Version Changes
Revised. Amendments from Version 1
In Version 2 of this data note we have added information about the genome annotation by Ensembl at the European Bioinformatics Institute. We have also added analysis of the final assembly in Merqury.FK, and expanded the information on the analysis. We have replaced the chromosome map in Figure 5 with a labelled map.
Abstract
We present a genome assembly from an individual female Tachina fera (Arthropoda; Insecta; Diptera; Tachinidae). The genome sequence is 752 megabases in span. Most of the assembly (99.98%) is scaffolded into 6 chromosomal pseudomolecules, with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 17.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 12 253 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland. The primary assembly achieves an Earth BioGenome Project quality code of 7.C.Q57.
Keywords: Tachina fera, genome sequence, chromosomal, Diptera
Species taxonomy
Eukaryota; Metazoa; Ecdysozoa; Arthropoda; Hexapoda; Insecta; Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha; Oestroidea; Tachinidae; Tachininae; Tachinini; Tachina; Tachina fera Linnaeus, 1761 (NCBI:txid631328).
Background
Tachina fera (Linnaeus, 1761) is one of the most striking flies commonly encountered in the UK countryside. With adults ranging between 9 and 14 mm in length, it is an easily noticeable fly. Spiky bristles, characteristic of the Tachinidae, adorn a chestnut abdomen with a dark central stripe. Tachina fera is abundant across Europe, North Africa and Asia ( Tschorsnig & Herting, 1994). In the UK, T. fera is bivoltine, with adults in flight from May to June, and from July to September ( Belshaw, 1993). Adults feed at a range of flowers throughout the landscape. Tachina fera has mainly been recorded emerging from Noctuid moth caterpillars ( Belshaw, 1993). The method of parasitism utilised by T. fera is notable as the egg is not placed into the host by the mother but laid pre-incubated onto leaves close to it. The larva, once hatched, will make its own way to the host, stimulated by vibration ( Belshaw, 1993; Stireman et al., 2006). The parasitic nature of Tachinid species such as T. fera mean they are important, but underappreciated, regulators of insect herbivory in our ecosystem ( Stireman et al., 2006), as well as playing important roles in pollination (e.g. Martel et al., 2021).
The chromosome-level genome assembly presented here is, to our knowledge, the first high-quality resource developed for a Tachinid and is the only genome publicly available for Tachina fera. It represents a key step in understanding the complex ecology of these beautiful and spiky flies. This assembly was generated 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 ( Blaxter et al., 2022).
Genome sequence report
The genome was sequenced from a single female T. fera collected from Wytham Woods, Oxfordshire (Biological vice-county: Berkshire), UK (latitude 51.770, longitude -1.338) ( Figure 1). A total of 41-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 46-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 246 missing/misjoins and removed 60 haplotypic duplications, reducing the assembly size by 1.88% and the scaffold number by 94.81%, and increasing the scaffold N50 by 120.51%.
Figure 1. Image of the Tachina fera specimen taken during preservation and processing.
The final assembly has a total length of 752 Mb in 12 sequence scaffolds with a scaffold N50 of 142 Mb ( Table 1). The majority, 99.98%, of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 5 autosomes (numbered by sequence length), and the X sex chromosome ( Figure 2– Figure 5; Table 2). The order and orientation of contigs within the centromere of chromosome 2 are not known. Lots of apparent haplotypic duplication was excised from this region owing to a divergent Hi-C pattern and seeming low coverage (which was somewhat ambiguous due to read coverage levels in this repetitive region).
Figure 2. Genome assembly of Tachina fera, idTacFera2.1: 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 751,737,434 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 (191,818,649 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 chromosome lengths (141,997,299 and 125,182,871 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 diptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idTacFera2.1/dataset/CAJMZS01/snail.
Figure 5. Genome assembly of Tachina fera, idTacFera2.1: Hi-C contact map.
Hi-C contact map of the idTacFera2.1 assembly, visualised in PretextView. Chromosomes are arranged in size order from left to right and top to bottom.
Table 1. Genome data for Tachina fera, idTacFera2.1.
| Project accession data | |
|---|---|
| Assembly identifier | idTacFera2.1 |
| Species | Tachina fera |
| Specimen | idTacFera2 |
| NCBI taxonomy ID | 631328 |
| BioProject | PRJEB42946 |
| BioSample ID | SAMEA7520333 |
| Isolate information | Female, thorax/abdomen (idTacFera2, genome
assembly, Hi-C); unknown sex, abdomen (idTacFera1, RNA-Seq) |
| Raw data accessions | |
| PacificBiosciences SEQUEL II | ERR6608654 |
| 10X Genomics Illumina | ERR6054375-ERR6054378 |
| Hi-C Illumina | ERR6054379-ERR6054381 |
| PolyA RNA-Seq Illumina | ERR6054382 |
| Genome assembly | |
| Assembly accession | GCA_905220375.1 |
| Accession of alternate haplotype | GCA_905220395.1 |
| Span (Mb) | 752 |
| Number of contigs | 322 |
| Contig N50 length (Mb) | 16.2 |
| Number of scaffolds | 12 |
| Scaffold N50 length (Mb) | 142 |
| Longest scaffold (Mb) | 192 |
| BUSCO * genome score | C:98.4%[S:97.9%,D:0.5%],
F:0.5%,M:1.1%,n:3285 |
*BUSCO scores based on the diptera_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/idTacFera2.1/dataset/CAJMZS01/busco.
Figure 3. Genome assembly of Tachina fera, idTacFera2.1: 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/idTacFera2.1/dataset/CAJMZS01/blob.
Figure 4. Genome assembly of Tachina fera, idTacFera2.1: 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/idTacFera2.1/dataset/CAJMZS01/cumulative.
Table 2. Chromosomal pseudomolecules in the genome assembly of Tachina fera, idTacFera2.1.
| INSDC accession | Chromosome | Size (Mb) | GC% |
|---|---|---|---|
| LR999963.1 | 1 | 191.82 | 30.1 |
| LR999964.1 | 2 | 150.60 | 30.7 |
| LR999965.1 | 3 | 142.00 | 30.2 |
| LR999966.1 | 4 | 127.83 | 30.2 |
| LR999967.1 | 5 | 125.18 | 30.0 |
| LR999968.1 | X | 14.18 | 31.7 |
| LR999969.1 | MT | 0.02 | 19.9 |
While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled (length 17.38 kb, LR999969.1). This sequence is included as a contig in the multifasta file of the genome submission and as a standalone record.
The assembly has a BUSCO v5.1.2 ( Manni et al., 2021) completeness of 98.4% (single 97.9%, duplicated 0.5%) using the diptera_odb10 reference set (n=3285). The combined primary and alternate assemblies achieve an estimated QV of 56.3. The k-mer completeness is 72.67% for the primary assembly, 71.02% for the alternate haplotype, and 98.88% for the combined assemblies. The quality code for the primary assembly is 7.C.Q57, calculated according to Earth BioGenome Project Report on Assembly Standards September 2024. This meets the recommended reference standard.
Genome annotation report
The Tachina fera genome assembly (GCA_905220375.1) was annotated by Ensembl at the European Bioinformatics Institute (EBI). This annotation includes 20 293 transcribed mRNAs from 12 253 protein-coding and 1 821 non-coding genes. The average transcript length is 18 139.12 bp, with an average of 1.44 coding transcripts per gene and 4.94 exons per transcript. For further information about the annotation, please refer to the annotation page on Ensembl.
Methods
Sample acquisition and DNA extraction
One female T. fera sample (idTacFera2), and a second sample of unknown sex (idTacFera1) were collected from Wytham Woods, Oxfordshire (Biological vice-county: Berkshire), UK (latitude 51.770, longitude -1.338) by Liam Crowley, University of Oxford, on 15 June 2020. The specimen was caught in woodland with a net, identified by the same individual, snap-frozen on dry ice and stored using a CoolRack.
DNA was extracted from the head/thorax of idTacFera2 at the Wellcome Sanger Institute (WSI) Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer’s instructions. RNA (from the abdomen of idTacFera1) was extracted in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer’s instructions. RNA was then eluted in 50 μl RNAse-free water and its concentration RNA assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit. Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics Chromium read cloud sequencing libraries were constructed according to the manufacturers’ instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq X (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were generated in the Tree of Life laboratory from remaining head/thorax tissue of idTacFera2 using the Arima v2 kit and sequenced on a HiSeq X instrument.
Genome assembly
Assembly was carried out with Hifiasm ( Cheng et al., 2021); haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). 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 et al., 2014) using SALSA2 ( Ghurye et al., 2019). The assembly was checked for contamination and corrected using gEVAL ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2021), which performs annotation using MitoFinder ( Allio et al., 2020).
Assembly quality assessment
The Merqury.FK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) 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 also analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores were generated. The BlobToolKit pipeline 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. Table 3 contains a list of all software tool versions used, where appropriate.
Table 3. Software tools used.
| Software tool | Version | Source |
|---|---|---|
| Hifiasm | 0.12 | 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 | 1.0 | Uliano-Silva et al., 2021 |
| gEVAL | N/A | Chow et al., 2016 |
| HiGlass | 1.11.6 | Kerpedjiev et al., 2018 |
| FastK | 1.1 | https://github.com/thegenemyers/FASTK |
| MerquryFK | 1.1.2 | https://github.com/thegenemyers/MERQURY.FK |
| PretextView | 0.1.x | https://github.com/wtsi-hpag/PretextView |
| BlobToolKit | 3.0.5 | 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: Tachina fera. Accession number PRJEB42946; https://identifiers.org/ena.embl/PRJEB42946.
The genome sequence is released openly for reuse. The T. fera 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. Raw data and assembly accession identifiers are reported in Table 1.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute (206194) and the Darwin Tree of Life Discretionary Award (218328).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 2; peer review: 2 approved, 1 approved with reservations]
Author information
Members of the University of Oxford and Wytham Woods Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.5746938.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.5744972.
Members of the Wellcome Sanger Institute Tree of Life programme are listed here: https://doi.org/10.5281/zenodo.6125027.
Members of Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective are listed here: https://doi.org/10.5281/zenodo.5746904.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.6125046.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.5638618.
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