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Nat Methods. 2008 Jul;5(7):597-600. doi: 10.1038/nmeth.1224. Epub 2008 Jun 15.

Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing.

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  • 1Center for Cancer Systems Biology, Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.


Describing the 'ORFeome' of an organism, including all major isoforms, is essential for a system-level understanding of any species; however, conventional cloning and sequencing approaches are prohibitively costly and labor-intensive. We describe a potentially genome-wide methodology for efficiently capturing new coding isoforms using reverse transcriptase (RT)-PCR recombinational cloning, 'deep-well' pooling and a next-generation sequencing platform. This ORFeome discovery pipeline will be applicable to any eukaryotic species with a sequenced genome.

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