A systematic evaluation of single cell RNA-seq analysis pipelines

Beate Vieth, Swati Parekh, Christoph Ziegenhain, Wolfgang Enard and Ines Hellmann ()
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Beate Vieth: Ludwig-Maximilians University
Swati Parekh: Max Planck Institute for Biology of Ageing
Christoph Ziegenhain: Karolinska Institutet
Wolfgang Enard: Ludwig-Maximilians University
Ines Hellmann: Ludwig-Maximilians University

Nature Communications, 2019, vol. 10, issue 1, 1-11

Abstract: Abstract The 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.

Date: 2019
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DOI: 10.1038/s41467-019-12266-7

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