Single-cell full-length transcriptome of lung cells reveals genetic effects on isoform regulation beyond eQTL

Background : Lung cancer is one of the most prevalent and life-threatening cancers worldwide. Genetic factors contribute to lung cancer risk in smokers and non-smokers, and genome-wide association studies (GWAS) identified > 50 genomic loci from diverse populations. Expression quantitative trait loci (eQTL) and splicing QTL (sQTL) analyses can reveal distinct genetic effects on gene- or transcript isoform-level regulation contributing to GWAS signals to identify target genes and elucidate etiology. However, current sQTL studies using short-read sequencing of bulk tissues fail to capture full-length and novel isoforms or cell-type-specific splicing events contributing to tumorigenesis. Methods : We present an isoform-level lung cell atlas from 129 never-smoking Korean women using single-cell long-read RNA-sequencing. Epithelial cells, where lung cancer originate, were enriched by FACS sorting. Isoform signatures of each lung cell type were identified by differential analysis. We mapped isoform-level QTLs (isoQTLs) using jaxQTL negative binomial model of pseudo-bulk counts. Colocalization and transcriptome-wide association study (TWAS) with GWAS were performed to prioritize susceptibility isoforms. Results: We identified 325,865 full-length isoforms from 360,133 lung cells, where 83% are novel isoforms not annotated in GENCODE v32. We identified isoform-level signatures of 37 lung cell types, where 67.2% of differential isoforms display larger differences than in gene levels. Isoform-QTLs (isoQTLs) identified unreported genes in bulk tissue-based sQTL studies attributed to cell-type-specific and unannotated isoforms. Compared to the barcode-matched short-read expression data, 46% of isoQTLs did not colocalize with eQTLs in the same cell type. Consistently, isoQTLs were distinctly enriched in the functional elements of splicing and post-transcriptional regulation. Colocalization of isoQTLs with lung cancer and trait GWAS signals nominated candidate isoforms, where 69% were previously unreported at the gene level. Moreover, 71% of GWAS-colocalized isoforms were independent from eQTLs, including PPIL6-207 for lung cancer. TWAS of ancestry-matched lung adenocarcinoma identified 12 isoforms from the sub-threshold GWAS regions, which include lineage or cell-type-specific genes. Conclusions: We established an isoform-level lung cell atlas using single-cell long-read sequencing and detected isoQTLs that are cell-type-specific and independent from eQTLs. This isoform-level resource advances our understanding of cell-type-specific isoform regulation and its contribution to lung cancer and diseases.