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Multiplex amplification of large sets of human exons

Nature Methods volume 4, pages 931–936 (2007)Cite this article

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Abstract

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.

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Figure 1: Schematic of multiplex exon capture.
Figure 2: Specificity of multiplex exon capture.
Figure 3: Quantification of uniformity by deep end-sequencing of captured amplicons.
Figure 4: Validation of exon resequencing data.

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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.

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Acknowledgements

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.

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Author notes

  1. Kun Zhang, Fredrik Dahl & Jay Shendure

    Present address: 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.).,

  2. Gregory J Porreca, Kun Zhang, George M Church and Jay Shendure: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, 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, 23284, Virginia, USA

    Bin Xie, Derek Austin & Yuan Gao

  3. Genomics Solution Unit, Agilent Technologies Inc., 5301 Stevens Creek Blvd., Santa Clara, 95051, California, USA

    Emily M LeProust & Bill J Peck

  4. Codon Devices Inc., One Kendall Square, Building 300, Third Floor, Cambridge, 02139, Massachusetts, USA

    Christopher J Emig

  5. Stanford Genome Technology Center, Clark Center W300, 318 Campus Drive, Stanford, 94305, California, USA

    Fredrik Dahl

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

    Yuan Gao

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  1. Gregory J Porreca
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Corresponding author

Correspondence to Jay Shendure.

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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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Table 1, Supplementary Methods (PDF 271 kb)

Supplementary Data 1

Sequences of targeting oligonucleotides and targets (55,000-plex). (TXT 12318 kb)

Supplementary Data 2

Sequences of targeting oligonucleotides and targets (480-plex). (TXT 93 kb)

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Porreca, G., Zhang, K., Li, J. et al. Multiplex amplification of large sets of human exons. Nat Methods 4, 931–936 (2007). https://doi.org/10.1038/nmeth1110

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