Subirana-Granes, M.; Nandi, S.; Zhang, H.; Chikina, M.; Pividori, M.

Gene expression analysis has long been fundamental for elucidating molecular pathways and gene-disease relationships, but traditional single-gene approaches cannot capture the coordinated regulatory networks underlying complex phenotypes; although unsupervised matrix factorization methods (e.g., PCA, NMF) reveal coexpression patterns, they lack the ability to incorporate prior biological knowledge and often struggle with interpretability and technical noise correction. Semi-supervised strategies such as PLIER have improved interpretability by integrating pathway annotations during latent variable extraction, yet the original PLIER implementation is prohibitively slow and memory-intensive, making it impractical for modern large-scale resources like ARCHS4 or recount3. Here, we introduce PLIERv2, which overcomes these constraints through a two-phase algorithmic design (an unsupervised \"PLIERbase\" initialization followed by a \"PLIERfull\" regression that incorporates priors via glmnet), rigorous internal cross-validation to tune regularization parameters for each latent variable, and efficient on-disk data handling using memory-mapped matrices from the bigstatsr package. Benchmarking on GTEx, recount2, and ARCHS4 demonstrates that PLIERv2 achieves 7x-41x speedups over PLIERv1, succeeds in modeling hundreds of thousands of samples that PLIERv1 cannot handle, and maintains or improves biological specificity of latent variables as shown by tissue-alignment and pathway enrichment analyses. By filling the gap in scalable, biologically informed latent variable extraction, PLIERv2 enables comprehensive analysis of modern transcriptomic compendia and paves the way for deeper insights into gene regulatory networks and downstream applications in translational genomics.