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Morgan, M.

Publications and source records attributed to Morgan, M..

At least 37 records · Page 2Linked to original sources

Whole body cell map tracks tissue-specific immune cell accumulation and plasticity loss through ageing

Understanding tissue biologys heterogeneity is crucial for advancing precision medicine. Despite the centrality of the immune system in tissue homeostasis, a detailed and comprehensive map of immune cell distribution and interactions across human tissues and demographics remains elusive. To fill this gap, we harmonised data from 12,981 single-cell RNA sequencing samples and curated 29 million cells from 45 anatomical sites to create a comprehensive compositional and transcriptional healthy map of the healthy immune system. We used this resource and a novel multilevel modelling approach to track immune ageing and test differences across sex and ethnicity. We uncovered conserved and tissue-specific immune-ageing programs, resolved sex-dependent differential ageing and identified ethnic diversity in clinically critical immune checkpoints. This study provides a quantitative baseline of the immune system, facilitating advances in precision medicine. By sharing our immune map, we hope to catalyse further breakthroughs in cancer, infectious disease, immunology and precision medicine.

immunology↗

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii

The phylum of Apicomplexa groups intracellular parasites that employ substratedependent gliding motility to invade host cells, egress from the infected cells and cross biological barriers. The glideosome associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the membrane binding interface within the C-terminal PH domain as well as an N-terminal fragment necessary for association with F-actin suggest that GAC adopts multiple conformations. A multi-conformational model for assembly of GAC within the glideosome is proposed.

biochemistry↗

JBrowse 2: A modular genome browser with views of synteny and structural variation

We present JBrowse 2, a general-purpose genome annotation browser offering enhanced visualization of complex structural variation and evolutionary relationships. JBrowse 2 retains the core features of the open-source JavaScript genome browser JBrowse while adding new views for synteny, dotplots, breakpoints, gene fusions, and whole-genome overviews. The software readily allows users to share sessions, open multiple genomes or views, and navigate quickly between these views. It can be embedded in a web page, used as a standalone desktop application, or run from Jupyter notebooks or R sessions. Using a plugin framework, developers can create new data adapters, track types, and visualizations. These improvements are enabled by a ground-up redesign of the JBrowse architecture using modern web technology. We describe application functionality, use cases, performance benchmarks, and implementation notes for web administrators and developers.

bioinformatics↗

Plasmid overlap and evolution between Enterobacterales isolates from bloodstream infections and non-human compartments

Plasmids enable the dissemination of antimicrobial resistance (AMR) in common Enterobacterales pathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution between Enterobacterales causing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is highly relevant to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets) Enterobacterales. Isolates were collected between 2008-2020 from sites <60km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared "backbones", with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid "backbones" were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies. FundingThis study was funded by the Antimicrobial Resistance Cross-council Initiative supported by the seven research councils and the NIHR, UK.

genomics↗

Curated Single Cell Multimodal Landmark Datasets for R/Bioconductor

BackgroundThe majority of high-throughput single-cell molecular profiling methods quantify RNA expression; however, recent multimodal profiling methods add simultaneous measurement of genomic, proteomic, epigenetic, and/or spatial information on the same cells. The development of new statistical and computational methods in Bioconductor for such data will be facilitated by easy availability of landmark datasets using standard data classes. ResultsWe collected, processed, and packaged publicly available landmark datasets from important single-cell multimodal protocols, including CITE-Seq, ECCITE-Seq, SCoPE2, scNMT, 10X Multiome, seqFISH, and G&T. We integrate data modalities via the MultiAssayExperiment Bioconductor class, document and re-distribute datasets as the SingleCellMultiModal package in Bioconductors Cloud-based ExperimentHub. The result is single-command actualization of landmark datasets from seven single-cell multimodal data generation technologies, without need for further data processing or wrangling in order to analyze and develop methods within Bioconductors ecosystem of hundreds of packages for single-cell and multimodal data. ConclusionsWe provide two examples of integrative analyses that are greatly simplified by SingleCellMultiModal. The package will facilitate development of bioinformatic and statistical methods in Bioconductor to meet the challenges of integrating molecular layers and analyzing phenotypic outputs including cell differentiation, activity, and disease. Author SummaryExperimental data packages that provide landmark datasets have historically played an important role in the development of new statistical methods in Bioconductor by lowering the barrier of access to relevant data, providing a common testing ground for software development and benchmarking, and encouraging interoperability around common data structures. In this manuscript, we review major classes of technologies for collecting multimodal data including genomics, transcriptomics, epigenetics, proteomics, and spatial information at the level of single cells. We present the SingleCellMultiModal R/Bioconductor package that provides single-command access to landmark datasets from seven different technologies, storing datasets using HDF5 and sparse arrays for memory efficiency and integrating data modalities via the MultiAssayExperiment class. We demonstrate two integrative analyses that are greatly simplified by SingleCellMultiModal. The package facilitates development and benchmarking of bioinformatic and statistical methods to integrate molecular layers at the level of single cells with phenotypic outputs including cell differentiation, activity, and disease, within Bioconductors ecosystem of hundreds of packages for single-cell and multimodal data.

bioinformatics↗

MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates -- splicing, export, localisation, decay, stability, and translation -- to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in the m6A pathway. MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A-containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3{beta} mRNA through m6A that stabilises the {beta}-CATENIN expression of a WNT/{beta}-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mouse embryonic stem cells (mESCs) state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

biochemistry↗

3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

cell biology↗

Inverting the model of genomics data sharing with the NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space (AnVIL)

The traditional model of genomic data analysis - downloading data from centralized warehouses for analysis with local computing resources - is increasingly unsustainable. Not only are transfers slow and cost prohibitive, but this approach also leads to redundant and siloed compute infrastructure that makes it difficult to ensure security and compliance of protected data. The NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space (AnVIL; https://anvilproject.org) inverts this model, providing a unified cloud computing environment for data storage, management, and analysis. AnVIL eliminates the need for data movement, allows for active threat detection and monitoring, and provides scalable, shared computing resources that can be acquired by researchers as needed. This presents many new opportunities for collaboration and data sharing that will ultimately lead to scientific discoveries at scales not previously possible.

bioinformatics↗

Cells of the human intestinal tract mapped across space and time

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprungs disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

cell biology↗

Tumor-infiltrating nerves create an electro-physiologically active microenvironment and contribute to treatment resistance

Patients with densely innervated tumors do poorly as compared to those with sparsely innervated disease. Why some tumors heavily recruit nerves while others do not, remains unknown as does the functional contribution of tumor-infiltrating nerves to cancer. Moreover, while patients receive chemotherapeutic treatment, whether these drugs affect nerve recruitment has not been tested. Using a murine model of ovarian cancer, we show that tumor-infiltrating sensory nerves potentiate tumor growth, decrease survival, and contribute to treatment resistance. Furthermore, matched patient samples show significantly increased tumor innervation following chemotherapy. In vitro analysis of tumor-released extracellular vesicles (sEVs) shows they harbor neurite outgrowth activity. These data suggest that chemotherapy may alter sEV cargo, endowing it with robust nerve recruiting capacity.

cancer biology↗

DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

Mycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99{degrees}C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes [&ge;]1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

microbiology↗

Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies

Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in AMR/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data has suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore (Oxford Nanopore Technologies [ONT]) flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 hours of sequencing, most chromosomes and plasmids had circularised and there was no benefit associated with longer running times. Quality was similar at 12 hours suggesting shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilising shorter run-times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per isolate sequencing cost. Ultimately this will facilitate large-scale studies utilising hybrid assembly advancing our understanding of the genomics of key human pathogens. Data SummaryO_LIRaw sequencing data is available via NCBI under project accession number PRJNA604975. Sample accession numbers are provided in table S1. C_LIO_LIAssemblies are available via Figshare https://doi.org/10.6084/m9.figshare.11816532.v1. C_LI Impact StatementMost existing sequencing data has been acquired from short-read platforms (eg. Illumina). For some species of bacteria, clinically important genes, such as those involved in antibiotic resistance and/or virulence, are carried on plasmids. Whilst Illumina sequencing is highly accurate, it is generally unable to resolve complete genomic structures due to repetitive regions. Hybrid assembly uses long reads to scaffold together short-read contigs, maximising the benefits of both technologies. A major limiting factor to using hybrid assemblies at scale is the cost of sequencing the same isolate with two different technologies. Here we show that high-quality hybrid assemblies can be created for most isolates using significantly shorter run-times than are currently standard. We demonstrate that a simple washing step allows several libraries to be run on the same flowcell, facilitating the ability to take advantage of shorter running times. Adding nuclease means that contamination between libraries is minimal and has no significant effect on the quality of subsequent hybrid assemblies. This approach reduces the cost of acquiring long reads by >30%, paving the way for large-scale studies utilising hybrid assemblies which will likely significantly enhance our understanding of the genomics of important human pathogens.

bioinformatics↗

A Single Nucleotide Polymorphism assay sheds light on the extent and distribution of genetic diversity, population structure and functional basis of key traits in cultivated North American Cannabis

BackgroundThe taxonomic classification of the Cannabis genus has been delineated through three main types: sativa (long and less branched plant with long and narrow leaves), indica (short but highly branched plant with broader leaves) and ruderalis (wild type with short stature, less branching and small thick leaves). While still under discussion, particularly whether the genus is polytypic or monotypic, this broad classification reflects putative geographical origin of each group and putative chemotypic and pharmacology. MethodsHere we describe a thorough investigation of cannabis accessions using a set of 22 highly informative and polymorphic SNP markers associated with important traits such as cannabinoid and terpenoid expression as well as fibre and resin production. The assay offers insight into cannabis population structure, phylogenetic relationship, population genetics and correlation to secondary metabolite concentrations and demonstrate the utility of this assay for rapid, repeatable and cost-efficient genotyping of commercial and industrial cannabis accessions for use in product traceability, breeding programs, regulatory compliance and consumer education. ResultsThe main outcomes are the identification of 5 clusters in the sample set available, including industrial hemp, resin hemp which likely underwent a bottleneck to stabilize CBDA accumulation (Type II & III). THC resin (type I) make up the other three clusters with terpinolene (colloquial "sativa" or "NLD"), myrcene/pinene and myrcene/limonene (colloquial "indica", "BLD"), which also putatively harbour an active CBCAS. ConclusionThe functional basis of key traits is also discussed as recently enabled by the NCBI Cannabis sativa Annotation Release 100, allowing for hypothesis testing with regards to secondary metabolite production as well as other key traits of importance for adaptable and compliant large-scale seed production under the new US Domestic Hemp Production Program.

plant biology↗

A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion - "X-shredding" and "X-meddling" - and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

synthetic biology↗

Tximeta: reference sequence checksums for provenance identification in RNA-seq

Correct annotation metadata is critical for reproducible and accurate RNA-seq analysis. When files are shared publicly or among collaborators with incorrect or missing annotation metadata, it becomes difficult or impossible to reproduce bioinformatic analyses from raw data. It also makes it more difficult to locate the transcriptomic features, such as transcripts or genes, in their proper genomic context, which is necessary for overlapping expression data with other datasets. We provide a solution in the form of an R/Bioconductor package tximeta that performs numerous annotation and metadata gathering tasks automatically on behalf of users during the import of transcript quantification files. The correct reference transcriptome is identified via a hashed checksum stored in the quantification output, and key transcript databases are downloaded and cached locally. The computational paradigm of automatically adding annotation metadata based on reference sequence checksums can greatly facilitate genomic workflows, by helping to reduce overhead during bioinformatic analyses, preventing costly bioinformatic mistakes, and promoting computational reproducibility. The tximeta package is available at https://bioconductor.org/packages/tximeta.

bioinformatics↗

Towards a gold standard for benchmarking gene set enrichment analysis

BackgroundAlthough gene set enrichment analysis has become an integral part of high-throughput gene expression data analysis, the assessment of enrichment methods remains rudimentary and ad hoc. In the absence of suitable gold standards, evaluations are commonly restricted to selected data sets and biological reasoning on the relevance of resulting enriched gene sets. However, this is typically incomplete and biased towards the goals of individual investigations.\n\nResultsWe present a general framework for standardized and structured benchmarking of enrichment methods based on defined criteria for applicability, gene set prioritization, and detection of relevant processes. This framework incorporates a curated compendium of 75 expression data sets investigating 42 different human diseases. The compendium features microarray and RNA-seq measurements, and each dataset is associated with a precompiled GO/KEGG relevance ranking for the corresponding disease under investigation. We perform a comprehensive assessment of 10 major enrichment methods on the benchmark compendium, identifying significant differences in (i) runtime and applicability to RNA-seq data, (ii) fraction of enriched gene sets depending on the type of null hypothesis tested, and (iii) recovery of the a priori defined relevance rankings. Based on these findings, we make practical recommendations on (i) how methods originally developed for microarray data can efficiently be applied to RNA-seq data, (ii) how to interpret results depending on the type of gene set test conducted, and (iii) which methods are best suited to effectively prioritize gene sets with high relevance for the phenotype investigated.\n\nConclusionWe carried out a systematic assessment of existing enrichment methods, and identified best performing methods, but also general shortcomings in how gene set analysis is currently conducted. We provide a directly executable benchmark system for straightforward assessment of additional enrichment methods.\n\nAvailabilityhttp://bioconductor.org/packages/GSEABenchmarkeR

bioinformatics↗

Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential of a diagnostic test for influenza which also provides insights on transmission, evolution and drug resistance, and simultaneously detects other viruses. We therefore set out to apply Oxford Nanopore Technology to metagenomic sequencing of respiratory samples. We generated influenza reads down to a limit of detection of 102-103 genome copies/ml in pooled samples, observing a strong relationship between the viral titre and the proportion of influenza reads (p = 4.7x10-5). Applying our methods to clinical throat swabs, we generated influenza reads for 27/27 samples with high-to-mid viral titres (Cycle threshold (Ct) values <30) and 6/13 samples with low viral titres (Ct values 30-40). No false positive reads were generated from 10 influenza-negative samples. Thus Nanopore sequencing operated with 83% sensitivity (95% CI 67-93%) and 100% specificity (95% CI 69-100%) compared to the current diagnostic standard. Coverage of full length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza titres, we were able to reconstruct >99% complete sequence for all eight gene segments. We also detected Human Coronavirus and generated a near complete Human Metapneumovirus genome from clinical samples. While further optimisation is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza and other respiratory viruses.

microbiology↗

Orchestrating Single-Cell Analysis with Bioconductor

Recent developments in experimental technologies such as single-cell RNA sequencing have enabled the profiling a high-dimensional number of genome-wide features in individual cells, inspiring the formation of large-scale data generation projects quantifying unprecedented levels of biological variation at the single-cell level. The data generated in such projects exhibits unique characteristics, including increased sparsity and scale, in terms of both the number of features and the number of samples. Due to these unique characteristics, specialized statistical methods are required along with fast and efficient software implementations in order to successfully derive biological insights. Bioconductor - an open-source, open-development software project based on the R programming language - has pioneered the analysis of such high-throughput, high-dimensional biological data, leveraging a rich history of software and methods development that has spanned the era of sequencing. Featuring state-of-the-art computational methods, standardized data infrastructure, and interactive data visualization tools that are all easily accessible as software packages, Bioconductor has made it possible for a diverse audience to analyze data derived from cutting-edge single-cell assays. Here, we present an overview of single-cell RNA sequencing analysis for prospective users and contributors, highlighting the contributions towards this effort made by Bioconductor.

genomics↗