identification. The manuscript states that the W and Z chromosomes were identified using copy number in the diploid assembly. It would be useful to provide a little more detail on the coverage/copy-number evidence used for these assignments.

Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes

Are the rationale for sequencing the genome and the species significance clearly described?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Entomology, Population Genomics, Comparative Genomics, Genome Assembly,

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.

Wellcome Open Res. 2026 Sep 11.

Reviewer response for version 1

Automated Benchmarking 1

As this article is a Genome Note, Automated Benchmarking has been used to determine whether the reported assembly passes three defined checks established by the Earth BioGenome Project: the raw and assembled data are publicly available, the Earth BioGenome Project three-part metric is met, and the BUSCO score passes the stated threshold. For Plutella hyperboreella (ilPluHype1), all three checks passed. Data available: pass. ENA study PRJEB92268, raw-read accessions ERR15282569 and ERR15286553, and assembly accessions GCA_966184305.1 and GCA_966185215.1 are public. Earth BioGenome Project three-part metric: pass. Haplotype 1 is reported as 7.C.Q61, meeting the 6.C.Q40 reference standard. BUSCO: pass. Haplotype 1, which is proposed as the reference assembly for this species, has 99.3% complete BUSCOs (97.5% single-copy and 1.8% duplicated). It meets the target of greater than 90% complete and single-copy BUSCOs. This decision uses values reported in the Genome Note and public accession records.

Reviewer Expertise:

NA

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.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Availability Statement

    European Nucleotide Archive: Plutella hyperboreella. Accession number: PRJEB92268; URL: https://identifiers.org/ena.embl/PRJEB92268. The genome sequence is released openly for reuse. The P. hyperboreella genome sequencing initiative is part of the Sanger Institute Tree of Life Programme ( PRJEB43745) and Project Psyche ( PRJEB71705). All raw sequence data and the haplotype assemblies have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.

    Pipelines used for genome assembly at the WSI Tree of Life are available at https://pipelines.tol.sanger.ac.uk/pipelines. Table 5 lists software versions used in this study.

    Table 5. Software versions and sources used for P. hyperboreella .

    Software Version Source
    BLAST 2.16.0+ https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/
    BlobToolKit 4.4.6 https://github.com/genomehubs/blobtoolkit
    BlobTk (hosted BlobDir) 0.5.1 https://github.com/genomehubs/blobtk
    BlobTk (snail and blob plot renderer) 0.8.3 https://github.com/genomehubs/blobtk
    BUSCO 6.0.0; 5.8.3 https://gitlab.com/ezlab/busco
    busco-alg-painter 0.1.0 https://github.com/Karenvn/busco-alg-painter
    bwa-mem2 2.2.1 https://github.com/bwa-mem2/bwa-mem2
    DIAMOND 2.1.8 https://github.com/bbuchfink/diamond
    fasta_windows 0.2.4 https://github.com/tolkit/fasta_windows
    FastK 1.2 https://github.com/thegenemyers/FASTK
    GenomeScope 2.1.0 https://github.com/tbenavi1/genomescope2.0
    Gfastats 1.3.6 https://github.com/vgl-hub/gfastats
    Hifiasm 0.19.8-r603 https://github.com/chhylp123/hifiasm
    HiGlass 1.13.4 https://github.com/higlass/higlass
    MerquryFK 1.1.0-c1 https://github.com/thegenemyers/MERQURY.FK
    Minimap2 2.28-r1209 https://github.com/lh3/minimap2
    MitoHiFi 3.2.2 https://github.com/marcelauliano/MitoHiFi
    MultiQC 1.14; 1.17 and 1.18 https://github.com/MultiQC/MultiQC
    Nextflow 24.10.4 https://github.com/nextflow-io/nextflow
    PretextSnapshot 0.0.6 https://github.com/sanger-tol/PretextSnapshot
    PretextView 1.0.3 https://github.com/sanger-tol/PretextView
    samtools 1.21 https://github.com/samtools/samtools
    sanger-tol/ascc 0.1.0 https://github.com/sanger-tol/ascc
    sanger-tol/blobtoolkit v0.9.0 https://github.com/sanger-tol/blobtoolkit
    sanger-tol/curationpretext 1.4.2 https://github.com/sanger-tol/curationpretext
    Seqtk 1.4-r122 https://github.com/lh3/seqtk
    Singularity 3.9.0 https://github.com/sylabs/singularity
    TreeVal v1.4.0 https://github.com/sanger-tol/treeval
    YaHS 1.2.2 https://github.com/c-zhou/yahs

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