Center for Cancer Systems Biology, Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA. kourosh_salehi-ashtiani@dfci.harvard.edu
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.