Comparison of alternative control types in time-course RNA-seq experiments

RNA-seq is a widely used approach to investigate gene expression and increasingly is used in time course studies to characterize transcriptomic changes over time. Two primary options are available as controls in time course experiments: samples collected at the first sampling time are used as controls (temporal control, TC) and samples collected in parallel at each individual sampling time are used as controls (biological control, BC). While both approaches are used in experimental studies, no analyses have been performed to date directly comparing effects of control type choices on identifying differentially expressed gene (DEG) and subsequent functional analysis. In the current study, we compare experimental results using these different control types for time course RNA-seq drought stress experiments in two wild grass species in the genus Paspalum. Our results showed BC assemblies obtained higher number of loci in both species. Moreover, the number of DEGs increased with increasing stress and then decreased dramatically at the recovery timepoint. High similarity in functional annotation of total assembled loci was observed, ranging between 90% and 96%. Expression levels of the same DEGs were highly correlated between control types in both species, ranging from r = 0.653 to r = 0.852. We also observed rank orders of shared enriched GO term lists using the two different control types were highly similar. Collectively, our findings suggest similar results in differential gene expression and functional annotation between control types. The ultimate choice of control type will rely on the experimental length and organism type. Labor time and sequencing costs are additional factors under consideration.