Statistical approaches for differential expression analysis in single-cell RNA sequencing studies

Single-cell RNA-sequencing (scRNA-seq) is a recent high-throughput sequencing technique for studying gene expressions at the cell level. Differential Expression (DE) analysis is a major downstream analysis of scRNA-seq data. DE analysis the in presence of noises from different sources remains a key challenge in scRNA-seq. Earlier practices for addressing this involved borrowing methods from bulk RNA-seq, which are based on non-zero differences in average expressions of genes across cell populations. Later, several methods specifically designed for scRNA-seq were developed. To provide guidance on choosing an appropriate tool or developing a new one, it is necessary to comprehensively study the performance of DE analysis methods.

University of Louisville researchers provide a review and classification of different DE approaches adapted from bulk RNA-seq practice as well as those specifically designed for scRNA-seq. The researchers also evaluate the performance of 19 widely used methods in terms of 13 performance metrics on 11 real scRNA-seq datasets. Their findings suggest that some bulk RNA-seq methods are quite competitive with the single-cell methods and their performance depends on the underlying models, DE test statistic(s), and data characteristics. Further, it is difficult to obtain the method which will be best-performing globally through individual performance criterion. However, the multi-criteria and combined-data analysis indicates that DECENT and EBSeq are the best options for DE analysis. The results also reveal the similarities among the tested methods in terms of detecting common DE genes. This evaluation provides proper guidelines for selecting the proper tool which performs best under particular experimental settings in the context of the scRNA-seq.

Schematic Representation of Classification of scRNA-seq DE Methods and Tools

Schematic overview illustrating the breakup of the methods that can be adapted from the RNA-seq practice to fit scRNA-seq data (Class I) as well as those specifically designed for single-cell (Classes II, III) based on the different distribution models that they fit. Different example tools belonging to each category are listed in pink color boxes. Methods use the external RNA spike-ins and Parametric approaches but can handle multi-modality of the data.