This article has been updated


A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Change history

  • 21 October 2007

    In the version of this article initially published online,the affiliation for Jay Shendure was listed as Department of Computer Science, Virginia Commonwealth University,601 West Main Street,Richmond,Virginia 23284,USA. The correct affiliation should be Department of Genome Sciences,University of Washington,1705 NE Pacific St.,Seattle,Washington 98195,USA. The error has been corrected for all versions of the article.


  1. 1.

    , , & Advanced sequencing technologies: methods and goals. Nat. Rev. Genet. 5, 335–344 (2004).

  2. 2.

    et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

  3. 3.

    , , & Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).

  4. 4.

    et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

  5. 5.

    , , , & Automating sequence-based detection and genotyping of SNPs from diploid samples. Nat. Genet. 38, 375–381 (2006).

  6. 6.

    et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006).

  7. 7.

    & Multiplex PCR: advantages, development, and applications. PCR Methods Appl. 3, S65–S75 (1994).

  8. 8.

    , & Multiplex polymerase chain reaction: a practical approach. J. Clin. Lab. Anal. 16, 47–51 (2002).

  9. 9.

    et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 21, 673–678 (2003).

  10. 10.

    et al. Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 15, 269–275 (2005).

  11. 11.

    et al. Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc. Natl. Acad. Sci. USA 104, 9387–9392 (2007).

  12. 12.

    et al. Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector. Nucleic Acids Res. 35, e47 (2007).

  13. 13.

    et al. Direct genomic selection. Nat. Methods 2, 63–69 (2005).

  14. 14.

    et al. Microarray-based genomic selection for high-throughput resequencing. Nat. Methods, advance online publication 14 October 2007 (doi:10.1038/nmeth1109).

  15. 15.

    et al. Direct selection of human genomic loci by microarray hybridization. Nat. Methods, advance online publication 14 October 2007 (doi:10.1038/nmeth1111).

  16. 16.

    et al. Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432, 1050–1054 (2004).

  17. 17.

    , , , & Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res. 33, e71 (2005).

  18. 18.

    et al. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat. Genet. 37, 161–165 (2005).

  19. 19.

    et al. Clinical and molecular genetic spectrum of congenital deficiency of the leptin receptor. N. Engl. J. Med. 356, 237–247 (2007).

  20. 20.

    et al. Population-based resequencing of ANGPTL4 uncovers variations that reduce triglycerides and increase HDL. Nat. Genet. 39, 513–516 (2007).

  21. 21.

    & The resequencing imperative. Nat. Genet. 39, 439–440 (2007).

  22. 22.

    et al. A census of human cancer genes. Nat. Rev. Cancer 4, 177–183 (2004).

  23. 23.

    et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153–158 (2007).

  24. 24.

    & Mapping the cancer genome. Pinpointing the genes involved in cancer will help chart anew course across the complex landscape of human malignancies. Sci. Am. 296, 50–57 (2007).

Download references


This work was supported by a Center for Excellence in Genome Sciences grant from the National Human Genome Research Institute, and a SPARC grant from the Broad Institute of Massachusetts Institute of Technology and Harvard University. We are grateful to G. Buck, M. Davis, N. Sheth, C. Childress, Jr. and J. Noble (Center for High Performance Computing and Center for the Study of Biological Complexity, Virginia Commonwealth University) for setting up the Illumina Genome Analyzer analysis pipeline. We thank H. Ji, S. Fredriksson, A. Gnirke, E. Lander, D. Jaffe and C. Nusbaum for discussions.

Author information

Author notes

    • Kun Zhang
    • , Fredrik Dahl
    •  & Jay Shendure

    Present addresses: Department of Bioengineering, University of California at San Diego, 9500 Gilman Dr., La Jolla, California 92093, USA (K.Z.), Complete Genomics Inc., 2071 Stierlin Court, Suite 100, Mountain View, California 94043, USA (F.D.), and Department of Genome Sciences, University of Washington, 1705 NE Pacific St., Seattle, Washington 98195, USA (J.S.).

    • Gregory J Porreca
    • , Kun Zhang
    • , George M Church
    •  & Jay Shendure

    These authors contributed equally to this work.


  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

    • Gregory J Porreca
    • , Kun Zhang
    • , Jin Billy Li
    • , Sara L Vassallo
    • , George M Church
    •  & Jay Shendure
  2. Center for the Study of Biological Complexity, Virginia Commonwealth University, 1000 W. Cary St. Richmond, Virginia 23284, USA.

    • Bin Xie
    • , Derek Austin
    •  & Yuan Gao
  3. Genomics Solution Unit, Agilent Technologies Inc., 5301 Stevens Creek Blvd., Santa Clara, California 95051, USA.

    • Emily M LeProust
    •  & Bill J Peck
  4. Codon Devices Inc., One Kendall Square, Building 300, Third Floor, Cambridge, Massachusetts 02139, USA.

    • Christopher J Emig
  5. Stanford Genome Technology Center, Clark Center W300, 318 Campus Drive, Stanford, California 94305, USA.

    • Fredrik Dahl
  6. Department of Computer Science, Virginia Commonwealth University, 601 West Main Street, Richmond, Virginia 23284, USA.

    • Yuan Gao


  1. Search for Gregory J Porreca in:

  2. Search for Kun Zhang in:

  3. Search for Jin Billy Li in:

  4. Search for Bin Xie in:

  5. Search for Derek Austin in:

  6. Search for Sara L Vassallo in:

  7. Search for Emily M LeProust in:

  8. Search for Bill J Peck in:

  9. Search for Christopher J Emig in:

  10. Search for Fredrik Dahl in:

  11. Search for Yuan Gao in:

  12. Search for George M Church in:

  13. Search for Jay Shendure in:

Competing interests

E.M.L. and B.J.P. are employed by Agilent Technologies, Inc., and Agilent reagents are used in the research presented in this article.

Corresponding authors

Correspondence to George M Church or Jay Shendure.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3, Supplementary Table 1, Supplementary Methods

Text files

  1. 1.

    Supplementary Data 1

    Sequences of targeting oligonucleotides and targets (55,000-plex).

  2. 2.

    Supplementary Data 2

    Sequences of targeting oligonucleotides and targets (480-plex).

About this article

Publication history





Further reading