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

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Short-read sequencing has limitations in detecting complex structural variations common in cancer.
• Long-read sequencing offers improved resolution for molecular profiling of cancer genomes and transcriptomes.

Purpose of the Study:

• To evaluate the utility of Pacific Biosciences long-read RNA sequencing (Iso-Seq) and the associated analysis pipeline (PB_FLIP) for complex structural variant and isoform detection.
• To characterize expressed fusion partners and isoforms in a pediatric and adolescent/young adult translational cancer research cohort.

Main Methods:

• Utilized Iso-Seq for full-length transcriptome characterization, including isoform discovery and allelic phasing.
• Applied the Pacific Biosciences Fusion and Long Isoform Pipeline (PB_FLIP) for identifying expressed fusion partners and isoforms.
• Sequenced a commercial reference (Spike-In RNA Variants) to benchmark the Iso-Seq and PB_FLIP workflow performance.

Main Results:

• Demonstrated high recall of the Iso-Seq and PB_FLIP workflow in identifying known isoforms.
• Successfully identified novel expressed fusion partners and resolved complex intragenic alterations in cancer samples.
• Discriminated between allele-specific expression profiles, providing deeper insights into cancer biology.

Conclusions:

• Iso-Seq and PB_FLIP analysis significantly improve the deconvolution of complex structural variants and isoform detection in cancer.
• This approach enhances the molecular profiling of pediatric and adolescent/young adult cancers, aiding in the discovery of novel biomarkers and therapeutic targets.