Orchestrating single-cell analysis with Bioconductor.
Ontology highlight
ABSTRACT:
SUBMITTER: Amezquita RA
PROVIDER: S-EPMC7358058 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Single-cell RNA sequencing (scRNA-seq) is widely used to profile the transcriptome of individual cells. This provides biological resolution that cannot be matched by bulk RNA sequencing, at the cost of increased technical noise and data complexity. The differences between scRNA-seq and bulk RNA-seq data mean that the analysis of the former cannot be performed by recycling bioinformatics pipelines for the latter. Rather, dedicated single-cell methods are required at various steps to exploit the cellular resolution while accounting for technical noise. This article describes a computational workflow for low-level analyses of scRNA-seq data, based primarily on software packages from the open-source Bioconductor project. It covers basic steps including quality control, data exploration and normalization, as well as more complex procedures such as cell cycle phase assignment, identification of highly variable and correlated genes, clustering into subpopulations and marker gene detection. Analyses were demonstrated on gene-level count data from several publicly available datasets involving haematopoietic stem cells, brain-derived cells, T-helper cells and mouse embryonic stem cells. This will provide a range of usage scenarios from which readers can construct their own analysis pipelines.
| S-EPMC5112579 | biostudies-literature
Project description:BACKGROUND:Single-cell RNA sequencing (scRNA-seq) enables the high-throughput quantification of transcriptional profiles in single cells. In contrast to bulk RNA-seq, additional preprocessing steps such as cell barcode identification or unique molecular identifier (UMI) deconvolution are necessary for preprocessing of data from single cell protocols. R packages that can easily preprocess data and rapidly visualize quality metrics and read alignments for individual cells across multiple samples or runs are still lacking. RESULTS:Here we present scruff, an R/Bioconductor package that preprocesses data generated from the CEL-Seq or CEL-Seq2 protocols and reports comprehensive data quality metrics and visualizations. scruff rapidly demultiplexes, aligns, and counts the reads mapped to genome features with deduplication of unique molecular identifier (UMI) tags. scruff also provides novel and extensive functions to visualize both pre- and post-alignment data quality metrics for cells from multiple experiments. Detailed read alignments with corresponding UMI information can be visualized at specific genome coordinates to display differences in isoform usage. The package also supports the visualization of quality metrics for sequence alignment files for multiple experiments generated by Cell Ranger from 10X Genomics. scruff is available as a free and open-source R/Bioconductor package. CONCLUSIONS:scruff streamlines the preprocessing of scRNA-seq data in a few simple R commands. It performs data demultiplexing, alignment, counting, quality report and visualization systematically and comprehensively, ensuring reproducible and reliable analysis of scRNA-seq data.
| S-EPMC6498700 | biostudies-literature
Project description:Single-cell RNA sequencing (scRNA-seq) technology allows researchers to profile the transcriptomes of thousands of cells simultaneously. Protocols that incorporate both designed and random barcodes have greatly increased the throughput of scRNA-seq, but give rise to a more complex data structure. There is a need for new tools that can handle the various barcoding strategies used by different protocols and exploit this information for quality assessment at the sample-level and provide effective visualization of these results in preparation for higher-level analyses. To this end, we developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols that include CEL-seq, MARS-seq, Chromium 10X, Drop-seq and Smart-seq. scPipe produces a count matrix that is essential for downstream analysis along with an HTML report that summarises data quality. These results can be used as input for downstream analyses including normalization, visualization and statistical testing. scPipe performs this processing in a few simple R commands, promoting reproducible analysis of single-cell data that is compatible with the emerging suite of open-source scRNA-seq analysis tools available in R/Bioconductor and beyond. The scPipe R package is available for download from https://www.bioconductor.org/packages/scPipe.
| S-EPMC6105007 | biostudies-literature
Project description:Novel single-cell transcriptome sequencing assays allow researchers to measure gene expression levels at the resolution of single cells and offer the unprecendented opportunity to investigate at the molecular level fundamental biological questions, such as stem cell differentiation or the discovery and characterization of rare cell types. However, such assays raise challenging statistical and computational questions and require the development of novel methodology and software. Using stem cell differentiation in the mouse olfactory epithelium as a case study, this integrated workflow provides a step-by-step tutorial to the methodology and associated software for the following four main tasks: (1) dimensionality reduction accounting for zero inflation and over dispersion and adjusting for gene and cell-level covariates; (2) cell clustering using resampling-based sequential ensemble clustering; (3) inference of cell lineages and pseudotimes; and (4) differential expression analysis along lineages.
| S-EPMC5558107 | biostudies-literature
Project description:Cell differentiation processes are achieved through a continuum of hierarchical intermediate cell states that might be captured by single-cell RNA seq. Existing computational approaches for the assessment of cell-state hierarchies from single-cell data can be formalized under a general framework composed of (i) a metric to assess cell-to-cell similarities (with or without a dimensionality reduction step) and (ii) a graph-building algorithm (optionally making use of a cell clustering step). The Sincell R package implements a methodological toolbox allowing flexible workflows under such a framework. Furthermore, Sincell contributes new algorithms to provide cell-state hierarchies with statistical support while accounting for stochastic factors in single-cell RNA seq. Graphical representations and functional association tests are provided to interpret hierarchies. The functionalities of Sincell are illustrated in a real case study, which demonstrates its ability to discriminate noisy from stable cell-state hierarchies.Sincell is an open-source R/Bioconductor package available at http://bioconductor.org/packages/sincell. A detailed manual and a vignette are provided with the package.antonio.rausell@isb-sib.chSupplementary data are available at Bioinformatics online.
| S-EPMC4595899 | biostudies-other
Project description:Mitochondria are highly dynamic organelles, the location, size and distribution of which are controlled by a family of proteins that modulate mitochondrial fusion and fission. Recent evidence indicates that mitochondrial morphology is crucial for cell physiology, as changes in mitochondrial shape have been linked to neurodegeneration, calcium signalling, lifespan and cell death. Because immune cells contain few mitochondria, these organelles have been considered to have only a marginal role in this physiological context-which is conversely well characterized from the point of view of signalling. Nevertheless, accumulating evidence shows that mitochondrial dynamics have an impact on the migration and activation of immune cells and on the innate immune response. Here, we discuss the roles of mitochondrial dynamics in cell pathophysiology and consider how studying dynamics in the context of the immune system could increase our knowledge about the role of dynamics in key signalling cascades.
| S-EPMC2933866 | biostudies-other
Project description:BackgroundNetwork-based analysis of gene expression through co-expression networks can be used to investigate modular relationships occurring between genes performing different biological functions. An extended description of each of the network modules is therefore a critical step to understand the underlying processes contributing to a disease or a phenotype. Biological integration, topology study and conditions comparison (e.g. wild vs mutant) are the main methods to do so, but to date no tool combines them all into a single pipeline.ResultsHere we present GWENA, a new R package that integrates gene co-expression network construction and whole characterization of the detected modules through gene set enrichment, phenotypic association, hub genes detection, topological metric computation, and differential co-expression. To demonstrate its performance, we applied GWENA on two skeletal muscle datasets from young and old patients of GTEx study. Remarkably, we prioritized a gene whose involvement was unknown in the muscle development and growth. Moreover, new insights on the variations in patterns of co-expression were identified. The known phenomena of connectivity loss associated with aging was found coupled to a global reorganization of the relationships leading to expression of known aging related functions.ConclusionGWENA is an R package available through Bioconductor ( https://bioconductor.org/packages/release/bioc/html/GWENA.html ) that has been developed to perform extended analysis of gene co-expression networks. Thanks to biological and topological information as well as differential co-expression, the package helps to dissect the role of genes relationships in diseases conditions or targeted phenotypes. GWENA goes beyond existing packages that perform co-expression analysis by including new tools to fully characterize modules, such as differential co-expression, additional enrichment databases, and network visualization.
| S-EPMC8152313 | biostudies-literature
Project description:Single-cell RNA sequencing is fast becoming one the standard method for gene expression measurement, providing unique insights into cellular processes. A number of methods, based on general dimensionality reduction techniques, have been suggested to help infer and visualise the underlying structure of cell populations from single-cell expression levels, yet their models generally lack proper biological grounding and struggle at identifying complex differentiation paths.Here we introduce cellTree: an R/Bioconductor package that uses a novel statistical approach, based on document analysis techniques, to produce tree structures outlining the hierarchical relationship between single-cell samples, while identifying latent groups of genes that can provide biological insights.With cellTree, we provide experimentalists with an easy-to-use tool, based on statistically and biologically-sound algorithms, to efficiently explore and visualise single-cell RNA data. The cellTree package is publicly available in the online Bionconductor repository at: http://bioconductor.org/packages/cellTree/ .
| S-EPMC5020541 | biostudies-literature
Project description:Inflammation is a key component of fibrosis, however, the immune processes that orchestrate kidney fibrosis are poorly understood. By performing single-cell sequencing of a mouse model of kidney fibrosis, we identified a subset of kidney tubule cells with a profibrotic-inflammatory phenotype, characterized by the expression of cytokines and chemokines associated with immune cell recruitment. Receptor-ligand interaction analysis and experimental validation indicated that CXCL1 secreted by profibrotic tubules recruits CXCR2+ basophils. In mice, these basophils were the key source of IL-6 and were responsible for the recruitment of Th17 cells. Genetic deletion or antibody-based depletion of basophils resulted in reduced renal fibrosis. Human kidney single-cell, bulk gene expression, and immunostaining results validated the role of basophils in patients with kidney fibrosis. Collectively, these studies identify basophils as key contributors to the development of renal fibrosis and suggest that targeting these cells may be a useful strategy for the management of chronic kidney disease.
2022-03-07 | GSE182256 | GEO
Project description: Not available
| S-EPMC2323405 | biostudies-literature