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The uncertainty of transcriptome reconstruction

Because most human genes are alternatively spliced1, knowing which isoform is expressed in a sample is critical for accurate research and analysis. A single gene may code for a surprising number of proteins — in some cases with opposing biological functions2. Splicing mutations have been associated with a variety of disease phenotypes, and numerous human diseases have been linked to changes in levels of alternative spliced isoforms3. However, tools for reconstructing complete isoforms from short-read data lack sensitivity and specificity, which complicates the interpretation of RNA sequencing data4.

Profile transcriptome complexity without assembly

Single Molecule, Real-Time (SMRT) Sequencing delivers the long reads needed to capture intact isoform information without assembly or complicated algorithms. The Iso-Seq application identifies transcripts and reveals novel gene isoforms, including gene fusions and full-length mRNA and lncRNA transcripts. You can use the Iso-Seq method to:

  • Directly sequence full-length transcripts and eliminate the need for transcript reconstruction
  • Perform broad or targeted surveys of transcript diversity to obtain key information about the frequency and types of alternative transcription
  • Observe allele-specific gene expression
  • Differentiate isoform expression between cells, tissues, and disease states

spotlight-human-icon

Workflow: from RNA to full-length transcripts

Featured research: identify novel full-length isoforms in castration-resistant prostate cancer (CRPR)

androgen receptor isoform sequencing (truncated)

Scientists used long-read sequencing to identify and quantify full-length androgen receptor (AR) isoforms expressed in CRPC. They identified a truncated isoform, AR-V9, which shows potential to be a more predictive biomarker than AR-V7 of primary resistance to AR-targeted therapies.5
Explore this research further

To learn more about how to profile the complexity of the transcriptome with the Iso-Seq application, contact us.


References

  1. Pan Q., et al., (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nature Genetics. 40, 1413-1415.
  2. Schwerk, C. and Schulze-Osthoff, K. (2005) Regulation of apoptosis by alternative pre-mRNA splicing. Molecular Cell. 19(1), 1-13.
  3. http://www.eurasnet.info/scientists/alternative-splicing-and-disease/list-of-diseases, accessed on 8/3/2015
  4. Korf, I. (2013) Genomics: the state of the art in RNA-seq analysis. Nature Method. 10(12), 1165-1166.
  5. Clark, T., et al., (April, 2017) SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants. Poster presented at American Association for Cancer Research. Washington D.C.

 

Selected Resources