Biology subjects

Swarbreck, D.

Publications and source records attributed to Swarbreck, D..

At least 19 recordsLinked to original sources

bioRxiv2026

EISCA and EISTA: Full-Spectrum Pipelines for Single-Cell and Spatial Transcriptomics Analysis

Single-cell and spatial transcriptomics are transforming our understanding of cellular heterogeneity and tissue organization, yet their analytical complexity remains a major bottleneck. Here, we present EISCA and EISTA, two standardized, end-to-end pipelines for single-cell RNA-seq and imaging-based spatial transcriptomics analysis. Built on the Nextflow nf-core framework, both pipelines implement modular, scalable, and reproducible workflows spanning primary, secondary, and tertiary analyses, from raw data processing to advanced downstream analyses. EISCA supports droplet- and plate-based scRNA-seq technologies, while EISTA is tailored for high-resolution spatial platforms including Vizgen MERFISH and 10x Xenium. Together, they integrate state-of-the-art methods for quality control, normalization, clustering, integration, cell-type annotation, differential expression, and cell-cell communication, with EISTA further enabling spatial statistical analyses. A central design principle is to balance standardization with flexibility: workflows can be executed end-to-end or modularly, enabling iterative, exploratory analyses with minimal overhead. Both pipelines deliver rapid preliminary results alongside an out-of-the-box report, facilitating immediate data assessment and accelerating downstream discovery. Case studies in plant immunity and human sepsis demonstrate that EISTA and EISCA reproducibly can be used to recover biologically meaningful insights. Collectively, these pipelines provide efficient, flexible, and scalable solutions for comprehensive single-cell and spatial transcriptomics analyses.

bioinformatics↗

bioRxiv2026

Long-read single-cell genomics: resolving chimeras in multiple displacement amplification

Multiple displacement amplification (MDA) enables whole-genome amplification from single cells, but introduces chimeric artifacts that severely compromise downstream analyses, particularly with long-read sequencing. Here, we systematically evaluate long-read PacBio HiFi sequencing of MDA amplified DNA from single cells using the model green alga Chlamydomonas reinhardtii. We show that MDA-derived libraries exhibit highly uneven coverage and extreme chimera rates impacting up to 70% of reads, leading to thousands of artefactual structural variants and misassemblies when assembled using algorithms designed for bulk sequencing. To overcome these challenges, we developed lrSAGA (long-read Single Amplified Genome Assembly), a novel tool to assemble long-read MDA sequencing datasets. Assemblies generated using lrSAGA are more complete, more contiguous, and have 75-95% fewer misassemblies compared to conventional assembly algorithms. Although overall contiguity is limited by MDA coverage dropouts, we demonstrate that up to 68% of the C. reinhardtii genome can be accurately assembled from just a single haploid cell. We further validated lrSAGA using published Oxford Nanopore and PacBio HiFi data from single or half Caenorhabditis elegans worms, generating accurate and highly complete assemblies. Applying our approach to single protist cells isolated from environmental water samples, we performed PacBio HiFi single-cell genome sequencing of four uncultivated microbial eukaryotes: an amoeboflagellate from the Naegleria genus, a flagellate from the Bodo genus, and two deep-branching flagellates from the enigmatic CRuMs supergroup, Collodictyon triciliatum and Diphylleia rotans. From single cells, we generated high-quality draft genome assemblies estimated to be 70-84% complete, demonstrating the potential of long-read single-cell genomics to unlock genome diversity from uncultivated microbial eukaryotes.

genomics↗

bioRxiv2026

Evolutionary Rewiring of Root Cell-Type Nitrogen Responses

Nitrate induces widespread transcriptional reprogramming in plants, but how transcription factors coordinate these responses across cell types and evolve across species with divergent root patterning remains unclear. Using single-cell transcriptomics in Arabidopsis thaliana and Solanum lycopersicum (tomato) under varying nitrate conditions, we demonstrate that many transcriptional responses are cell type and developmental stage-specific. Arabidopsis NIN-LIKE PROTEIN 7 (NLP7) directly modulates both broadly expressed and cell-type-specific targets, but is not required to establish the cell type specific nature of these patterns. Comparative analysis reveals evolutionary shifts; key nitrogen programs are localized to different cell types in tomato, with the exodermis as a major regulatory hub. Identification of tomato NLP7a and NLP7b direct targets at cellular resolution supports their role in direct modulation of cell type N-transcriptional programs. This is evidenced by NLP7-controlled exodermal cell wall remodeling in low nitrate, highlighting functional conservation despite divergent cellular topographies.

plant biology↗

bioRxiv2026

Chromosome-scale genome assembly of diploid halophyte Thinopyrum bessarabicum excludes J genome from polyploid Thinopyrum ancestry

Wild relatives of wheat harbour genetic diversity essential for improving resilience to climate-driven stresses, yet their deployment is hampered by unresolved evolutionary relationships and the absence of reference genomes. Here we present a chromosome-scale reference genome for Thinopyrum bessarabicum, a diploid halophyte and high-priority donor for wheat salt tolerance breeding. A key unresolved question is whether the diploid J genome contributed directly to the subgenome composition of extant polyploid Thinopyrum species, and which genomic features underpin its exceptional salt tolerance. Using this resource, we show that the diploid J genome of Th. bessarabicum is not represented among the subgenomes of polyploid Thinopyrum species, resolving a long-standing ambiguity in Triticeae genomics. Gene-level resolution of the reciprocal 4/5 chromosomal translocation across six related Triticeae species identifies conserved breakpoint gene pairs, supporting a single ancestral rearrangement. Genome-wide gene content analysis shows that halophytic capacity is underpinned by quantitative expansion of conserved stress-response gene families. Salt tolerance phenotyping validates chromosome 5J as a tolerance locus in both Th. bessarabicum and wheat introgression lines. A physically anchored marker framework and dual-reference skim-sequencing pipeline enable precise megabase-resolution characterisation of Th. bessarabicum introgressions in wheat, providing a genomic foundation for deploying J-genome diversity in crop improvement.

plant biology↗

bioRxiv2025

Single-cell sequencing reveals unexpected genetic diversity among Bodo spp. flagellates and their bacterial endosymbionts

Bodo is a cosmopolitan genus of free living bacterivorous single-celled flagellates in the class Kinetoplastea. Members of genus Bodo are considered the closest free-living relatives to the parasitic lineages Trypanosoma and Leishmania, the causative agents of the human diseases sleeping sickness, Chagas disease, and leishmaniasis. Currently, a single genome exists for the one formally described species in the genus, Bodo saltans. Previous studies on B. saltans have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, "Candidatus Bodocaedibacter vickermanii". Using single cell-sequencing, we isolated, sequenced, and assembled genomes for seven uncultured Bodo spp. cells from a single freshwater sample from Royal Leamington Spa, UK. By using comparative genomics, we show that these seven cells represent three potentially novel Bodo species and exhibit unexpected levels of diversity at the genome level. Our results indicate that Small Subunit (SSU) rDNA sequencing, often used to classify Bodo flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven Bodo spp. cells. Surprisingly, these seven bacterial endosymbionts also represent three potentially novel species and one novel genus of Holosporales bacteria. This diversity would be indistinguishable in routinely-used SSU ribosomal DNA (rDNA) metabarcoding or bulk sequencing pipelines, thus demonstrating the utility of using single-cell sequencing to reveal the level of genomic diversity within lineages of microbial eukaryotes and their cobionts.

microbiology↗

bioRxiv2025

A comparison of long-read single-cell transcriptomic approaches

Long-read sequencing enables the incorporation of isoform-level expression into single-cell transcriptomic studies, offering detail beyond those accessible with short-read methods. Although insightful, these approaches have typically been costly and yielded limited data for each individual cell. Recent advances in library preparation approaches and sequencing throughput have brought long-read single-cell studies closer to the mainstream. Here, we present a comparative analysis of commercial approaches for single-cell long-read sequencing. We have performed parallel analyses of the same cDNA material, generated using the 10X genomics platform, on Illumina short-read, and PacBio and Oxford Nanopore long-read platforms. We also demonstrate the impact of CRISPR-based depletion of libraries, to remove highly expressed transcripts, prior to long-read sequencing in these experiments. By analysing single-source cDNA libraries in parallel, we enable a direct comparison of each platform, evaluating standard metrics alongside concordance in clustering and cell type identification. While each approach generates usable gene and isoform expression data, we identify limitations common across platforms, primarily linked to cDNA synthesis inefficiencies and read filtering strategies. Our work demonstrates the increasing utility of single-cell long-read sequencing for isoform-resolved analyses, such as direct immunoglobulin chain reconstruction without additional amplification, and the detection of alternative splicing patterns across immune cell subtypes in CD45, a key gene for immune cell activation and differentiation. Our benchmarking of current platform options provides a foundation for researchers looking to adopt single-cell long-read sequencing into their transcriptomic studies, providing a framework for its integration into diverse biological questions.

cell biology↗

bioRxiv2025

Leveraging existing data to maximise quality and consistency across gene model annotations: a Fusarium pan-annotation

Comparative genomics analyses are frequently used to inform our understanding of how organisms have evolved and how genetics contributes to phenotypic traits. Thanks to the considerable growth in the number of sequenced and assembled genomes, there is increasingly abundant data with which to perform such analyses. However, the appropriate use of genomic data can be highly dependent on genome annotation. Genome annotation is the critical step for providing biological context to genome sequences, but due to the complexity of the task it remains a major bottleneck in analyses. Different methods, and a lack of widely accepted standards, can also result in a great diversity of completeness and accuracy across genome annotations. Accordingly, comparative genomics analyses are susceptible to errors which can be misinterpreted as biological variation, yet efforts to revise and update existing annotations have not kept pace with advances in technology and expanding data resources. We have developed a workflow to utilise existing genome annotations alongside de novo gene predictions to improve both the collective consistency and individual quality of genome annotations of a closely related group of genomes. In this work we apply this new workflow on a dataset of 82 genomes from the economically, ecologically and clinically important fungal genus Fusarium. We show that both individual as well as collective annotation quality can be improved. The development of reannotation approaches such as we present here will be essential if we are to capitalise on the huge investment that has gone into generating existing genome data. The Fusarium pan-annotation is available from Zenodo at https://zenodo.org/doi/10.5281/zenodo.13829922. Workflow code and sample commands are available from https://github.com/EI-CoreBioinformatics/FusariumPanAnno. KEY POINTSO_LIComparative genomics is a fundamental approach to understand the contributions of genetic features to biological questions. C_LIO_LITo take advantage of existing data, most comparative genomics studies compare gene models produced using a variety of annotation methodologies, which introduces computational bias that can be misinterpreted as biological signal. C_LIO_LIWe present a bioinformatics workflow to improve consistency across a set of gene model annotations and minimise computational bias for downstream comparative genomics analyses. C_LIO_LIReannotation of a previously published Fusarium dataset reaffirms the finding that comparing annotations generated from a mix of methodologies can underestimate core genes, overestimate taxon-specific genes and confound patterns of gene presence/absence. C_LI

genomics↗

bioRxiv2025

High-quality genome assembly for the genetically improved Abbassa Nile tilapia enables the reconstruction of X and Y haplotypes

The success of the Nile tilapia (Oreochromis niloticus) as an aquaculture species is partly the result of continuous selective breeding leading to high performing strains. These elite strains have been derived from breeding populations of diverse origins and crosses with other Oreochromis species. Owing to the complex and unique evolutionary histories of each strain, existing reference genomes of wild populations are unsuitable to implement genomic selection for beneficial traits such as growth or environmental resilience in aquaculture programmes. Here we generated a high-quality genome assembly and annotation of the WorldFish Genetically Improved Abbassa Nile tilapia (GIANT) elite strain using a combination of PacBio HiFi, and Omni-C Illumina sequencing. As a male Abbassa Nile tilapia was used for the generation of the genome assembly, we reconstructed both X and Y haplotypes, identifying both amhY and amh{Delta}y on LG23 indicating that Abbassa likely shares the same sex determination system as GIFT, and thereby differs from the existing reference genome, whose sex determination loci are located on LG1.

genomics↗

bioRxiv2025

Whole genome sequencing reveals how plasticity and genetic differentiation underlie sympatric morphs of Arctic charr

Salmonids have a remarkable ability to form sympatric morphs after postglacial colonization of freshwater lakes. These morphs often exhibit differences in morphology, feeding, and spawning behaviour. Here we explore the genetics of morph differentiation by establishing a high-quality, annotated reference genome for the Arctic charr and use this as a resource for population genomic analysis of morphs from two Norwegian and two Icelandic lakes. The four lakes represent the spectrum of genetic differentiation between morphs from one lake with no genetic differentiation between morphs, implying phenotypic plasticity only, to two lakes with locus-specific genetic differentiation, implying incomplete reproductive isolation, and one lake with strong genome-wide divergence consistent with complete reproductive isolation. As many as 12 putative inversions ranging from 0.45 to 3.25 Mbp in size segregated among the four morphs present in one lake, Thingvallavatn, and these contributed significantly to the genetic differentiation among morphs. None of the putative inversions was found in any of the other lakes, but there were cases of partial haplotype sharing in similar morph contrasts in other lakes. The results are consistent with a highly polygenic basis of morph differentiation with limited genetic parallelism between lakes. The results support a model where morph differentiation is usually first established due to phenotypic plasticity that results in niche expansion and separation, followed by gradual development of reproductive isolation and locus-specific differentiation and eventually complete reproductive isolation and genome-wide divergence. A major explanation for salmonids ability to diversify into multiple sympatric morphs is likely the genome complexity caused by their ancient whole genome duplication that enhances evolvability.

evolutionary biology↗

bioRxiv2025

Biosynthesis and Bioactivity of Anti-Inflammatory Triterpenoids in Calendula officinalis (pot marigold)

Plants have been central to traditional medicine for millennia, yet the precise metabolites responsible for their therapeutic properties often remain unidentified. Moreover, the low natural abundance and structural complexity of bioactive molecules can hinder their accessibility and chemical synthesis. Here, we investigate the widely reported anti-inflammatory properties of Calendula officinalis (pot marigold), an ancient medicinal herb. We confirm C16-hydroxylated triterpenoids as key contributors to the anti-inflammatory activity of C. officinalis floral extracts and uncover a novel mechanism by which they act in modulating interleukin 6 release. Through biosynthetic pathway elucidation, we demonstrate that the oxidosqualene synthase catalysing the first committed step emerged early in Asteraceae evolution and identify residues governing product specificity. Further, we functionally characterise cytochrome P450s and acyltransferases responsible for downstream modifications. By reconstructing the complete biosynthetic pathway in the heterologous plant chassis Nicotiana benthamiana, we provide a platform for production of the anti-inflammatory components. Our work highlights how integrated studies of bioactivity and biosynthesis can unlock the therapeutic potential of medicinal plants.

plant biology↗

bioRxiv2024

Chromosome-scale genome assembly and de novo annotation of Alopecurus aequalis.

Alopecurus aequalis is a winter annual or short-lived perennial bunchgrass which has in recent years emerged as the dominant agricultural weed of barley and wheat in certain regions of China and Japan, causing significant yield losses. Its robust tillering capacity and high fecundity, combined with the development of both target and non-target-site resistance to herbicides means it is a formidable challenge to food security. Here we report on a chromosome-scale assembly of A. aequalis with a genome size of 2.83 Gb. The genome contained 33,758 high-confidence protein-coding genes with functional annotation. Comparative genomics revealed that the genome structure of A. aequalis is more similar to Hordeum vulgare rather than the more closely related Alopecurus myosuroides and has undergone an expansion of cytochrome P450 genes, a gene family involved in non-target-site herbicide resistance.

plant biology↗

bioRxiv2024

A haplotype-resolved chromosome-level genome assembly of Urochloa decumbens cv. Basilisk resolves its allopolyploid ancestry and composition

Haplotyped-resolved phased assemblies aim to capture the full allelic diversity in heterozygous and polyploid species to enable accurate genetic analyses. However, building non-collapsed references still presents a challenge. Here, we used long-range interaction Hi-C reads (high-throughput chromatin conformation capture) and HiFi PacBio reads to assemble the genome of the apomictic cultivar Basilisks from Urochloa decumbens (2n = 4x = 36), an outcrossed tetraploid Paniceae grass widely cropped to feed livestock in the tropics. We identified and removed Hi-C reads between homologous unitigs to facilitate their scaffolding and employed methods for the manual curation of rearrangements and misassemblies. Our final phased assembly included the four haplotypes in 36 chromosomes. We found that 18 chromosomes originated from diploid U. brizantha and the other 18 from either U. ruziziensis or diploid U. decumbens. We also identified a chromosomal translocation between chromosomes 5 and 32, as well as evidence of pairing exclusively within subgenomes, except for a homoeologous exchange in chromosome 21. Our results demonstrate that haplotype-aware assemblies accurately capture the allelic diversity in heterozygous species, making them the preferred option over collapsed-haplotype assemblies.

plant biology↗

bioRxiv2024

Multiple Independent Genetic Code Reassignments of the UAG Stop Codon in Phyllopharyngean Ciliates

The translation of nucleotide sequences into amino acid sequences, governed by the genetic code, is one of the most conserved features of molecular biology. The standard genetic code, which uses 61 sense codons to encode one of the 20 standard amino acids and 3 stop codons (UAA, UAG, and UGA) to terminate translation, is used by most extant organisms. The protistan phyla Ciliophora (the ciliates) are an unusual exception to this norm, exhibiting the greatest diversity of non-canonical nuclear genetic code variants and evidence of repeated changes in code. In this study, we report the discovery of multiple independent genetic code changes within the Phyllopharyngea class of ciliates. By mining publicly available ciliate genome datasets, we discovered that three ciliate species from the TARA Oceans eukaryotic metagenome dataset use the UAG codon to putatively encode leucine. We identified novel suppressor tRNA genes in two of these genomes. Phylogenomics analysis revealed that these three uncultivated taxa form a monophyletic lineage within the Phyllopharyngea class. Expanding our analysis by reassembling published phyllopharyngean genome datasets led to the discovery that the UAG codon had also been reassigned to putatively code for glutamine in Hartmannula sinica and Trochilia petrani. Phylogenomics analysis suggests that this occurred via two independent genetic code change events. These data demonstrate that the reassigned UAG codons have widespread usage as sense codons within the phyllopharyngean ciliates. Furthermore, we show that the function of UAA is firmly fixed as the preferred stop codon. These findings shed light on the evolvability of the genetic code in understudied microbial eukaryotes.

evolutionary biology↗

bioRxiv2024

The genome sequence of the Violet Carpenter Bee, Xylocopa violacea (Linnaeus, 1785): a hymenopteran species undergoing range expansion.

We present a reference genome assembly from an individual male Violet Carpenter Bee (Xylocopa violacea, Linnaeus, 1758). The assembly is 1.02 gigabases in span. 48% of the assembly is scaffolded into 17 pseudo-chromosomal units. The mitochondrial genome has also been assembled and is 21.8 kilobases in length. The genome is highly repetitive, likely representing a highly heterochromatic architecture expected of bees from the genus Xylocopa. We also use an evidence-based methodology to annotate 10,152 high confidence coding genes. This genome was sequenced as part of the pilot project of the European Reference Genome Atlas (ERGA) and represents an important addition to the genomic resources available for Hymenoptera.

genomics↗

bioRxiv2024

Evolutionary genomics reveals variation in structure and genetic content implicated in virulence and lifestyle in the genus Gaeumannomyces

Gaeumannomyces tritici is responsible for take-all disease, one of the most important wheat root threats worldwide. High-quality annotated genome resources are sorely lacking for this pathogen, as well as for the closely related antagonist and potential wheat take-all biocontrol agent, G. hyphopodioides. As such, we know very little about the genetic basis of the interactions in this host-pathogen-antagonist system. Using PacBio HiFi sequencing technology we have generated nine near-complete assemblies, including two different virulence lineages for G. tritici and the first assemblies for G. hyphopodioides and G. avenae (oat take-all). Genomic signatures support the presence of two distinct virulence lineages in G. tritici (types A and B), with A strains potentially employing a mechanism to prevent gene copy-number expansions. The CAZyme repertoire was highly conserved across Gaeumannomyces, while candidate secreted effector proteins and biosynthetic gene clusters showed more variability and may distinguish pathogenic and non-pathogenic lineages. A transition from self-sterility (heterothallism) to self-fertility (homothallism) may also be a key innovation implicated in lifestyle. We did not find evidence for transposable element and effector gene compartmentalisation in the genus, however the presence of Starship giant transposable elements likely contributes to genomic plasticity in the genus. Our results depict Gaeumannomyces as an ideal system to explore interactions within the rhizosphere, the nuances of intraspecific virulence, interspecific antagonism, and fungal lifestyle evolution. The foundational genomic resources provided here will enable the development of diagnostics and surveillance of understudied but agriculturally important fungal pathogens.

evolutionary biology↗

bioRxiv2024

Chromosome-scale genome assembly of bread wheat's wild relative Triticum timopheevii

Wheat (Triticum aestivum) is one of the most important food crops with an urgent need for increase in its production to feed the growing world. Triticum timopheevii (2n = 4x = 28) is an allotetraploid wheat wild relative species containing the At and G genomes that has been exploited in many pre-breeding programmes for wheat improvement. In this study, we report the generation of a chromosome-scale reference genome assembly of T. timopheevii accession PI 94760 based on PacBio HiFi reads and chromosome conformation capture (Hi-C). The assembly comprised a total size of 9.35 Gb, featuring a contig N50 of 42.4 Mb, and 166,325 predicted gene models. DNA methylation analysis showed that the G genome had on average more methylated bases than the At genome. The G genome was also more closely related to the S genome of Aegilops speltoides than to the B genome of hexaploid or tetraploid wheat. In summary, the T. timopheevii genome assembly provides a valuable resource for genome-informed discovery of agronomically important genes for food security.

plant biology↗

bioRxiv2024

De novo annotation of the wheat pangenome reveals complexity and diversity of the hexaploid wheat pan-transcriptome

Wheat is the most widely cultivated crop in the world with over 215 million hectares grown annually. However, to meet the demands of a growing global population, breeders face the challenge of increasing wheat production by approximately 60% within the next 40 years. The 10+ Wheat Genomes Project recently sequenced and assembled to chromosome level the genomes of nine wheat cultivars to develop our understanding of genetic diversity and selection within the pan-genome of wheat. Here, we provide a wheat pan-transcriptome with de novo annotation and differential expression analysis for these wheat cultivars, across multiple different tissues and whole seedlings sampled at dusk/dawn. Analysis of these de novo annotations facilitated the discovery of genes absent from the Chinese Spring reference, identified genes specific to particular cultivars and defined the core and dispensable genomes. Expression analysis across cultivars and tissues revealed conservation in expression between a large core set of homeologous genes, but also widespread changes in subgenome homeolog expression bias between cultivars. Co-expression network analysis revealed the impact of divergence of sub-genome homeolog expression and identified cultivar-specific expression profiles. In a case study utilising both the newly constructed wheat pan-genome and pan-transcriptome we demonstrate prevalent variation in the prolamin superfamily and immune-reactive proteins across the pan-cultivars.In summary, this work provides both a valuable resource for the wider wheat community and reveals diversity in gene content and expression patterns between global wheat cultivars.

genomics↗

bioRxiv2023

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report the assembly of an improved N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated 3-4 times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and opens the possibility that BBLs are the sought-after coupling enzymes in nicotine biosynthesis.

plant biology↗