Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 have been identified with short-read RNA sequencing, a critical barrier in understanding the functional consequences of these splicing changes is that we lack the full transcript context in which these changes are occurring. Using nanopore sequencingtechnology, we have resequenced full-length cDNA from CLL samples with and without the hotspot SF3B1 K700E mutation, and a normal B cell. We have developed a workflow called FLAIR (Full-Length Alternative Isoform analysis of RNA), leveraging the full-length transcript sequencing data that nanoporeaffords. We report results from nanopore sequencingthat are concordant with known SF3B1 biology from short read sequencing as well as altered intronretention events more confidently observed using long reads. Splicing analysis of nanoporereads between the SF3B1 WT and SF3B1 K700E samples identifies alternative upstream 39 splice sites associated with SF3B1 K700E . We also find downregulation of intronretention events in SF3B1 K700E relative to SF3B1 WT and no difference between CLL SF3B1 MUT and B cell, suggesting an aberrant intronretention landscape in CLL samples lacking SF3B1 mutation. With full-length isoforms, we are able to better estimate the abundance of RNA transcripts that are productive and will likely be translated versus those that are unproductive. Validation from short-read data also reveals a strong branch pointsequence in these downregulated intronretention events, consistent with previously reported branch pointsassociated with mutated SF3B1 . As nanopore sequencinghas yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencingfor cancer and splicing research.