Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq method developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. The Iso-Seq library preparation protocol has recently been optimized for the higher-throughput PacBio Sequel platform, decreasing the need for size fractionation steps, reducing sample input requirements, and increasing the throughput per SMRT cell. We will review the simplified protocol and share results from applying the new method to both a cancer cell line and fresh frozen tumor samples. We will then review several examples of how researchers are using the Iso-Seq method to undercover new cancer biology, including the identification of candidate biomarkers for early detection of colorectal cancer and the discovery of a novel biomarker for drug resistance in prostate cancer. Researchers are encouraged to consider how the Iso-Seq method can provide new insights into their own cancer research and submit a brief application to win a SMRT Grant via our website.
February 5, 2021 | Webinar