Differential expression (DE) analysis of RNA-seq data still poses inferential challenges, such as handling of transcripts characterized by low expression levels. In this study, researchers from Kansas State University used a plasmode-based approach to assess the relative performance of alternative inferential strategies on RNA-seq transcripts, with special emphasis on transcripts characterized by a small number of read counts, so-called low-count transcripts, as motivated by an ecological application in prairie grasses.
Focused on low-count transcripts, they compare alternative ad-hoc data filtering techniques commonly used in RNA-seq pipelines and assess the inferential performance of recently developed statistical methods for DE analysis, namely DESeq2 and edgeR robust. These methods attempt to overcome the inherently noisy behavior of low-count transcripts by either shrinkage or differential weighting of observations, respectively.
Both DE methods seemed to properly control family-wise type 1 error on low-count transcripts, whereas edgeR robust showed greater power and DESeq2 showed greater precision and accuracy. However, specification of the degree of freedom parameter under edgeR robust had a non-trivial impact on inference and should be handled carefully. When properly specified, both DE methods showed overall promising inferential performance on low-count transcripts, suggesting that ad-hoc data filtering steps at arbitrary expression thresholds may be unnecessary. A note of caution is in order regarding the approximate nature of DE tests under both methods.
Frequency of transcripts declared differentially expressed (DE) using edgeR robust and DESeq2.
Venn diagrams of all transcripts and of low-count transcripts declared DE using edgeR robust (with degrees of freedom specified based on the corresponding estimate obtained using classical edgeR software) and DESeq2 on the complete dataset (i.e. no data filtering) or following data filtering based on a reads-present (RP) criterion or a counts per million (CPM) criterion