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The challenge of isoform reconstruction

In eukaryotic organisms, the majority of genes are alternatively spliced to produce multiple transcript isoforms, dramatically increasing the protein-coding potential of a genome1. Alternatively spliced isoforms from the same gene can have significantly different, even antagonistic, effects2. To study gene expression, researchers have looked at fragments of an organism’s genes utilizing next-generation sequencing methods, commonly referred to as RNA sequencing (RNA-seq). However, short-read RNA-seq cannot span full-length transcripts, making it difficult to accurately characterize the diverse landscape of isoforms3.

Produce full-length transcripts without assembly

The isoform sequencing (Iso-Seq) application generates full-length cDNA sequences — from the 5’ end of transcripts to the poly-A tail — eliminating the need for transcriptome reconstruction using isoform-inference algorithms. The Iso-Seq method generates accurate information about alternatively spliced exons and transcriptional start sites. It also delivers information about poly-adenylation sites for transcripts up to 10 kb in length across the full complement of isoforms within targeted genes or the entire transcriptome.

 

PacBio delivers confident characterization of isoform diversity with the Sequel System

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. Boise, LH., et al., (1993) Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 74(4), 597-608.
  3. Steijger, T., et al. (2013) Assessment of transcript reconstruction methods for RNA-seq. Nature Methods. 10(12), 1177-1184.

Selected Resources