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      Linnorm: improved statistical analysis for single cell RNA-seq expression data

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          Abstract

          Linnorm is a novel normalization and transformation method for the analysis of single cell RNA sequencing (scRNA-seq) data. Linnorm is developed to remove technical noises and simultaneously preserve biological variations in scRNA-seq data, such that existing statistical methods can be improved. Using real scRNA-seq data, we compared Linnorm with existing normalization methods, including NODES, SAMstrt, SCnorm, scran, DESeq and TMM. Linnorm shows advantages in speed, technical noise removal and preservation of cell heterogeneity, which can improve existing methods in the discovery of novel subtypes, pseudo-temporal ordering of cells, clustering analysis, etc. Linnorm also performs better than existing DEG analysis methods, including BASiCS, NODES, SAMstrt, Seurat and DESeq2, in false positive rate control and accuracy.

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          Accounting for technical noise in single-cell RNA-seq experiments.

          Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.
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            Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq.

            Our understanding of the development and maintenance of tissues has been greatly aided by large-scale gene expression analysis. However, tissues are invariably complex, and expression analysis of a tissue confounds the true expression patterns of its constituent cell types. Here we describe a novel strategy to access such complex samples. Single-cell RNA-seq expression profiles were generated, and clustered to form a two-dimensional cell map onto which expression data were projected. The resulting cell map integrates three levels of organization: the whole population of cells, the functionally distinct subpopulations it contains, and the single cells themselves-all without need for known markers to classify cell types. The feasibility of the strategy was demonstrated by analyzing the transcriptomes of 85 single cells of two distinct types. We believe this strategy will enable the unbiased discovery and analysis of naturally occurring cell types during development, adult physiology, and disease.
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              Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data

              A large number of computational methods have been developed for analyzing differential gene expression in RNA-seq data. We describe a comprehensive evaluation of common methods using the SEQC benchmark dataset and ENCODE data. We consider a number of key features, including normalization, accuracy of differential expression detection and differential expression analysis when one condition has no detectable expression. We find significant differences among the methods, but note that array-based methods adapted to RNA-seq data perform comparably to methods designed for RNA-seq. Our results demonstrate that increasing the number of replicate samples significantly improves detection power over increased sequencing depth.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                15 December 2017
                18 September 2017
                18 September 2017
                : 45
                : 22
                : e179
                Affiliations
                [1 ]Centre for Genomic Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
                [2 ]Department of Health Sciences Research and Center for Individualized Medicine, Mayo Clinic, Scottsdale, AZ 85259, USA
                [3 ]School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
                [4 ]Department of Psychiatry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
                [5 ]State Key Laboratory in Cognitive and Brain Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
                [6 ]Department of Biomedical Informatics, Arizona State University, Scottsdale, AZ 85259, USA
                Author notes
                To whom correspondence should be addressed. Tel: +1 480 301 4644; Fax: +1 480 301 8387; Email: wang.junwen@ 123456mayo.edu
                Article
                gkx828
                10.1093/nar/gkx828
                5727406
                28981748
                ab7e33bc-322c-490c-bf63-eb0d0901a7b2
                © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                : 06 September 2017
                : 31 August 2017
                : 17 July 2017
                Page count
                Pages: 12
                Categories
                Methods Online

                Genetics
                Genetics

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