E-MTAB-7334
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.
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.