Quantification method affects replicability of eQTL analysis, colocalization, and TWAS

Expression quantitative trait loci (eQTL) mapping, colocalization, and transcriptome-wide association studies (TWAS) are widely used to contextualize GWAS loci, yet the impact of upstream RNA-seq processing choices on these analyses remains critically unexplored. Two poorly characterized decisions include the transcriptomic reference annotation used for quantification of short-read RNA-sequencing samples, which defines transcript composition and structure, and the quantification method itself. Through a comprehensive analysis spanning 48 tissues in GTEx and seven widely studied and diverse phenotypes, here we find that these choices substantially affect eQTL detection and gene expression prediction with significant downstream impact on colocalization and TWAS results. Concordant results across pipelines are further enriched for longer genes, genes with more isoforms, and greater expression heritability. Our findings demonstrate that seemingly minor methodological decisions substantially affect these common analyses, highlighting the need for standardized practices to ensure reproducible genetic association studies.