Version Compatibility

Reference examples tested with: edgeR 4.0+, limma 3.58+, statmod 1.5+ (for voom internals), tximport 1.30+ (for catchSalmon path)

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.

edgeR Basics

"Find differentially expressed genes between conditions" -> Fit a negative-binomial GLM per gene with empirical-Bayes-moderated dispersions, test coefficients with the quasi-likelihood F-test (proper finite-sample FPR control), and report ranked DE lists.

The Single Most Important Modern Insight -- edgeR v4 changed the QL framework and legacy=FALSE is now default

Chen, Chen, Lun, Baldoni, Smyth (2025) Nucleic Acids Res 53(2):gkaf018 introduced a bias-corrected adjusted profile likelihood (APL) for dispersion estimation that handles small/zero counts properly, and reworked the QL framework. glmQLFit() in v4 takes legacy=FALSE by default. Old (v3) pipelines that worked perfectly in 2023 now produce subtly different numbers in 2025 -- not wrong, just different. If a tutorial result doesn't match, check which version was used; set legacy=TRUE to reproduce v3 exactly OR (preferred) re-run with v4 defaults and accept the new -- better -- numbers.

Two other v4 changes worth knowing: (1) calcNormFactors() is deprecated in favor of normLibSizes() (same function, new name); (2) method='TMM' remains the documented default; method='TMMwsp' (TMM with singleton pairing) is an alternative introduced for samples with many zeros and is the preferred choice for sparse / low-count data. Pass method= explicitly for reproducibility. Old names still work, so old scripts run, but new code should use the new names.