Mutations in the splicing factor SF3B1 in various cancers have been associated with characteristic alterations in splicing. SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 on a junction level have been observed, these patterns have not been systematically examined on an isoform level. Isoform-level analyses are imperative for building a better understanding of altered splicing in a cell; exon connectivity is best resolved using long reads. As such, we have resequenced cDNA from CLL samples with and without K700E mutation in SF3B1, as well as a normal B cell, with nanopore sequencing technology. By converting changes in current caused by blockage of DNA threading through a nanopore into sequence, nanopore sequencing can sequence complete molecules of DNA that exceed 200kb in length. We have developed a novel workflow to perform isoform-level differential splicing analyses leveraging the full-length transcript data that nanopore affords. We report results from nanopore sequencing data that are concordant with known SF3B1 biology from short read sequencing as well as altered splicing patterns that are only observed definitively using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E identifies a bias toward increased alternative 3' splice sites than alternative 5'. In addition, we observe a relative decrease in the expression of genes with intron retention events in CLL samples compared to B cell. We also discover a previously unreported finding of enrichment in intron retention events in SF3B1WT relative to SF3B1K700E and no enrichment between CLL SF3B1MT and B cell, suggesting that mutated SF3B1 in CLL modifies the intron retention landscape to be more similar to a normal phenotype. Gene ontology analyses reveal enrichment in nuclear export in genes associated with intron retention events, and we postulate that many of these are detained introns of transcripts that are kept in the nucleus. With full-length cDNA sequence, we are also able to better detect premature termination codons and estimate the proportion of transcripts subject to nonsense-mediated mRNA decay. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the utility of nanopore sequencing for cancer and splicing research.

Citation Format: Alison Tang. Full-length characterization of transcript isoforms to investigate cancer-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3443.