“Microdroplet-Based PCR Enrichment for Large-Scale Targeted Sequencing”
A paper published in the November 2009 issue of Nature Biotechnology finds that data generated using microdroplet PCR technology as an enrichment method are particularly well suited for performing sequence-based association studies.
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.
“Microdroplet-based PCR Enrichment for Large-Scale Targeted Sequencing,” authors: Ryan Tewhey1,2, Jason Warner3, Masakazu Nakano1,4, Brian Libby3, Martina Medkova3, Patricia David3, Steve Kotsopoulos3, Michael Samuels3, J. Brian Hutchinson3, Jonathan W. Larson3, Eric J. Topol1, Michael P. Weiner3, Olivier Harismendy1,4, Jeff Olson3, Darren R. Link3, and Kelly A. Frazer1,4. DOI: 10.1038/nbt.1583.
1Scripps Genomic Medicine, Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, California, U.S.A.
2Division of Biological Sciences, University of California at San Diego, La Jolla, California, U.S.A.
3RainDance Technologies, Lexington, Massachusetts, U.S.A.
4Present address: Moores UCSD Cancer Center, La Jolla, California, U.S.A.
Paper co-author Kelly A. Frazer, Ph.D., Professor of Pediatrics and Chief, Division of Genome Information Sciences, UC San Diego Department of Pediatrics, said they were impressed with how well the technology worked. “Compared to other types of sequence enrichment, we were able to generate greater uniform coverage of targeted sequences. In addition, the problems we typically encounter with standard PCR, such as difficult primer design and high allelic bias, were not observed with the microdroplet-based PCR workflow. These benefits translated into a much higher efficiency of sequencing and a much more cost-effective way to do population studies,” Dr. Frazer said.
“I believe this new technology will be used to study the basic underpinnings of diseases and to understand their root causes,” said co-author Eric J. Topol, M.D., Director, Scripps Translational Science Institute, in commenting on the paper. “For instance, we’re now using it to understand the causes of Anorexia Nervosa. We also can use the technology to comprehend a disease’s response to a therapy or to determine a drug’s side effects. We even can employ it to study the healthy aging process. These are all applications for microdroplet PCR that will help us understand the vital aspects of human health and disease,” said Dr. Topol.
