Biology subjects

Thorpe, J.

Publications and source records attributed to Thorpe, J..

7 recordsLinked to original sources

bioRxiv2026

A Plasmodium falciparum Pangenome Resource to Drive Structural Variant Discovery and to assist Malaria Control

Plasmodium falciparum, the deadliest causative agent of malaria, harbours extensive structural variation that underlies key biological processes including drug resistance and diagnostic evasion. Here, we present PfPan, a P. falciparum pangenome constructed from 13 geographically diverse high-quality reference genomes using Minigraph-Cactus, adding 4.7 Mb of sequence beyond the 3D7 linear reference. We identify over 5,000 structural variants across the reference genomes and demonstrate improved genotyping using the vg toolkit compared to linear reference-based approaches, with comparable performance for small variant discovery. Benchmarking against assembly-derived truth sets confirms pangenome superiority for structural variant detection, particularly at complex and hypervariable loci. Applying PfPan to 878 globally sampled P. falciparum whole-genome sequences, we characterise the population-level frequency of clinically relevant structural variants, including a high-frequency 10.4 kb insertion at the drug resistance-linked gch1 locus, and explore deletions upstream of the gene encoding the diagnostic target HRP2. PfPan provides a foundational resource for reducing reference bias in P. falciparum genomic surveillance and offers a framework for improved detection of variants relevant to drug resistance and malaria control.

genomics↗

bioRxiv2026

Protocol-dependent cardiomyocyte states determine disease modelling capacity of human iPSCs

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are widely used to model cardiovascular disease, yet numerous differentiation protocols generate cardiomyocytes with heterogeneous molecular and functional properties, complicating experimental design. Here we systematically compare sixteen commonly used cardiomyocyte differentiation protocols and characterize their resulting cell states using single-nucleus RNA sequencing, functional phenotyping and computational integration with human genetic data. Despite similar cardiomyocyte yields, protocols produced distinct transcriptional programs, subtype compositions and physiological properties. By integrating protocol-specific gene expression signatures with genome-wide association studies of cardiovascular traits, we identify cardiomyocyte states enriched for genetic architectures underlying specific diseases. These analyses accurately predict protocols most suitable for modelling particular disease contexts, including electrophysiological defects associated with Brugada syndrome and metabolic vulnerability relevant to myocardial infarction. Our results demonstrate that differentiation protocols encode biologically distinct cardiomyocyte states with differential disease relevance and establish a framework for aligning stem-cell differentiation strategies with human complex trait genetics to guide model selection. This approach enables rational design of iPSC-based disease models and highlights how population-scale genetic data can inform experimental systems in stem cell biology.

systems biology↗

bioRxiv2025

Robotic Handling Preserves Induced Pluripotent Stem Cell Derived Vascular Smooth Muscle Cells Differentiation using a Weekend-free, Automatable, Low-variability, Low-cost (WALL) Protocol

Vascular disease modeling and tissue engineering require scalable, efficient, and reproducible methods for differentiating cells, however no studies have validated whether automation, essential for scale-up, preserves differentiation quality in cells. Here, we developed a differentiation protocol for induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iVSMCs) that is applicable to both manual, and automated robotic culture. iPSC lines generated from male and female donors across various ages were used to optimize the generation of iVSMCs. iVSMCs had consistent morphology and protein expression was largely similar to primary VSMCs, including expression of myosin heavy chain-11 (MYH11), -smooth muscle actin (-SMA), transgelin (TAGLN) and calponin (CNN1). Functionally, these iVSMCs could respond to the vasoconstrictor carbachol. Coupling this protocol with an automated Hamilton liquid-handling robotics system allowed the generation of iVSMCs in large quantities, morphologically similar to manually differentiated iVSMCs. Comparative proteomic analysis confirmed protein expression did not differ between automated and manual differentiation methods. Automation markedly reduced manual labor and facilitated increased production without sacrificing cell quality. This study demonstrates the feasibility of automating iVSMC differentiation, marking a significant step towards scalable VSMC manufacturing for three-dimensional applications, and for the ever-growing demands of organoid and tissue engineering applications. Statement of significanceThe rapidly expanding fields of tissue engineering and high-throughput disease modeling require large quantities of viable cells, making automation essential. Robotic liquid handling systems are increasingly employed for generating large quantities of induced pluripotent stem cell (iPSC)-derived cells, requiring the development of scalable protocols. No studies have yet evaluated whether the use of robotic liquid handling alters the differentiation of cells compared to traditional manual handling. Here, we report a scalable differentiation protocol that robustly generates iPSC-derived VSMCs (iVSMCs) from a cohort of female and male donors of varying age (n=5 lines). We demonstrate for the first time that iVSMCs, differentiated through an automated robotic pipeline, had no significant differences in morphological or proteomic phenotype compared to manual differentiation. The ability to automate this protocol facilitates robust and reproducible differentiation of large quantities of iVSMC, setting a precedent for the application of automation in generating iPSC-derived cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/690512v1_ufig1.gif" ALT="Figure 1000"> View larger version (18K): org.highwire.dtl.DTLVardef@673d61org.highwire.dtl.DTLVardef@1eead89org.highwire.dtl.DTLVardef@c3089eorg.highwire.dtl.DTLVardef@6af2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

The First Chromosome-Scale Genome Assembly for Dermacentor reticulatus: A Key Vector of Tick-Borne Pathogens of Public and Veterinary Health Importance in Europe

BackgroundDermacentor reticulatus is a key tick species across Europe and an established vector of multiple pathogens that affect both human and animal health. Despite its significant role in disease transmission, genomic data for this species remain limited. Here, we present the first chromosome-scale genome assembly of D. reticulatus, constructed using Oxford Nanopore long-read sequencing data. MethodsHigh molecular weight DNA was extracted from a female Dermacentor reticulatus collected in Devon, UK, and sequenced using Oxford Nanopore long-read technology. Genome assemblies were generated using both guided and unguided approaches, followed by repeat masking, gene prediction, and functional annotation. Genome completeness was assessed using BUSCO, and comparative, phylogenetic, and functional analyses were performed against other Dermacentor and tick species. ResultsThis chromosome-scale genome assembly revealed a repeat-rich genome, with approximately 61.9% of the total sequence consisting of repetitive elements. Benchmarking universal single-copy ortholog (BUSCO) analysis demonstrated strong genome completeness, with guided assembly (chromosome) achieving a score of 97.1%, closely matching those of related Dermacentor species. For comparison, unguided assembly (scaffold) yielded a BUSCO score of 96.7%. Gene annotation following repeat masking resulted in BUSCO completeness scores of 95.1% (guided) and 95.0% (unguided). Functional characterisation included Pfam domain assignment and Gene Ontology analysis. Additionally, we assembled the mitochondrial genome (15,103 bp), comprising 38 genes, providing further insight into D. reticulatus phylogenetic placement. ConclusionsThis genomic resource establishes a foundation for functional genomics, evolutionary studies and supports future research in vector biology and the control of tick-borne diseases.

genomics↗

bioRxiv2025

A novel tool for designing targeted gene amplicons and an optimised set of primers for high-throughput sequencing in tuberculosis genomic studies

Amplicon sequencing (Amp-Seq) of Mycobacterium tuberculosis genes associated with drug resistance and strain typing offers a cost-effective approach for profiling infections and tailoring the clinical management of tuberculosis. However, Amp-Seq assays require continual updates to incorporate new loci and mutations linked to drug resistance. We introduce TOAST (Tuberculosis Optimised Amplicon Sequencing Tool), a customisable software tool that optimises amplicon design across any loci and sequencing platforms (e.g., Illumina, Oxford Nanopore Technology (ONT)), informed by an integrated and expanding database of mutations from >50K M. tuberculosis isolates. TOAST software allows users to define parameters such as melting temperature, amplicon length, and GC content, while accounting for potential primer interactions like homodimer formation and non-specific binding. To demonstrate its robustness, we designed 33 amplicons in a single multiplex group, prioritising coverage of resistance-associated mutations in the form of insertions, deletions and single nucleotide polymorphisms (SNPs) for 13 different drugs. An efficient experimental protocol was established, resulting in a minimum depth coverage exceeding 50-fold for each amplicon region as validated by ONT sequencing of two clinical samples with multi-drug resistance. TOAST software enables the development of Amp-Seq assays for the rapid detection of drug-resistant TB, enhancing treatment strategies, improving outcomes, and curbing resistant infections. The cost-effectiveness and adaptability of Amp-Seq approaches make it crucial for managing TB in resource-limited settings, advancing global control efforts.

bioinformatics↗

bioRxiv2020

Comprehensive identification of somatic nucleotide variants in human brain tissue

Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. Here, the Brain Somatic Mosaicism Network conducted a coordinated, multi-institutional study to: (i) examine the ability of existing methods to detect simulated somatic single nucleotide variants (SNVs) in DNA mixing experiments; (ii) generate multiple replicates of whole genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual; (iii) devise strategies to discover somatic SNVs; and (iv) apply various approaches to validate somatic SNVs. These efforts led to the identification of 43 bona fide somatic SNVs that ranged in variant allele fractions from ~0.005 to ~0.28. Guided by these results, we devised best practices for calling mosaic SNVs from 250X whole genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrated that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. Thus, this study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

genomics↗

bioRxiv2020

Discovery of a Novel Polymer for Xeno-free, Long-term Culture of Human Pluripotent Stem Cell Expansion

Human pluripotent stem cells (hPSCs) can be expanded and differentiated in vitro into almost any adult tissue cell type, and thus have great potential as a source for cell therapies with biomedical application. In this study, a fully-defined polymer synthetic substrate is identified for hPSC culture in completely defined, xeno-free conditions. This system can overcome the cost, scalability and reproducibility limitations of current hPSC culture strategies, and facilitate large-scale production. A high-throughput, multi-generational polymer microarray platform approach was used to test over 600 unique polymers and rapidly assess hPSC-polymer interactions in combination with the fully defined xeno-free medium, Essential 8TM (E8). This study identifies as novel nanoscale phase separated blend of poly(tricyclodecane-dimethanol diacrylate) and poly(butyl acrylate) (2:1 v/v), which supports long-term expansion of hPSCs and can be readily coated onto standard cultureware. Analysis of cell-polymer interface interactions through mass spectrometry and integrin blocking studies provides novel mechanistic insight into the role of the E8 proteins in promoting integrin-mediated hPSC attachment and maintaining hPSC signaling, including ability to undergo multi-lineage differentiation. This study therefore identifies a novel substrate for long-term serial passaging of hPSCs in serum-free, commercial chemically-defined E8, which provides a promising and economic hPSC expansion platform for clinical-scale application.

synthetic biology↗