Soulette, C. M. | Search bioRxiv

bioRxiv2018

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 sequencing technology, 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 nanopore affords. We report results from nanopore sequencing that are concordant with known SF3B1 biology from short read sequencing as well as altered intron retention events more confidently observed using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E samples identifies alternative upstream 3 splice sites associated with SF3B1K700E. We also find downregulation of intron retention events in SF3B1K700E relative to SF3B1WT and no difference between CLL SF3B1MT and B cell, suggesting an aberrant intron retention 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 point sequence in these downregulated intron retention events, consistent with previously reported branch points associated with mutated SF3B1. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

genomics↗

bioRxiv2017

Pan-cancer study of heterogeneous RNA aberrations

We present the most comprehensive catalogue of cancer-associated gene alterations through characterization of tumor transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes project. Using matched whole-genome sequencing data, we attributed RNA alterations to germline and somatic DNA alterations, revealing likely genetic mechanisms. We identified 444 associations of gene expression with somatic non-coding single-nucleotide variants. We found 1,872 splicing alterations associated with somatic mutation in intronic regions, including novel exonization events associated with Alu elements. Somatic copy number alterations were the major driver of total gene and allele-specific expression (ASE) variation. Additionally, 82% of gene fusions had structural variant support, including 75 of a novel class called \"bridged\" fusions, in which a third genomic location bridged two different genes. Globally, we observe transcriptomic alteration signatures that differ between cancer types and have associations with DNA mutational signatures. Given this unique dataset of RNA alterations, we also identified 1,012 genes significantly altered through both DNA and RNA mechanisms. Our study represents an extensive catalog of RNA alterations and reveals new insights into the heterogeneous molecular mechanisms of cancer gene alterations.

genomics↗