Quantitative measurement of full-length transcript isoforms reveals precise regulation of splicing in human heart cells

Alternative splicing generates various RNA isoforms that form the complexity of eukaryotic transcriptomes, and mis-splicing of RNA isoforms are involved in numerous diseases including cardiovascular disease. Yet, an accurate and quantitative measurement of human isoforms at genome-wide level is still missing. Here, using Oxford Nanopore (ONT) sequencing, we developed high-throughput experimental and computational analysis pipelines that accurately quantify and compare genome-wide full-length isoform expression. The data is used to generate a global view of human full-length transcriptome including numerous novel transcript isoforms. This wasn't possible by short read sequencing based technology due to its length limitation. We also conducted unbiased co-association test for distant and adjacent exon pairs. Using our pipelines, we are able to conduct, for the first time, differential expression tests of full-length isoforms between samples. We applied this to iPSC-CMs containing RBM20 WT and R634Q mutation known to be associated with dilated cardiomyopathy (DCM), and pinpointed the full-length isoforms which are mis-spliced in RBM20 mutant. The fact that for the mis-spliced genes, only specific but not all isoforms are differentially expressed suggests that splicing is precisely regulated at the isoform level.