Introduction

Normalization of RNA-sequencing (RNA-seq) data has proven essential to ensure accurate inference of expression levels. Here, we show that usual normalization approaches mostly account for sequencing depth and fail to correct for library preparation and other more complex unwanted technical effects. We evaluate the performance of the External RNA Control Consortium (ERCC) spike-in controls and investigate the possibility of using them directly for normalization. We show that the spike-ins are not reliable enough to be used in standard global-scaling or regression-based normalization procedures. We propose a normalization strategy, called remove unwanted variation (RUV), that adjusts for nuisance technical effects by performing factor analysis on suitable sets of control genes (e.g., ERCC spike-ins) or samples (e.g., replicate libraries). Our approach leads to more accurate estimates of expression fold-changes and tests of differential expression compared to state-of-the-art normalization methods. In particular, RUV promises to be valuable for large collaborative projects involving multiple laboratories, technicians, and/or sequencing platforms.

Publications

  1. Normalization of RNA-seq data using factor analysis of control genes or samples.
    Cite this
    Risso D, Ngai J, Speed TP, Dudoit S, 2014-09-01 - Nature biotechnology

Credits

  1. Davide Risso
    Developer

    Department of Statistics, University of California, United States of America

  2. John Ngai
    Developer

    1] Department of Molecular and Cell Biology, University of California, United States of America

  3. Terence P Speed
    Developer

    1] Department of Statistics, University of California, United States of America

  4. Sandrine Dudoit
    Investigator

    1] Department of Statistics, University of California, United States of America

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Summary
AccessionBT001707
Tool TypeApplication
Category
PlatformsLinux/Unix
TechnologiesR
User InterfaceTerminal Command Line
Download Count0
Country/RegionUnited States of America
Submitted BySandrine Dudoit