Long-read sequencing reveals novel isoform-specific eQTLs and regulatory mechanisms of isoform expression

Genetic variations linked to changes in gene expression are known as expression quantitative loci (eQTLs). The identification of eQTLs provides a profound understanding of the mechanisms governing gene expression. However, prior studies have primarily utilized short-read sequencing techniques, and the analysis of eQTLs on isoforms has been relatively limited. In this study, we employed long-read sequencing technology (Oxford Nanopore) on B cells from 67 healthy Japanese individuals to explore genetic variations associated with isoform expression levels, referred to as isoform eQTLs (ieQTLs). Our analysis revealed 33,928 ieQTLs, with 69.0% remaining undetected by a gene-level analysis. Additionally, we identified ieQTLs that have significantly different effects on isoform expression levels within a gene. A functional feature analysis demonstrated a significant enrichment of ieQTLs at splice sites and specific histone marks, such as H3K36me3, H3K4me1, H3K4me3, and H3K79me3. Through an experimental validation using genome editing, we observed that a distant genomic region can modulate isoform-specific expression. Moreover, an ieQTL analysis and minigene splicing assays unveiled functionally crucial variants in splicing, which software-based predictions failed to anticipate. A comparison with GWAS data revealed a higher number of colocalizations between ieQTLs and GWAS findings compared to gene eQTLs. These findings highlight the substantial contribution of ieQTLs identified through long-read analysis in our understanding of the functional implications of genetic variations and the regulatory mechanisms governing isoforms.