RESUMEN
Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of â¼10, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterize, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions (ICRs) as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new ICRs.
Asunto(s)
Genoma , Impresión Genómica , Técnicas de Genotipaje , Haplotipos , Análisis de Secuencia de ADN/estadística & datos numéricos , Alelos , Animales , Mapeo Cromosómico , Islas de CpG , Metilación de ADN , Embrión de Mamíferos/química , Embrión de Mamíferos/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Placenta/química , Placenta/metabolismo , EmbarazoRESUMEN
With the growing number of single-cell analysis tools, benchmarks are increasingly important to guide analysis and method development. However, a lack of standardisation and extensibility in current benchmarks limits their usability, longevity, and relevance to the community. We present Open Problems, a living, extensible, community-guided benchmarking platform including 10 current single-cell tasks that we envision will raise standards for the selection, evaluation, and development of methods in single-cell analysis.
RESUMEN
The incidence of cutaneous T-cell lymphoma (CTCL) increases with age, and blood involvement portends a worse prognosis. To advance our understanding of the development of CTCL and identify potential therapeutic targets, we performed integrative analyses of paired single-cell RNA and T-cell receptor (TCR) sequencing of peripheral blood CD4+ T cells from patients with CTCL to reveal disease-unifying features. The malignant CD4+ T cells of CTCL showed highly diverse transcriptomic profiles across patients, with most displaying a mature Th2 differentiation and T-cell exhaustion phenotype. TCR-CDR3 peptide prediction analysis suggested limited diversity between CTCL samples, consistent with a role for a common antigenic stimulus. Potential of heat diffusion for affinity-based trajectory embedding transition analysis identified putative precancerous circulating populations characterized by an intermediate stage of gene expression and mutation level between the normal CD4+ T cells and malignant CTCL cells. We further revealed the therapeutic potential of targeting CD82 and JAK that endow the malignant CTCL cells with survival and proliferation advantages.
Asunto(s)
Linfoma Cutáneo de Células T , Neoplasias Cutáneas , Humanos , Transcriptoma , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Linfoma Cutáneo de Células T/patología , Linfocitos T CD4-Positivos/metabolismo , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Due to commonalities in pathophysiology, age-related macular degeneration (AMD) represents a uniquely accessible model to investigate therapies for neurodegenerative diseases, leading us to examine whether pathways of disease progression are shared across neurodegenerative conditions. Here we use single-nucleus RNA sequencing to profile lesions from 11 postmortem human retinas with age-related macular degeneration and 6 control retinas with no history of retinal disease. We create a machine-learning pipeline based on recent advances in data geometry and topology and identify activated glial populations enriched in the early phase of disease. Examining single-cell data from Alzheimer's disease and progressive multiple sclerosis with our pipeline, we find a similar glial activation profile enriched in the early phase of these neurodegenerative diseases. In late-stage age-related macular degeneration, we identify a microglia-to-astrocyte signaling axis mediated by interleukin-1ß which drives angiogenesis characteristic of disease pathogenesis. We validated this mechanism using in vitro and in vivo assays in mouse, identifying a possible new therapeutic target for AMD and possibly other neurodegenerative conditions. Thus, due to shared glial states, the retina provides a potential system for investigating therapeutic approaches in neurodegenerative diseases.
Asunto(s)
Degeneración Macular , Enfermedades Neurodegenerativas , Humanos , Ratones , Animales , Degeneración Macular/metabolismo , Retina/metabolismo , Neuroglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Análisis de la Célula IndividualRESUMEN
As the biomedical community produces datasets that are increasingly complex and high dimensional, there is a need for more sophisticated computational tools to extract biological insights. We present Multiscale PHATE, a method that sweeps through all levels of data granularity to learn abstracted biological features directly predictive of disease outcome. Built on a coarse-graining process called diffusion condensation, Multiscale PHATE learns a data topology that can be analyzed at coarse resolutions for high-level summarizations of data and at fine resolutions for detailed representations of subsets. We apply Multiscale PHATE to a coronavirus disease 2019 (COVID-19) dataset with 54 million cells from 168 hospitalized patients and find that patients who die show CD16hiCD66blo neutrophil and IFN-γ+ granzyme B+ Th17 cell responses. We also show that population groupings from Multiscale PHATE directly fed into a classifier predict disease outcome more accurately than naive featurizations of the data. Multiscale PHATE is broadly generalizable to different data types, including flow cytometry, single-cell RNA sequencing (scRNA-seq), single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), and clinical variables.
Asunto(s)
COVID-19 , Análisis de la Célula Individual , Cromatina , Humanos , Análisis de la Célula Individual/métodos , Transposasas , Secuenciación del ExomaRESUMEN
Current methods for comparing single-cell RNA sequencing datasets collected in multiple conditions focus on discrete regions of the transcriptional state space, such as clusters of cells. Here we quantify the effects of perturbations at the single-cell level using a continuous measure of the effect of a perturbation across the transcriptomic space. We describe this space as a manifold and develop a relative likelihood estimate of observing each cell in each of the experimental conditions using graph signal processing. This likelihood estimate can be used to identify cell populations specifically affected by a perturbation. We also develop vertex frequency clustering to extract populations of affected cells at the level of granularity that matches the perturbation response. The accuracy of our algorithm at identifying clusters of cells that are enriched or depleted in each condition is, on average, 57% higher than the next-best-performing algorithm tested. Gene signatures derived from these clusters are more accurate than those of six alternative algorithms in ground truth comparisons.
Asunto(s)
Biología Computacional , Análisis de Secuencia de ARN/tendencias , Análisis de la Célula Individual/tendencias , Transcriptoma/genética , Algoritmos , Análisis por Conglomerados , Simulación por Computador , Humanos , Funciones de VerosimilitudRESUMEN
We propose a novel framework for combining datasets via alignment of their intrinsic geometry. This alignment can be used to fuse data originating from disparate modalities, or to correct batch effects while preserving intrinsic data structure. Importantly, we do not assume any pointwise correspondence between datasets, but instead rely on correspondence between a (possibly unknown) subset of data features. We leverage this assumption to construct an isometric alignment between the data. This alignment is obtained by relating the expansion of data features in harmonics derived from diffusion operators defined over each dataset. These expansions encode each feature as a function of the data geometry. We use this to relate the diffusion coordinates of each dataset through our assumption of partial feature correspondence. Then, a unified diffusion geometry is constructed over the aligned data, which can also be used to correct the original data measurements. We demonstrate our method on several datasets, showing in particular its effectiveness in biological applications including fusion of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) data measured on the same population of cells, and removal of batch effect between biological samples.
RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The high-dimensional data created by high-throughput technologies require visualization tools that reveal data structure and patterns in an intuitive form. We present PHATE, a visualization method that captures both local and global nonlinear structure using an information-geometric distance between data points. We compare PHATE to other tools on a variety of artificial and biological datasets, and find that it consistently preserves a range of patterns in data, including continual progressions, branches and clusters, better than other tools. We define a manifold preservation metric, which we call denoised embedding manifold preservation (DEMaP), and show that PHATE produces lower-dimensional embeddings that are quantitatively better denoised as compared to existing visualization methods. An analysis of a newly generated single-cell RNA sequencing dataset on human germ-layer differentiation demonstrates how PHATE reveals unique biological insight into the main developmental branches, including identification of three previously undescribed subpopulations. We also show that PHATE is applicable to a wide variety of data types, including mass cytometry, single-cell RNA sequencing, Hi-C and gut microbiome data.
Asunto(s)
Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Animales , Macrodatos , Diferenciación Celular , Células Cultivadas , Simulación por Computador , Bases de Datos Genéticas , Microbioma Gastrointestinal , Humanos , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Oxford Nanopore Technologies' (ONT's) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.