Abstract
We present a genome assembly from a female specimen of Cupido argiades (Short-tailed Blue; Arthropoda; Insecta; Lepidoptera; Lycaenidae). The assembly contains two haplotypes with total lengths of 445.87 megabases and 384.46 megabases. Most of haplotype 1 (99.85%) is scaffolded into 25 chromosomal pseudomolecules, including the W, Z 1, and Z 2 sex chromosomes. Haplotype 2 was assembled to scaffold level. The mitochondrial genome has also been assembled, with a length of 15.41 kilobases. This work is part of Project Psyche, a collaborative programme generating genomes for European butterflies and moths.
Keywords: Cupido argiades; Short-tailed Blue; genome sequence; chromosomal; Lepidoptera
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; Obtectomera; Papilionoidea; Lycaenidae; Polyommatinae; Cupido; Cupido argiades (Pallas, 1771) (NCBI:txid596724)
Background
The Short-tailed Blue or Tailed Cupid ( Cupido argiades) is a relatively small butterfly with short tails on the hindwings, with at least two prominent orange lunules close to the tails on the wing undersides. The wing undersides are otherwise whitish with black spots. The uppersides of the wings are blue (males) or brownish (females). The species lives in variable grassland habitats, but mostly close to shrublands or forest margins. The species is vagrant, distributed throughout most of the Palaearctic region, extending to the east in India, China and south-east Asia ( Wang & Fan, 2002).
The larval host plants are various genera of the family Fabaceae such as Lotus, Medicago, Trifolium, or Ulex. C. argiades has only a weak association with ants ( Fiedler, 2021). The butterfly is multivoltine and usually has two or three generations per year ( Beneš et al., 2002). While it is not threatened and even extending its range in south-eastern and central Europe, the species declined in western Europe in the second half of the 20th century with extinction in Belgium ( Maes et al., 2019). The species does not exhibit any mitochondrial variability across Europe ( Suchácková Bartonová et al., 2024).
We present a chromosome-level genome sequence for C. argiades, sequenced as part of Project Psyche. The sequence data were derived from a female specimen ( Figure 1) collected from Gréixer, Guardiola de Berguedà, Barcelona, Catalonia, Spain.
Figure 1. Voucher photographs of the Cupido argiades (ilCupArgi2) specimen used for genome sequencing.
Top: dorsal view; bottom: ventral view.
Methods
Sample acquisition
The specimen used for genome sequencing was an adult female Cupido argiades (specimen ID SAN28000284, ToLID ilCupArgi2; Figure 1), collected from Gréixer, Guardiola de Berguedà, Barcelona, Catalonia, Spain (latitude 42.2884, longitude 1.841) on 2018-08-19. Another specimen (male) was used for RNA sequencing (specimen ID SAN28000283, ToLID ilCupArgi1). The specimens were collected and identified by Roger Vila (Institut de Biologia Evolutiva).
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). The ilCupArgi2 sample was weighed and triaged to determine the appropriate extraction protocol. Tissue from the whole organism 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 FemtoPulse system. For this sample, the final post-shearing DNA had a Qubit concentration of 32.93 ng/μL and a yield of 1 547.71 ng, with a fragment size of 17.2 kb.
RNA was extracted from whole organism tissue of ilCupArgi1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol. 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.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core. Libraries were prepared using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, California, USA), 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 using a Qubit Fluorometer v4.0 (ThermoFisher 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 sample was sequenced on a Revio instrument (Pacific Biosciences). The prepared library was normalised to 2 nM, and 15 μL was used for making complexes. Primers were annealed and polymerases bound to generate circularised complexes, following the manufacturer’s instructions. Complexes were purified using 1.2X SMRTbell beads, then diluted to the Revio loading concentration (200–300 pM) and spiked with a Revio sequencing internal control. The sample was sequenced on a Revio 25M SMRT cell. The SMRT Link software (Pacific Biosciences), a web-based workflow manager, was used to configure and monitor the run and to carry out primary and secondary data analysis. Specimen details, sequencing platforms, and data yields are summarised in Table 1.
Table 1. Specimen and sequencing data for BioProject PRJEB80915.
| Platform | PacBio HiFi | Hi-C | RNA-seq |
|---|---|---|---|
| ToLID | ilCupArgi2 | ilCupArgi2 | ilCupArgi1 |
| Specimen ID | SAN28000284 | SAN28000284 | SAN28000283 |
| BioSample (source individual) | SAMEA115768736 | SAMEA115768736 | SAMEA115768734 |
| BioSample (tissue) | SAMEA115768853 | SAMEA115768853 | SAMEA115768851 |
| Tissue | whole organism | whole organism | whole organism |
| Instrument | Revio | Illumina NovaSeq X | Illumina NovaSeq X |
| Run accessions | ERR13800475 | ERR13802619 | ERR15551481 |
| Read count total | 1.32 million | 649.13 million | 95.23 million |
| Base count total | 14.83 Gb | 98.02 Gb | 14.38 Gb |
Hi-C
Sample preparation and crosslinking
The Hi-C sample was prepared from 20–50 mg of frozen tissue from the whole organism of the ilCupArgi2 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. Library amplification was performed using 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. Sequencing was performed using paired-end 150 bp reads on the Illumina NovaSeq X. Specimen details, sequencing platforms, and data yields are summarised in Table 1.
RNA-seq library preparation and sequencing
Libraries were prepared using the NEBNext ® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Poly(A) mRNA in the total RNA solution was isolated using oligo(dT) beads, converted to cDNA, and uniquely indexed; 14 PCR cycles were performed. Libraries were size-selected to produce fragment lengths of 100–300 bp. Libraries were quantified, normalised, pooled to a final concentration of 2.8 nM, and diluted to 150 pM for loading. Sequencing was carried out on the Illumina NovaSeq X to generate 150-bp paired-end 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. GenomeScope2 ( Ranallo-Benavidez et al., 2020) was used to analyse the k-mer frequency distributions, providing estimates of genome size, heterozygosity, and repeat content.
The HiFi reads were assembled using Hifiasm in Hi-C phasing mode ( Cheng et al., 2021; Cheng et al., 2022), producing two haplotypes. Hi-C reads ( Rao et al., 2014) were mapped to the primary 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 MERQURY.FK ( 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. TreeVal was used to generate the flat files and maps for use in curation. 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). Manual corrections included nine breaks and 46 joins. This reduced the scaffold count by 36.6%, increased scaffold N50 by 1.8%, and increased the total assembly length by 3.9%. The curation process is described at https://gitlab.com/wtsi-grit/rapid-curation. PretextSnapshot was used to generate a Hi-C contact map of the final assembly.
Assembly quality assessment
The Merqury.FK 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 database ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
The genome was analysed using the BlobToolKit pipeline, a Nextflow ( Di Tommaso et al., 2017) implementation of the earlier Snakemake version ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. It runs BUSCO ( Manni et al., 2021) using lineages identified from the NCBI Taxonomy ( Schoch et al., 2020). For the three domain-level lineages, BUSCO genes are aligned to the UniProt Reference Proteomes database ( Bateman et al., 2023) using DIAMOND blastp ( Buchfink et al., 2021). The genome is divided into chunks based on the density of BUSCO genes from the closest taxonomic lineage, and each chunk is aligned to the UniProt Reference Proteomes database with DIAMOND blastx. Sequences without hits are chunked using seqtk and aligned to the NT database with blastn ( Altschul et al., 1990). The BlobToolKit suite consolidates all outputs into a blobdir for visualisation. The BlobToolKit pipeline was developed using nf-core tooling ( Ewels et al., 2020) and MultiQC ( Ewels et al., 2016), with containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).
We used lep_busco_painter to paint Merian elements along chromosomes ( Wright et al., 2024). Merian elements represent the 32 ancestral linkage groups in Lepidoptera. The painting process utilised BUSCO gene locations from the lepidoptera_odb10 set ( Kriventseva et al., 2019) and chromosome lengths from NCBI Datasets. Each complete BUSCO gene (both single-copy and duplicated) was assigned to a Merian element based on a reference database, then plotted along chromosomes drawn to scale.
Genome sequence report
Sequence data
PacBio sequencing of the Cupido argiades specimen generated 14.83 Gb (gigabases) from 1.32 million reads, which were used to assemble the genome. GenomeScope2.0 analysis estimated the haploid genome size at 410.61 Mb, with a heterozygosity of 1.72% and repeat content of 36.25% ( Figure 2). These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 35× coverage. Hi-C sequencing produced 98.02 Gb from 649.13 million reads, which were used to scaffold the assembly. RNA sequencing data were also generated and are available in public sequence repositories. Table 1 summarises the specimen and sequencing details.
Figure 2. Frequency distribution of k-mers generated using GenomeScope2.
The plot shows observed and modelled k-mer spectra, providing estimates of genome size, heterozygosity, and repeat content based on unassembled sequencing reads.
Assembly statistics
The genome was assembled into two haplotypes using Hi-C phasing. Haplotype 1 was curated to chromosome level, while haplotype 2 was assembled to scaffold level. The final assembly has a total length of 445.87 Mb in 44 scaffolds, with 101 gaps, and a scaffold N50 of 18.36 Mb ( Table 2).
Table 2. Genome assembly statistics.
| Assembly name | ilCupArgi2.hap1.1 | ilCupArgi2.hap2.1 |
| Assembly accession | GCA_964277285.1 | GCA_964277125.1 |
| Assembly level | chromosome | scaffold |
| Span (Mb) | 445.87 | 384.46 |
| Number of chromosomes | 25 | scaffold-level |
| Number of contigs | 145 | 112 |
| Contig N50 | 8.09 Mb | 7.63 Mb |
| Number of scaffolds | 44 | 38 |
| Scaffold N50 | 18.36 Mb | 18.09 Mb |
| Longest scaffold length (Mb) | 25.31 | - |
| Sex chromosomes | W, Z 1, and Z 2 | - |
| Organelles | Mitochondrion: 15.41 kb | - |
Most of the assembly sequence (99.85%) was assigned to 25 chromosomal-level scaffolds, representing 22 autosomes and the W, Z 1, and Z 2 sex chromosomes. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 3; Table 3). Chromosome painting with Merian elements illustrates the distribution of orthologues along chromosomes and highlights patterns of chromosomal evolution relative to Lepidopteran ancestral linkage groups ( Figure 4). Chromosomes W, Z 1 and Z 2 were assigned based on the Hi-C signal.
Figure 3. Hi-C contact map of the Cupido argiades genome assembly.
Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.
Figure 4. Merian elements painted across chromosomes in the ilCupArgi2.hap1.1 assembly of Cupido argiades.
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.
Table 3. Chromosomal pseudomolecules in the haplotype 1 genome assembly of Cupido argiades ilCupArgi2.
| INSDC
accession |
Molecule | Length
(Mb) |
GC% | Assigned
Merian elements |
|---|---|---|---|---|
| OZ195111.1 | 1 | 25.31 | 37 | M1;M19 |
| OZ195114.1 | 2 | 21.70 | 37 | M18;M30 |
| OZ195115.1 | 3 | 21.64 | 37 | M11;M23 |
| OZ195116.1 | 4 | 21.20 | 36.50 | M25;M5 |
| OZ195117.1 | 5 | 20.21 | 36.50 | M12;M29 |
| OZ195118.1 | 6 | 19.75 | 36.50 | M14;M26 |
| OZ195119.1 | 7 | 18.66 | 37 | M17;M20 |
| OZ195120.1 | 8 | 18.55 | 37 | M2 |
| OZ195121.1 | 9 | 18.36 | 36.50 | M9 |
| OZ195122.1 | 10 | 18.04 | 37 | M8 |
| OZ195123.1 | 11 | 17.34 | 37 | M3 |
| OZ195124.1 | 12 | 16.36 | 37 | M4 |
| OZ195125.1 | 13 | 16.31 | 37 | M7 |
| OZ195126.1 | 14 | 15.85 | 36.50 | M16 |
| OZ195127.1 | 15 | 15.83 | 37 | M6 |
| OZ195128.1 | 16 | 15.55 | 37 | M21 |
| OZ195129.1 | 17 | 15.39 | 37 | M15 |
| OZ195130.1 | 18 | 15.10 | 37 | M13 |
| OZ195132.1 | 19 | 14.92 | 37 | M10 |
| OZ195133.1 | 20 | 14.39 | 37.50 | M28;M31 |
| OZ195134.1 | 21 | 11.46 | 37 | M24 |
| OZ195135.1 | 22 | 10.51 | 37 | M27 |
| OZ195113.1 | W | 23.53 | 37.50 | M22 |
| OZ195112.1 | Z 1 | 24.22 | 37.50 | MZ |
| OZ195131.1 | Z 2 | 15.04 | 37 | M22 |
The mitochondrial genome was also assembled (length 15.41 kb, OZ195136.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
Assembly quality metrics
For haplotype 1, the estimated QV is 65.8, and for haplotype 2, 65.9. When the two haplotypes are combined, the assembly achieves an estimated QV of 65.8. The k-mer completeness is 73.28% for haplotype 1, 65.73% for haplotype 2, and 99.24% for the combined haplotypes ( Figure 5).
Figure 5. Evaluation of k-mer completeness using MerquryFK.
This plot illustrates the recovery of k-mers from the original read data in the final assemblies. 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 (the homozygous peak) corresponds to k-mers shared by both haplotypes and the red and blue curves (the heterozygous peaks) show k-mers found only in one of the haplotypes.
BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5 286) identified 97.1% of the expected gene set (single = 94.2%, duplicated = 3.0%) in haplotype 1. For haplotype 2, BUSCO analysis identified 90.7% of the expected gene set (single = 90.2%, duplicated = 0.5%). The snail plot in Figure 6 summarises the scaffold length distribution and other assembly statistics for haplotype 1. The blob plot in Figure 7 shows the distribution of scaffolds by GC proportion and coverage for haplotype 1.
Figure 6. Assembly metrics for ilCupArgi2.hap1.1.
The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the whole genome sequence, 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. The longest scaffold is indicated by the red arc, and the deeper orange and pale orange arcs represent the N50 and N90 lengths. A 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 set is presented at the top right. An interactive version of this figure can be accessed on the BlobToolKit viewer.
Figure 7. BlobToolKit GC-coverage plot for ilCupArgi2.hap1.1.
Blob plot showing sequence coverage (vertical axis) and GC content (horizontal axis). The circles represent scaffolds, with the size proportional to scaffold length and the colour representing phylum membership. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. An interactive version of this figure is available on the BlobToolKit viewer.
Table 4 lists the assembly metric benchmarks adapted from Rhie et al. (2021) the Earth BioGenome Project Report on Assembly Standards September 2024. The EBP metric, calculated for the haplotype 1, is 6.C.Q65, meeting the recommended reference standard.
Table 4. Earth Biogenome Project summary metrics for the Cupido argiades assembly.
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (haplotype 1) | 6.C.Q65 | 6.C.Q40 |
| Contig N50 length | 8.09 Mb | ≥ 1 Mb |
| Scaffold N50 length | 18.36 Mb | = chromosome N50 |
| Consensus quality (QV) | Haplotype 1: 65.8; haplotype 2:
65.9; combined: 65.8 |
≥ 40 |
| k-mer completeness | Haplotype 1: 73.28%; Haplotype 2:
65.73%; combined: 99.24% |
≥ 95% |
| BUSCO | C:97.1% [S:94.2%; D:3.0%]; F:0.5%;
M:2.4%; n:5 286 |
S > 90%; D < 5% |
| Percentage of assembly
assigned to chromosomes |
99.85% | ≥ 90% |
Notes: EBP summary uses log10(Contig N50); chromosome-level (C) or log10(Scaffold N50); Q (Merqury QV). BUSCO: C=complete; S=single-copy; D=duplicated; F=fragmented; M=missing; n=orthologues
Wellcome Sanger Institute – Legal and Governance
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator. 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 undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Tree of Life collaborator, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances, other Tree of Life collaborators.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540). RV was funded by Grant PID2022-139689NB-I00 (MICIU/ AEI/ 10.13039/501100011033 and ERDF, EU) and grant 2021-SGR-00420 (Departament de Recerca i Universitats, Generalitat de Catalunya).
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: Cupido argiades. Accession number PRJEB80915. The genome sequence is released openly for reuse. The Cupido argiades genome sequencing initiative is part of the Sanger Institute Tree of Life Programme (PRJEB43745) and Project Psyche (PRJEB71705). 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 Ensembl at the European Bioinformatics Institute. 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.
Author information
Contributors are listed at the following links:
Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team
Members of Wellcome Sanger Institute Scientific Operations – Sequencing Operations
Members of the Wellcome Sanger Institute Tree of Life Core Informatics team
Members of the Tree of Life Core Informatics collective
Members of the Project Psyche Community
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