Abstract
Abstract The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information can be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment. The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, single molecule cDNA sequences, allowing for the unambiguous, direct determination of splice variants. This has led to the identification of novel cancer biomarker candidates and has yielded new insights into gene fusion events that contribute to cancer progression.1,2,3 Recent improvements to the Iso-Seq bioinformatics pipeline increases the speed and scalability of data analysis while boosting the reliability of isoform detection and cross-platform usability. Here we report evaluation of Iso-Seq runs of human UHRR samples with spiked-in synthetic RNA controls on the Sequel System and show that the new pipeline recovers both more human and synthetic isoforms while reducing the number of false positives. We also share the results of sequencing the well-characterized HCC-1954 breast cancer and matched normal cell lines, which will be made publicly available. Combined with the recent simplification of the Iso-Seq sample preparation protocol, the new analysis pipeline completes a streamlined workflow for revealing the most comprehensive picture of transcriptomes at the throughput needed to characterize cancer samples. 1. Kohli M, et. al. (2017) Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance. Clin Cancer Res. ePub ahead of print. doi: 10.1158/1078-0432.CCR-17-0017 2. Komor MA, et. al. (2017) Identification of differentially expressed splice variants by the proteogenomic pipeline Splicify. Mol Cell Prot. ePub ahead of print. doi: 10.1074/mcp.TIR117.000056 3. Weirather JL, et. al. (2015) Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing. Nucleic Acids Res, 43 (18), e116. Citation Format: Elizabeth Tseng, Tyson Clark, Meredith H. Ashby. Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2278.
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