Abstract
We present a genome assembly from a male specimen of Philophylla caesio (tephritid fly; Arthropoda; Insecta; Diptera; Tephritidae). The genome sequence has a total length of 777.43 megabases. Most of the assembly (98.51%) is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled, with a length of 19.03 kilobases. Gene annotation of this assembly on Ensembl identified 31,396 protein-coding genes.
Keywords: Philophylla caesio, tephritid fly, genome sequence, chromosomal, Diptera
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha; Eremoneura; Cyclorrhapha; Schizophora; Acalyptratae; Tephritoidea; Tephritidae; Trypetinae; Trypetini; Trypetina; Philophylla; Philophylla caesio (Harris, 1780) (NCBI:txid472889)
Background
As part of the Darwin Tree of Life Project – which aims to generate high-quality reference genomes for all named eukaryotic species in Britain and Ireland to support research, conservation, and the sustainable use of biodiversity – we present a chromosomally complete genome sequence for the tephritid fly, Philophylla caesio. This genome was assembled using the Tree of Life pipeline from a specimen collected in Wytham Woods, Oxfordshire, United Kingdom ( Figure 1).
Figure 1. Photograph of the Philophylla caesio (idPhiCaes1) specimen used for genome sequencing.
Genome sequence report
Sequencing data
The genome of a specimen of Philophylla caesio ( Figure 1) was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating 37.46 Gb (gigabases) from 3.86 million reads, which were used to assemble the genome. GenomeScope analysis estimated the haploid genome size at 681.23 Mb, with a heterozygosity of 1.24% and repeat content of 37.67%. These estimates guided expectations for the assembly. Based on the estimated genome size, the sequencing data provided approximately 51 coverage. Hi-C sequencing produced 85.38 Gb from 565.43 million reads, used to scaffold the assembly. Table 1 summarises the specimen and sequencing details.
Table 1. Specimen and sequencing data for Philophylla caesio.
| Project information | |||
|---|---|---|---|
| Study title | Philophylla caesio | ||
| Umbrella BioProject | PRJEB70504 | ||
| Species | Philophylla caesio | ||
| BioSpecimen | SAMEA112232776 | ||
| NCBI taxonomy ID | 472889 | ||
| Specimen information | |||
| Technology | ToLID |
BioSample
accession |
Organism part |
| PacBio long read sequencing | idPhiCaes1 | SAMEA112233277 | whole organism |
| Hi-C sequencing | idPhiCaes1 | SAMEA112233277 | whole organism |
| Sequencing information | |||
| Platform |
Run
accession |
Read count | Base count (Gb) |
| Hi-C Illumina NovaSeq 6000 | ERR12321264 | 5.65e+08 | 85.38 |
| PacBio Revio | ERR12340372 | 3.86e+06 | 37.46 |
Assembly statistics
The primary haplotype was assembled, and contigs corresponding to an alternate haplotype were also deposited in INSDC databases. The assembly was improved by manual curation, which corrected 174 misjoins or missing joins and removed 6 haplotypic duplications. These interventions reduced the total assembly length by 0.51%, decreased the scaffold count by 61.24%, and increased the scaffold N50 by 26.58%. The final assembly has a total length of 777.43 Mb in 80 scaffolds, with 445 gaps, and a scaffold N50 of 124.27 Mb ( Table 2).
Table 2. Genome assembly data for Philophylla caesio.
| Genome assembly | ||
|---|---|---|
| Assembly name | idPhiCaes1.1 | |
| Assembly accession | GCA_963971545.1 | |
| Alternate haplotype accession | GCA_963971555.1 | |
| Assembly level for primary assembly | chromosome | |
| Span (Mb) | 777.43 | |
| Number of contigs | 525 | |
| Number of scaffolds | 80 | |
| Longest scaffold (Mb) | 222.32 | |
| Assembly metric | Measure | Benchmark |
| Contig N50 length | 3.97 Mb | ≥ 1 Mb |
| Scaffold N50 length | 124.27 Mb | = chromosome N50 |
| Consensus quality (QV) | Primary: 63.6; alternate: 63.3;
combined: 63.4 |
≥ 40 |
| k-mer completeness | Primary: 80.87%; alternate:
75.69%; combined: 97.28% |
≥ 95% |
| BUSCO * | C:98.6%[S:97.8%,D:0.8%],
F:0.4%,M:0.9%,n:3,285 |
S > 90%; D < 5% |
| Percentage of assembly assigned to
chromosomes |
98.51% | ≥ 90% |
| Sex chromosomes | X |
localised
homologous pairs |
| Organelles | Mitochondrial genome: 19.03 kb |
complete single
alleles |
* BUSCO scores based on the diptera_odb10 BUSCO set using version 5.5.0. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison.
The snail plot in Figure 2 provides a summary of the assembly statistics, indicating the distribution of scaffold lengths and other assembly metrics. Figure 3 shows the distribution of scaffolds by GC proportion and coverage. Figure 4 presents a cumulative assembly plot, with separate curves representing different scaffold subsets assigned to various phyla, illustrating the completeness of the assembly.
Figure 2. Genome assembly of Philophylla caesio, idPhiCaes1.1: metrics.
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 diptera_odb10 set is presented at the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/GCA_963971545.1/dataset/GCA_963971545.1/snail.
Figure 3. Genome assembly of Philophylla caesio, idPhiCaes1.1: BlobToolKit GC-coverage plot.
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 at https://blobtoolkit.genomehubs.org/view/GCA_963971545.1/dataset/GCA_963971545.1/blob.
Figure 4. Genome assembly of Philophylla caesio, idPhiCaes1.1: 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/GCA_963971545.1/dataset/GCA_963971545.1/cumulative.
Most of the assembly sequence (98.51%) was assigned to 6 chromosomal-level scaffolds, representing 5 autosomes and the X sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size ( Figure 5; Table 3). During curation, chromosome X was identified based on read coverage statistics. The heterogametic sex was sampled, and this individual is XO. The following regions of the assembly have uncertain order and orientation: Chromosome 1 (~49–158 Mbp), Chromosome 2 (~46–56.5 Mbp), Chromosome 3 (~79.5–85 Mbp), Chromosome 4 (~47–54 Mbp), and Chromosome 5 (~18.4–25 Mbp). The mitochondrial genome was also assembled. This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
Figure 5. Genome assembly of Philophylla caesio: Hi-C contact map of the idPhiCaes1.1 assembly, generated using HiGlass Chromosomes are shown in order of size and labelled along the axes.
An interactive version is available here: https://genome-note-higlass.tol.sanger.ac.uk/l/?d=K7cLJ8rfT_WDPDVssux0Yw.
Table 3. Chromosomal pseudomolecules in the genome assembly of Philophylla caesio, idPhiCaes1.
| INSDC
accession |
Name | Length
(Mb) |
GC% |
|---|---|---|---|
| OZ020604.1 | 1 | 222.32 | 36 |
| OZ020606.1 | 2 | 126.41 | 34.5 |
| OZ020607.1 | 3 | 124.27 | 35 |
| OZ020608.1 | 4 | 120.0 | 34.5 |
| OZ020609.1 | 5 | 102.65 | 34 |
| OZ020605.1 | X | 70.21 | 38 |
| OZ020610.1 | MT | 0.02 | 19 |
Assembly quality metrics
The estimated Quality Value (QV) and k-mer completeness metrics, along with BUSCO completeness scores, were calculated for each haplotype and the combined assembly. The QV reflects the base-level accuracy of the assembly, while k-mer completeness indicates the proportion of expected k-mers identified in the assembly. BUSCO scores provide a measure of completeness based on benchmarking universal single-copy orthologues.
The combined primary and alternate assemblies achieve an estimated QV of 63.4. The k-mer completeness is 80.87% for the primary haplotype and 75.69% for the alternate haplotype; and 97.28% for the combined primary and alternate assemblies. BUSCO v.5.5.0 analysis using the diptera_odb10 reference set ( n = 3,285) identified 98.6% of the expected gene set (single = 97.8%, duplicated = 0.8%).
Table 2 provides assembly metric benchmarks adapted from Rhie et al. (2021) and the Earth BioGenome Project Report on Assembly Standards September 2024. The primary assembly achieves the EBP reference standard of 6.C.Q63.
Genome annotation report
The Philophylla caesio genome assembly (GCA_963971545.1) was annotated externally by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 32,282 transcribed mRNAs from 31,396 protein-coding genes. The average transcript length is 3,386.81 bp, with 2.97 exons per transcript. For further information about the annotation, please refer to https://beta.ensembl.org/species/f9b5431c-2506-4b4b-be38-92000fb439db.
Methods
Sample acquisition and DNA barcoding
The specimen used for genome sequencing was an adult male Philophylla caesio (specimen ID Ox002589, ToLID idPhiCaes1), collected from Wytham Woods, Oxfordshire, United Kingdom (latitude 51.772, longitude -1.338) on 2022-07-29 by netting. The specimen was collected and identified by James McCulloch (University of Oxford) and preserved on dry ice.
The initial identification was verified by an additional DNA barcoding process according to the framework developed by Twyford et al. (2024). A small sample was dissected from the specimen and stored in ethanol, while the remaining parts were shipped on dry ice to the Wellcome Sanger Institute (WSI) ( Pereira et al., 2022). The tissue was lysed, the COI marker region was amplified by PCR, and amplicons were sequenced and compared to the BOLD database, confirming the species identification ( Crowley et al., 2023). Following whole genome sequence generation, the relevant DNA barcode region was also used alongside the initial barcoding data for sample tracking at the WSI ( Twyford et al., 2024). The standard operating procedures for Darwin Tree of Life barcoding have been deposited on protocols.io ( Beasley et al., 2023).
Metadata collection for samples adhered to the Darwin Tree of Life project standards described by Lawniczak et al. (2022).
Nucleic acid extraction
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification ( Howard et al., 2025). Detailed protocols are available on protocols.io ( Denton et al., 2023b). The idPhiCaes1 sample was prepared for DNA extraction by weighing and dissecting it on dry ice ( 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., 2023a). For ultra-low input (ULI) PacBio sequencing, DNA was fragmented using the Covaris g-TUBE method ( Oatley et al., 2023b). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA ( Strickland et al., 2023). 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.
Hi-C sample preparation and crosslinking
Hi-C data were generated from the whole organism of the idPhiCaes1 sample using the Arima-HiC v2 kit (Arima Genomics) with 20–50 mg of frozen tissue (stored at –80 °C). As per manufacturer’s instructions, tissue was fixed, and the DNA crosslinked using a TC buffer with 22% formaldehyde concentration, and a final formaldehyde concentration of 2%. The tissue was then homogenised using the Diagnocine Power Masher-II. The crosslinked DNA was digested using a restriction enzyme master mix, then biotinylated and ligated. A clean up was performed with SPRIselect beads prior to library preparation. DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and Qubit HS Assay Kit, and sample biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
Library preparation and sequencing
Library preparation and sequencing were performed at the WSI Scientific Operations core.
PacBio HiFi
A ULI library was prepared using PacBio SMRTbell® Express Template Prep Kit 2.0 and PacBio SMRTbell® gDNA Sample Amplification Kit. To begin, samples were normalised to 20 ng of DNA. Initial removal of single-strand overhangs, DNA damage repair, and end repair/A-tailing were performed per manufacturer’s instructions. From the SMRTbell® gDNA Sample Amplification Kit, amplification adapters were then ligated. A 0.85X pre-PCR clean-up was performed with Promega ProNex beads and the sample was then divided into two for a dual PCR. PCR reactions A and B each followed the PCR programs as described in the manufacturer’s protocol. A 0.85X post-PCR clean-up was performed with ProNex beads for PCR reactions A and B and DNA concentration was quantified using the Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and Qubit HS Assay Kit and fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) and gDNA 55kb BAC analysis kit. PCR reactions A and B were then pooled, ensuring the total mass was ≥500 ng in 47.4 μl. The pooled sample then repeated the process for DNA damage repair, end repair/A-tailing and additional hairpin adapter ligation. A 1X clean-up was performed with ProNex beads and DNA concentration was quantified using the Qubit and fragment size analysis was carried out using the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies). Size selection was performed using the PippinHT system (Sage Science) with target fragment size determined by analysis from the Femto Pulse, usually a value between 4000 and 9000 bp. Size-selected libraries were then cleaned-up using1.0X ProNex beads and normalised to 2 nM before proceeding to sequencing.
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.
Hi-C
For Hi-C library preparation, the biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size-selected to 400–600 bp using SPRISelect beads. DNA was then enriched using Arima-HiC v2 Enrichment beads. The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) was used for end repair, A-tailing, and adapter ligation, following a modified protocol in which library preparation is carried out while the DNA remains bound to the enrichment beads. PCR amplification was performed using KAPA HiFi HotStart mix and custom dual-indexed adapters (Integrated DNA Technologies) in a 96-well plate format. Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, samples were amplified for 10–16 PCR cycles. Post-PCR clean-up was carried out using SPRISelect beads. The libraries were quantified using the Accuclear Ultra High Sensitivity dsDNA Standards Assay kit (Biotium) and normalised to 10 ng/μL before sequencing. Hi-C sequencing was performed on the Illumina NovaSeq 6000 instrument using 150 bp paired-end reads.
Genome assembly, curation and evaluation
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 first assembled using Hifiasm ( Cheng et al., 2021) with the --primary option. Haplotypic duplications were identified and removed using purge_dups ( Guan et al., 2020). The Hi-C reads ( Rao et al., 2014) were mapped to the primary contigs using bwa-mem2 ( Vasimuddin et al., 2019), and the contigs were scaffolded 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. Flat files and maps used in curation were generated via the TreeVal pipeline ( Pointon et al., 2023). Manual curation was conducted primarily in PretextView ( Harry, 2022) and HiGlass ( Kerpedjiev et al., 2018), with additional insights provided by JBrowse2 ( Diesh et al., 2023). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). Any identified contamination, missed joins, and mis-joins were amended, and duplicate sequences were tagged and removed. Sex chromosomes were identified by read coverage statistics. The curation process is documented at https://gitlab.com/wtsi-grit/rapid-curation.
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 the primary and alternate haplotypes using the k-mer databases ( 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 BlobToolKit pipeline ( Challis et al., 2020). The pipeline aligns PacBio reads using minimap2 ( Li, 2018) and SAMtools ( Danecek et al., 2021) to generate coverage tracks. Simultaneously, it queries the GoaT database ( Challis et al., 2023) to identify relevant BUSCO lineages and runs BUSCO ( Manni et al., 2021). For the three domain-level BUSCO 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 package management via Conda and Bioconda ( Grüning et al., 2018), and containerisation through Docker ( Merkel, 2014) and Singularity ( Kurtzer et al., 2017).
Table 4 contains a list of relevant software tool versions and sources.
Table 4. Software tools: versions and sources.
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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (220540) and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].
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]
Data availability
European Nucleotide Archive: Philophylla caesio. Accession number PRJEB70504; https://identifiers.org/ena.embl/PRJEB70504. The genome sequence is released openly for reuse. The Philophylla caesio genome sequencing initiative is part of the Darwin Tree of Life Project (PRJEB40665) and Sanger Institute Tree of Life Programme (PRJEB43745). All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.12157525.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.12158331.
Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.12162482.
Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.14870789.
Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.12160324.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.12205391.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.
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