Nature Methods
- 5, 597 - 600 (2008)
Published online: 15 June 2008; | doi:10.1038/nmeth.1224
Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencingKourosh Salehi-Ashtiani1, 2, 5, Xinping Yang1, 2, 5, Adnan Derti1, 3, 5, Weidong Tian1, 3, 5, Tong Hao1, 2, 5, Chenwei Lin1, 2, Kathryn Makowski4, Lei Shen4, Ryan R Murray1, 2, David Szeto1, 2, Nadeem Tusneem4, Douglas R Smith4, Michael E Cusick1, 2, David E Hill1, 2, Frederick P Roth1, 3 & Marc Vidal1, 21
Center for Cancer Systems Biology, Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.
2
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 3
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA. 4
Agencourt Bioscience Corporation, 500 Cummings Center, Beverly, Massachusetts 01915, USA. 5
These authors contributed equally to this work.
Correspondence should be addressed to Marc Vidal marc_vidal@dfci.harvard.edu or Kourosh Salehi-Ashtiani kourosh_salehi-ashtiani@dfci.harvard.edu or Frederick P Roth fritz_roth@hms.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.
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