Version Compatibility

Reference examples tested with: DESeq2 1.42+, edgeR 4.0+, limma 3.58+, splines (base R), maSigPro 1.74+, ImpulseDE2 1.10+ (Bioconductor archive; verify availability), variancePartition / dream 1.32+, Mfuzz 2.62+, TCseq 1.26+, ggplot2 3.5+, pheatmap 1.0+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Time-Series Differential Expression

"Find genes that change over time" -> Define what "change" means -- any non-zero time effect (LRT), a smooth nonlinear trend (splines), a transient impulse (ImpulseDE2), or differing trajectories between groups (interaction LRT) -- and choose the model that asks that specific question while handling repeated-measures correctly.

The Single Most Important Modern Insight -- Most dedicated time-course tools UNDERPERFORM pairwise + LRT on short series

Spies, Renz, Beyer, Ciaudo 2019 Brief Bioinform 20:288 benchmarked dedicated time-course tools (ImpulseDE2, splineTC, maSigPro, EBSeqHMM, TimeReg) against naive DESeq2/edgeR pairwise comparisons + LRT for omnibus, on simulated and real time-courses. Finding: on short series (<8 time points), naive pairwise pattern-of-significance OUTPERFORMS dedicated TC tools because of high false-positive rates in the latter. The exception is ImpulseDE2, which holds up better than the others -- IF its impulse assumption (rise-then-plateau or fall-then-plateau) actually fits the biology.

For most experimental time courses (3-6 time points, common in pharmacology and developmental biology), the right tool is DESeq2 with test='LRT' and a sensible reduced model. Reserve splines for >5 evenly-spaced time points; reserve ImpulseDE2 for monotonic-then-saturating dynamics; reserve DREAM for repeated measures.