RNA-Seq has become a key technology in transcriptome studies because it can quantify overall expression levels and the degree of alternative splicing for each gene simultaneously. To interpret high-throughout transcriptome profiling data, functional enrichment analysis is critical. However, existing functional analysis methods can only account for differential expression, leaving differential splicing out altogether.

In this work, we present a novel approach to derive biological insight by integrating differential expression and splicing from RNA-Seq data with functional gene set analysis. This approach designated SeqGSEA, uses count data modelling with negative binomial distributions to first score differential expression and splicing in each gene, respectively, followed by two strategies to combine the two scores for integrated gene set enrichment analysis. Method comparison results and biological insight analysis on an artificial data set and three real RNA-Seq data sets indicate that our approach outperforms alternative analysis pipelines and can detect biological meaningful gene sets with high confidence, and that it has the ability to determine if transcription or splicing is their predominant regulatory mechanism.

By integrating differential expression and splicing, the proposed method SeqGSEA is particularly useful for efficiently translating RNA-Seq data to biological discoveries.