Research abstract
Article abstract
Nature Biotechnology 27, 1025 - 1031 (2009)
Published online: 1 November 2009 | Corrected online: 11 November 2009 | doi:10.1038/nbt.1583
Microdroplet-based PCR enrichment for large-scale targeted sequencing
Ryan Tewhey1,2, Jason B Warner3, Masakazu Nakano1,4, Brian Libby3, Martina Medkova3, Patricia H David3, Steve K Kotsopoulos3, Michael L Samuels3, J Brian Hutchison3, Jonathan W Larson3, Eric J Topol1, Michael P Weiner3,4, Olivier Harismendy1,4, Jeff Olson3, Darren R Link3 & Kelly A Frazer1,4
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.
- Scripps Genomic Medicine, Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, California, USA
- Division of Biological Sciences, University of California at San Diego, La Jolla, California, USA
- RainDance Technologies, Lexington, Massachusetts, USA
- Present addresses: Moores UCSD Cancer Center, La Jolla, California, USA (M.N.) and Affomix, Branford, Connecticut, USA (M.P.W.).
Correspondence to: Kelly A Frazer1,4 e-mail: kafrazer@ucsd.edu
Correspondence to: Darren R Link3 e-mail: dlink@raindancetech.com
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