Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3

Background Clinical sequencing has traditionally focused on genomic DNA through the use of targeted panels and exome sequencing, rather than investigating the potential transcriptomic consequences of disease-associated variants. RNA sequencing has recently been shown to be an effective additional tool for identifying disease-causing variants. We here use targeted long-read genome and transcriptome sequencing to efficiently and economically identify molecular consequences of a rare, disease-associated variant in hypertrophic cardiomyopathy (HCM). Methods and Results Our study, which employed both Pacific Biosciences SMRT sequencing and Oxford Nanopore Technologies MinION sequencing, as well as two RNA targeting strategies, identified alternatively-spliced isoforms that resulted from a splice-site variant containing allele in HCM. These included a predicted in-frame exon-skipping event, as well as an abundance of additional isoforms with unexpected intron-inclusion, exon-extension, and pseudo-exon events. The use of long-read RNA sequencing allowed us to not only investigate full length alternatively-spliced transcripts but also to phase them back to the variant-containing allele. Conclusions We suggest that targeted, long-read RNA sequencing in conjunction with genome sequencing may provide additional molecular evidence of disease for rare or de novo variants in cardiovascular disease, as well as providing new information about the consequence of these variants on downstream RNA and protein expression.