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

Nature Methods volume 5, pages 597600 (2008) | Download Citation

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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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.

Author information

Author notes

    • Kourosh Salehi-Ashtiani
    • , Xinping Yang
    • , Adnan Derti
    • , Weidong Tian
    •  & Tong Hao

    These authors contributed equally to this work.

Affiliations

  1. Center for Cancer Systems Biology, Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

    • Kourosh Salehi-Ashtiani
    • , Xinping Yang
    • , Adnan Derti
    • , Weidong Tian
    • , Tong Hao
    • , Chenwei Lin
    • , Ryan R Murray
    • , David Szeto
    • , Michael E Cusick
    • , David E Hill
    • , Frederick P Roth
    •  & Marc Vidal
  2. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

    • Kourosh Salehi-Ashtiani
    • , Xinping Yang
    • , Tong Hao
    • , Chenwei Lin
    • , Ryan R Murray
    • , David Szeto
    • , Michael E Cusick
    • , David E Hill
    •  & Marc Vidal
  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.

    • Adnan Derti
    • , Weidong Tian
    •  & Frederick P Roth
  4. Agencourt Bioscience Corporation, 500 Cummings Center, Beverly, Massachusetts 01915, USA.

    • Kathryn Makowski
    • , Lei Shen
    • , Nadeem Tusneem
    •  & Douglas R Smith

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Competing interests

K.M., L.S., N.T. and D.R.S. are employed by Agencourt Corp.

Corresponding authors

Correspondence to Kourosh Salehi-Ashtiani or Frederick P Roth or Marc Vidal.

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    Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Note, Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth.1224

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