Pacific Biosciences Fusion and Long Isoform Pipeline for Cancer Transcriptome-Based Resolution of Isoform Complexity

J Mol Diagn. 2022 Dec;24(12):1292-1306. doi: 10.1016/j.jmoldx.2022.09.003. Epub 2022 Sep 30.


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 the resolution of such events. For example, the Pacific Biosciences long-read RNA-sequencing (Iso-Seq) transcriptome protocol provides full-length isoform characterization, discernment of allelic phasing, and isoform discovery, and identifies expressed fusion partners. The Pacific Biosciences Fusion and Long Isoform Pipeline (PB_FLIP) incorporates a suite of RNA-sequencing software analysis tools and scripts to identify expressed fusion partners and isoforms. In addition, sequencing of a commercial reference (Spike-In RNA Variants) with known isoform complexity was performed and demonstrated high recall of the Iso-Seq and PB_FLIP workflow to benchmark our protocol and analysis performance. This study describes 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 translational cancer research cohort. The exemplar case studies demonstrate that Iso-Seq and PB_FLIP discover novel expressed fusion partners, resolve complex intragenic alterations, and discriminate between allele-specific expression profiles.

MeSH terms

  • Adolescent
  • Alternative Splicing
  • Child
  • Gene Expression Profiling / methods
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Neoplasms* / genetics
  • Protein Isoforms / genetics
  • RNA / genetics
  • Sequence Analysis, RNA
  • Transcriptome*
  • Young Adult


  • Protein Isoforms
  • RNA