PacBio Fusion and Long Isoform Pipeline (PB_FLIP) for Cancer Transcriptome-based Resolution of Isoform Complexity

Genomic profiling using short-read sequencing has utility in detecting disease-associated variation in both DNA and RNA. However, given the frequent occurrence of structural variation in cancer, molecular profiling using long-read sequencing improves resolution of such events. For example, the Pacific Biosciences (PacBio) Iso-Seq transcriptome protocol provides full-length isoform characterization, discernment of allelic phasing, isoform discovery, and identifies expressed fusion partners. Our PacBio Fusion and Long Isoform Pipeline (PB_FLIP) incorporates a suite of RNA-Seq software analysis tools and scripts to identify expressed fusion partners and isoforms. We sequenced a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity and demonstrated high recall of the Iso-Seq and PB_FLIP workflow to benchmark our protocol and analysis performance. Herein, we describe the utility of Iso-Seq and PB_FLIP analysis in improving deconvolution of complex structural variants and isoform detection within an institutional pediatric and adolescent/young adult (AYA) translational cancer research cohort. Using exemplar case studies, we demonstrate that Iso-Seq and PB_FLIP discover novel expressed fusion partners, resolve complex intragenic alterations, and discriminate between allele-specific expression.