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Brief Communication

Nature Methods 5, 597–600 (1 July 2008) | doi:10.1038/nmeth.1224

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

Kourosh Salehi-Ashtiani , Xinping Yang , Adnan Derti , Weidong Tian , Tong Hao , Chenwei Lin , Kathryn Makowski , Lei Shen , Ryan R Murray , David Szeto , Nadeem Tusneem , Douglas R Smith , Michael E Cusick , David E Hill , Frederick P Roth & Marc Vidal

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.