RESUMO
The normalization of RNA-seq data is essential for accurate downstream inference, but the assumptions upon which most normalization methods are based are not applicable in the single-cell setting. Consequently, applying existing normalization methods to single-cell RNA-seq data introduces artifacts that bias downstream analyses. To address this, we introduce SCnorm for accurate and efficient normalization of single-cell RNA-seq data.
Assuntos
Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala/normas , RNA/genética , Análise de Sequência de RNA/normas , Análise de Célula Única/normas , Transcriptoma/genética , Interpretação Estatística de Dados , Valores de Referência , SoftwareRESUMO
Oscillatory gene expression is fundamental to development, but technologies for monitoring expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applying Oscope to a number of data sets, we demonstrated its utility and also identified a potential artifact in the Fluidigm C1 platform.
Assuntos
Interpretação Estatística de Dados , Modelos Genéticos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Algoritmos , Análise de Variância , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , SoftwareRESUMO
MOTIVATION: Messenger RNA expression is important in normal development and differentiation, as well as in manifestation of disease. RNA-seq experiments allow for the identification of differentially expressed (DE) genes and their corresponding isoforms on a genome-wide scale. However, statistical methods are required to ensure that accurate identifications are made. A number of methods exist for identifying DE genes, but far fewer are available for identifying DE isoforms. When isoform DE is of interest, investigators often apply gene-level (count-based) methods directly to estimates of isoform counts. Doing so is not recommended. In short, estimating isoform expression is relatively straightforward for some groups of isoforms, but more challenging for others. This results in estimation uncertainty that varies across isoform groups. Count-based methods were not designed to accommodate this varying uncertainty, and consequently, application of them for isoform inference results in reduced power for some classes of isoforms and increased false discoveries for others. RESULTS: Taking advantage of the merits of empirical Bayesian methods, we have developed EBSeq for identifying DE isoforms in an RNA-seq experiment comparing two or more biological conditions. Results demonstrate substantially improved power and performance of EBSeq for identifying DE isoforms. EBSeq also proves to be a robust approach for identifying DE genes. AVAILABILITY AND IMPLEMENTATION: An R package containing examples and sample datasets is available at http://www.biostat.wisc.edu/kendzior/EBSEQ/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Perfilação da Expressão Gênica/métodos , Isoformas de RNA/metabolismo , Análise de Sequência de RNA/métodos , Teorema de Bayes , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Genoma , Modelos Estatísticos , RNA Mensageiro/metabolismo , SoftwareRESUMO
MOTIVATION: RNA-Seq is a promising new technology for accurately measuring gene expression levels. Expression estimation with RNA-Seq requires the mapping of relatively short sequencing reads to a reference genome or transcript set. Because reads are generally shorter than transcripts from which they are derived, a single read may map to multiple genes and isoforms, complicating expression analyses. Previous computational methods either discard reads that map to multiple locations or allocate them to genes heuristically. RESULTS: We present a generative statistical model and associated inference methods that handle read mapping uncertainty in a principled manner. Through simulations parameterized by real RNA-Seq data, we show that our method is more accurate than previous methods. Our improved accuracy is the result of handling read mapping uncertainty with a statistical model and the estimation of gene expression levels as the sum of isoform expression levels. Unlike previous methods, our method is capable of modeling non-uniform read distributions. Simulations with our method indicate that a read length of 20-25 bases is optimal for gene-level expression estimation from mouse and maize RNA-Seq data when sequencing throughput is fixed.