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ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms

View ORCID ProfileGloria M. Sheynkman, Katharine S. Tuttle, Elizabeth Tseng, Jason G. Underwood, Liang Yu, Da Dong, Melissa L. Smith, Robert Sebra, Tong Hao, Michael A. Calderwood, David E. Hill, Marc Vidal
doi: https://doi.org/10.1101/604157
Gloria M. Sheynkman
1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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  • ORCID record for Gloria M. Sheynkman
  • For correspondence: gloriam_sheynkman@dfci.harvard.edu david_hill@dfci.harvard.edu
Katharine S. Tuttle
1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
3Department of Biochemistry, Northeastern University, Boston, MA 02115, USA
4Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
5Icahn Institute of Data Science and Genomic Technology, New York, NY 10029, USA
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Elizabeth Tseng
6Pacific Biosciences, Menlo Park, CA 94025, USA
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Jason G. Underwood
6Pacific Biosciences, Menlo Park, CA 94025, USA
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Liang Yu
7School of Computer Science and Technology, Xidian University, Xi’an 710071, China
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Da Dong
7School of Computer Science and Technology, Xidian University, Xi’an 710071, China
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Melissa L. Smith
4Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
5Icahn Institute of Data Science and Genomic Technology, New York, NY 10029, USA
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Robert Sebra
4Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
5Icahn Institute of Data Science and Genomic Technology, New York, NY 10029, USA
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Tong Hao
1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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Michael A. Calderwood
1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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David E. Hill
1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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  • For correspondence: gloriam_sheynkman@dfci.harvard.edu david_hill@dfci.harvard.edu
Marc Vidal
1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
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Abstract

Most human protein-coding genes are expressed as multiple isoforms. This in turn greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every gene, the majority of alternative isoforms remains uncharacterized experimentally. This is primarily due to: i) vast differences of overall levels between different isoforms expressed from common genes, and ii) the difficulty of obtaining contiguous full-length ORF sequences. Here, we present ORF Capture-Seq (OCS), a flexible and cost-effective method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude, compared to unenriched sample. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will allow mapping of the full set of human isoforms at reasonable cost.

Footnotes

  • Updated acknowledgements.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 25, 2019.
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ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms
Gloria M. Sheynkman, Katharine S. Tuttle, Elizabeth Tseng, Jason G. Underwood, Liang Yu, Da Dong, Melissa L. Smith, Robert Sebra, Tong Hao, Michael A. Calderwood, David E. Hill, Marc Vidal
bioRxiv 604157; doi: https://doi.org/10.1101/604157
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ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms
Gloria M. Sheynkman, Katharine S. Tuttle, Elizabeth Tseng, Jason G. Underwood, Liang Yu, Da Dong, Melissa L. Smith, Robert Sebra, Tong Hao, Michael A. Calderwood, David E. Hill, Marc Vidal
bioRxiv 604157; doi: https://doi.org/10.1101/604157

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