Robustly detecting differential expression in RNA sequencing data using observation weights


Xiaobei Zhou1,2, Helen Lindsay1,2, Mark D. Robinson1,2,*

1 Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190 CH-8057 Zurich, Switzerland
2 SIB Swiss Institute of Bioinformatics, University of Zurich, Zurich, Switzerland

* Corresponding authors: mark.robinson@imls.uzh.ch.

Summary

A popular approach for comparing gene expression levels between (replicated) conditions of RNA sequencing data relies on counting reads that map to features of interest. Within such count-based methods, many flexible and advanced statistical approaches now exist and offer the ability to adjust for covariates (e.g. batch effects). Often, these methods include some sort of sharing of information across features to improve inferences in small samples. It is important to achieve an appropriate tradeoff between statistical power and protection against outliers. Here, we study the robustness of existing approaches for count-based differential expression analysis and propose a new strategy based on observation weights that can be used within existing software frameworks. The results suggest that outliers can have a global effect on differential analyses. We demonstrate the effectiveness of our new approach with real data and simulated data that reflects many of the properties of such datasets (e.g., dispersion-mean trend) and develop an extensible framework for extensive testing of current and future methods. In addition, we explore the origin of such outliers, in some cases highlighting additional biology within the experiment.


Supplementary Data, R Code for simulations and analyses: