March 20, 2024  |  Cancer research

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

Authors: Saranga Wijeratne, Maria E. Hernandez Gonzalez, Kelli Roach, Katherine E. Miller, Kathleen M. Schieffer, James R. Fitch, Jeffrey Leonard, Peter White, Benjamin J. Kelly, Catherine E. Cottrell, Elaine R. Mardis, Richard K. Wilson & Anthony R. Miller

Cancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.

Journal: BMC Genomics
DOI: 10.1186/s12864-024-10021-x
Year: 2024

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