A systematic evaluation of single cell RNA-seq analysis pipelines

• DOI: 10.1038/s41467-019-12266-7
• Publication Date: 2019-10-11T10:04:04Z
• Authors: Beate Vieth; Swati Parekh; Christoph Ziegenhain; Wolfgang Enard; Ines Hellmann
• Keywords: Electronic Data Processing; Science; Q; 0206 medical engineering; Chromosome Mapping; 02 engineering and technology; Article; Mice; Animals; Computer Simulation; RNA-Seq; Single-Cell Analysis

AbstractThe recent rapid spread of single cell RNA sequencing (scRNA-seq) methods has created a large variety of experimental and computational pipelines for which best practices have not yet been established. Here, we use simulations based on five scRNA-seq library protocols in combination with nine realistic differential expression (DE) setups to systematically evaluate three mapping, four imputation, seven normalisation and four differential expression testing approaches resulting in ~3000 pipelines, allowing us to also assess interactions among pipeline steps. We find that choices of normalisation and library preparation protocols have the biggest impact on scRNA-seq analyses. Specifically, we find that library preparation determines the ability to detect symmetric expression differences, while normalisation dominates pipeline performance in asymmetric DE-setups. Finally, we illustrate the importance of informed choices by showing that a good scRNA-seq pipeline can have the same impact on detecting a biological signal as quadrupling the sample size.