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Microdroplet-based PCR enrichment for large-scale targeted sequencing

Nature Biotechnology volume 27, pages 1025–1031 (2009)Cite this article

A Corrigendum to this article was published on 01 February 2010

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Abstract

Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across ∼90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r2 = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing.

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Figure 1: Microdroplet PCR workflow.
Figure 2: Coverage plots of targeted sequences.
Figure 3: Normalized coverage distribution plots.
Figure 4: Intersample reproducibility of amplicon coverage.

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Change history

  • 11 November 2009

    In the version of this article initially published, the email address for K.A.F. should have been kafrazer@ucsd.edu. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank X. Wang, K. Post (STSI), O. Iartchouk (Partners HealthCare Center for Personalized Genetic Medicine, K. Makowski (Agencourt Bioscience Corporation) for excellent technical assistance, N. Schork (STSI) for helpful conversations, N. Hafez (seqWise) for assistance with data analysis, and the US National Institutes of Health (CTSA grant 1U54RR025204-01; Innovative Technologies for Molecular Analysis of Cancer grant 1R21CA125693-01) and Japan Foundation for Aging and Health (MN fellowship) for support for this effort.

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Author notes

  1. Masakazu Nakano, Michael P Weiner, Olivier Harismendy & Kelly A Frazer

    Present address: Present addresses: Moores UCSD Cancer Center, La Jolla, California, USA (M.N.) and Affomix, Branford, Connecticut, USA (M.P.W.).,

Authors and Affiliations

  1. Scripps Genomic Medicine, Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, California, USA

    Ryan Tewhey, Masakazu Nakano, Eric J Topol, Olivier Harismendy & Kelly A Frazer

  2. Division of Biological Sciences, University of California at San Diego, La Jolla, California, USA

    Ryan Tewhey

  3. RainDance Technologies, Lexington, Massachusetts, USA

    Jason B Warner, Brian Libby, Martina Medkova, Patricia H David, Steve K Kotsopoulos, Michael L Samuels, J Brian Hutchison, Jonathan W Larson, Michael P Weiner, Jeff Olson & Darren R Link

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Contributions

K.A.F., E.J.T., M.P.W., and D.R.L. conceived the project; K.A.F., O.H., J.O. and D.R.L. designed the experiments, J.W., M.N., R.T., B.L., M.M., P.D., S.K., M.S., J.B.H., J.W.L., and O.H. performed the experiments; R.T. and J.W. performed the data analysis; R.T., J.W., J.O., D.R.L. and K.A.F. wrote the manuscript.

Corresponding authors

Correspondence to Darren R Link or Kelly A Frazer.

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Competing interests

J.W., B.L., M.M., P.D., S.K., M.S., J.B.H., J.W.L., J.O., M.P.W. and D.R.L. are employed by RainDance Technologies, Inc. RainDance Technologies is commercializing microdroplet PCR for targeted sequencing applications.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–5, Supplementary Tables 1–8 and Supplementary Discussion (PDF 2752 kb)

Supplementary Movie 1

Droplet merging on a microfluidic chip (MOV 137 kb)

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Tewhey, R., Warner, J., Nakano, M. et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27, 1025–1031 (2009). https://doi.org/10.1038/nbt.1583

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