Single-cell expression quantitative trait loci (sc-eQTL) studies have advanced our understanding of how genetic variation influences gene expression in a cell-type-specific manner. However, to date, these have focused on gene-level signals for each cell. Alternative splicing, which generates multiple RNA isoforms from a single gene, is one such mechanism that can further modulate RNA products and thus protein sequences. Genetic effects on splicing (isoform-specific expression) can thus offer critical insights into genotype–phenotype relationships by revealing regulatory links invisible to standard gene-level eQTL analyses.
As part of the TenK10K project, we generated long-read single-cell RNA-seq data from >400,000 immune cells from 372 individuals, along with matched whole genome sequencing and short-read single-cell RNA data. Through the implementation of a novel splice-QTL method, developed on top of SAIGE-QTL, we were able to map isoform- and splice-specific eQTL across 28 cell types and transient cell states. We identified a total of 4,515 isoform-specific eQTLs, of which 38 showed isoform-specific regulatory effects in opposite directions, underscoring a complexity missed by gene-level analyses. For instance, we identified a cell-type specific sQTL in the PMF1 gene, where the variant 1:156236330:G>A in central memory CD4+ T cells drives opposing expression of two isoforms: upregulation of ENST00000368279 and downregulation of the canonical ENST00000368277. These isoforms encode structurally distinct proteins, the latter producing a full-length transcriptional coactivator, while the former results in a truncated version lacking key functional domains. This splicing event has been previously implicated in COVID-19 susceptibility, pointing to relevance in immune-related diseases. Notably, this regulatory effect is not detectable at the gene level, underscoring the value of isoform-resolved QTL mapping. This work underscores the value of long-read transcriptomic profiling in capturing the full complexity of genetic regulation, ultimately enhancing our understanding of how genetic variation shapes complex human traits.