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Nat Methods. 2008 Jul;5(7):587-9.
Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing.
Salehi-Ashtiani K,
Yang X,
Derti A,
Tian W,
Hao T,
Lin C,
Makowski K,
Shen L,
Murray RR,
Szeto D,
Tusneem N,
Smith DR,
Cusick ME,
Hill DE,
Roth FP,
Vidal M.
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.
PMID: 18552854 [PubMed - indexed for MEDLINE]