ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms
Gloria M. Sheynkman,
Katharine S Tuttle,
Jason G Underwood,
Melissa L. Smith,
Michael A. Calderwood,
David E. Hill,
Posted 11 Apr 2019
bioRxiv DOI: 10.1101/604157 (published DOI: 10.1038/s41467-020-16174-z)
Posted 11 Apr 2019
Most human protein-coding genes are expressed as multiple isoforms. This in turn greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every gene, the majority of alternative isoforms remains uncharacterized experimentally. This is primarily due to: i) vast differences of overall levels between different isoforms expressed from common genes, and ii) the difficulty of obtaining contiguous full-length ORF sequences. Here, we present ORF Capture-Seq (OCS), a flexible and cost-effective method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude, compared to unenriched sample. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will allow mapping of the full set of human isoforms at reasonable cost.
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