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Multiplex amplification of large sets of human exons

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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.

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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Correspondence to Jay Shendure.

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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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