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      diffcyt: Differential discovery in high-dimensional cytometry via high-resolution clustering

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          Abstract

          High-dimensional flow and mass cytometry allow cell types and states to be characterized in great detail by measuring expression levels of more than 40 targeted protein markers per cell at the single-cell level. However, data analysis can be difficult, due to the large size and dimensionality of datasets as well as limitations of existing computational methods. Here, we present diffcyt, a new computational framework for differential discovery analyses in high-dimensional cytometry data, based on a combination of high-resolution clustering and empirical Bayes moderated tests adapted from transcriptomics. Our approach provides improved statistical performance, including for rare cell populations, along with flexible experimental designs and fast runtimes in an open-source framework.

          Abstract

          Lukas Weber et al. present diffcyt, an open-source computational framework for differential analysis of high-dimensional flow and mass cytometry data. The diffcyt framework uses high-resolution clustering and empirical Bayes moderated tests to provide improved statistical performance, flexible experimental designs, and fast runtimes.

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          Most cited references44

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          limma powers differential expression analyses for RNA-sequencing and microarray studies

          limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
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            edgeR: a Bioconductor package for differential expression analysis of digital gene expression data

            Summary: It is expected that emerging digital gene expression (DGE) technologies will overtake microarray technologies in the near future for many functional genomics applications. One of the fundamental data analysis tasks, especially for gene expression studies, involves determining whether there is evidence that counts for a transcript or exon are significantly different across experimental conditions. edgeR is a Bioconductor software package for examining differential expression of replicated count data. An overdispersed Poisson model is used to account for both biological and technical variability. Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference. The methodology can be used even with the most minimal levels of replication, provided at least one phenotype or experimental condition is replicated. The software may have other applications beyond sequencing data, such as proteome peptide count data. Availability: The package is freely available under the LGPL licence from the Bioconductor web site (http://bioconductor.org). Contact: mrobinson@wehi.edu.au
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              Integrating single-cell transcriptomic data across different conditions, technologies, and species

              Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple data sets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq data sets based on common sources of variation, enabling the identification of shared populations across data sets and downstream comparative analysis. We apply this approach, implemented in our R toolkit Seurat (http://satijalab.org/seurat/), to align scRNA-seq data sets of peripheral blood mononuclear cells under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across data sets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq data sets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution.
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                Author and article information

                Contributors
                mark.robinson@imls.uzh.ch
                Journal
                Commun Biol
                Commun Biol
                Communications Biology
                Nature Publishing Group UK (London )
                2399-3642
                14 May 2019
                14 May 2019
                2019
                : 2
                : 183
                Affiliations
                [1 ]ISNI 0000 0004 1937 0650, GRID grid.7400.3, Institute of Molecular Life Sciences, , University of Zurich, ; CH-8057 Zurich, Switzerland
                [2 ]ISNI 0000 0004 1937 0650, GRID grid.7400.3, SIB Swiss Institute of Bioinformatics, , University of Zurich, ; CH-8057 Zurich, Switzerland
                [3 ]ISNI 0000 0004 0374 1269, GRID grid.417570.0, Present Address: F. Hoffmann-La Roche AG, ; CH-4070 Basel, Switzerland
                [4 ]ISNI 0000 0001 2110 3787, GRID grid.482245.d, Present Address: Friedrich Miescher Institute for Biomedical Research and SIB Swiss Institute of Bioinformatics, ; CH-4058 Basel, Switzerland
                Author information
                http://orcid.org/0000-0002-3282-1730
                http://orcid.org/0000-0003-3833-2169
                http://orcid.org/0000-0002-3048-5518
                Article
                415
                10.1038/s42003-019-0415-5
                6517415
                31098416
                925b3839-02b6-40d6-8d59-e2723eddf3a1
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 20 December 2018
                : 5 April 2019
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                © The Author(s) 2018

                statistical methods,cell signalling,software
                statistical methods, cell signalling, software

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