Abstract
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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References
Johnson, J.M. et al. Science 302, 2141–2144 (2003).
Zahler, A.M. WormBook 2005, 1–13 (2005).
Schuster, S.C. Nat. Methods 5, 16–18 (2008).
Margulies, M. et al. Nature 437, 376–380 (2005).
Shendure, J. et al. Science 309, 1728–1732 (2005).
Moore, M.J. et al. BMC Plant Biol. 6, 17 (2006).
Oh, J.D. et al. Proc. Natl. Acad. Sci. USA 103, 9999–10004 (2006).
Torres, T.T., Metta, M., Ottenwalder, B. & Schlotterer, C. Genome Res. 18, 172–177 (2008).
Porreca, G.J. et al. Nat. Methods 4, 931–936 (2007).
Emrich, S.J., Barbazuk, W.B., Li, L. & Schnable, P.S. Genome Res. 17, 69–73 (2007).
Wicker, T. et al. BMC Genomics 7, 275 (2006).
Walhout, A.J. et al. Methods Enzymol. 328, 575–592 (2000).
The MGC Project Team. Genome Res. 14, 2121–2127 (2004).
Rual, J.F. et al. Genome Res. 14, 2128–2135 (2004).
Lamesch, P. et al. Genomics 89, 307–315 (2007).
Hamosh, A., Scott, A.F., Amberger, J.S., Bocchini, C.A. & McKusick, V.A. Nucleic Acids Res. 33, D514–D517 (2005).
Clamp, M. et al. Proc. Natl. Acad. Sci. USA 104, 19428–19433 (2007).
Yamasaki, C. et al. Nucleic Acids Res. 36, D793–D799 (2008).
Acknowledgements
This work was funded in part by a grant from the Ellison Foundation (awarded to M.V.) and in part by the Dana Farber Cancer Institute Strategic Initiative in support of Center for Cancer Systems Biology. F.P.R. acknowledges support from US National Institutes of Health grants NS054052, HG003224, HL081341. W.T. was supported in part by National Institutes of Health grant DK070078. We thank the West Quad Computing Group at Harvard Medical School as well as Research Computing at Massachusetts General Hospital for assistance with computational resources. We thank G. Temple for helpful comments on the manuscript.
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K.M., L.S., N.T. and D.R.S. are employed by Agencourt Corp.
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Salehi-Ashtiani, K., Yang, X., Derti, A. et al. Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing. Nat Methods 5, 597–600 (2008). https://doi.org/10.1038/nmeth.1224
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DOI: https://doi.org/10.1038/nmeth.1224
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