Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays (original) (raw)

Nature Biotechnology volume 18, pages 630–634 (2000) Cite this article

A Corrigendum to this article was published on 01 October 2000

Abstract

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3 × 106 microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16–20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

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Acknowledgements

The authors thank Steve Macevicz of Lynx Therapeutics for preparing the manuscript; Mel Kronick of Agilent Technologies and Dan Pinkel of the Cancer Center, Department of Laboratory Medicine, University of California, San Francisco, for helpful comments; and Larry DeDionisio and Victor Quijano of Lynx Therapeutics for technical assistance.

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Authors and Affiliations

  1. Lynx Therapeutics, Inc., 25861 Industrial Blvd., Hayward, 94545, California
    Sydney Brenner, Maria Johnson, John Bridgham, George Golda, David H. Lloyd, Davida Johnson, Shujun Luo, Sarah McCurdy, Michael Foy, Mark Ewan, Rithy Roth, Dave George, Sam Eletr, Glenn Albrecht, Eric Vermaas, Steven R. Williams, Keith Moon, Timothy Burcham, Michael Pallas, Robert B. DuBridge, James Kirchner, Karen Fearon, Jen-i Mao & Kevin Corcoran

Authors

  1. Sydney Brenner
  2. Maria Johnson
  3. John Bridgham
  4. George Golda
  5. David H. Lloyd
  6. Davida Johnson
  7. Shujun Luo
  8. Sarah McCurdy
  9. Michael Foy
  10. Mark Ewan
  11. Rithy Roth
  12. Dave George
  13. Sam Eletr
  14. Glenn Albrecht
  15. Eric Vermaas
  16. Steven R. Williams
  17. Keith Moon
  18. Timothy Burcham
  19. Michael Pallas
  20. Robert B. DuBridge
  21. James Kirchner
  22. Karen Fearon
  23. Jen-i Mao
  24. Kevin Corcoran

Corresponding author

Correspondence toSydney Brenner.

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Brenner, S., Johnson, M., Bridgham, J. et al. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.Nat Biotechnol 18, 630–634 (2000). https://doi.org/10.1038/76469

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