Abstract

Short-read sequencing for genomic profiling is useful for identifying disease-related variation in both DNA and RNA. However, molecular profiling utilising long-read sequencing enhances the resolution of such events because structural variation in cancer occurs often. For instance, the Pacific Biosciences long-read RNA-sequencing (Iso-Seq) transcriptome technique finds expressed fusion partners and offers full-length isoform characterisation, discernment of allelic phasing, and isoform identification. To find expressed fusion partners and isoforms, the Pacific Biosciences Fusion and Long Isoform Pipeline (PB FLIP) uses a variety of RNA-sequencing software analysis tools and scripts. In order to test our methodology and analytical performance, sequencing of a commercial reference (Spike-In RNA Variants) with known isoform complexity was carried out. This sequencing showed strong recall of the Iso-Seq and PB FLIP workflow. This work explains how Iso-Seq and PB FLIP analysis can help with isoform recognition and difficult structural variant deconvolution in a cohort of institutional paediatric and adolescent/young adult cancer research participants. The exemplary case studies show that Iso-Seq and PB FLIP can distinguish between allele-specific expression patterns, resolve complex intragenic changes, and find novel expressed fusion partners.