Benchmarking algorithms for pathway activity transformation of single-cell RNA-seq data.

Biological pathway analysis provides new insights for cell clustering and functional annotation from single-cell RNA sequencing (scRNA-seq) data. Many pathway analysis algorithms have been developed to transform gene-level scRNA-seq data into functional gene sets representing pathways or biological processes. Here, we collected seven widely-used pathway activity transformation algorithms and 32 available datasets based on 16 scRNA-seq techniques. We proposed a comprehensive framework to evaluate their accuracy, stability and scalability. The assessment of scRNA-seq preprocessing showed that cell filtering had the less impact on scRNA-seq pathway analysis, while data normalization of sctransform and scran had a consistent well impact across all tools. We found that Pagoda2 yielded the best overall performance with the highest accuracy, scalability, and stability. Meanwhile, the tool PLAGE exhibited the highest stability, as well as moderate accuracy and scalability.