Microdroplet-based PCR enrichment for large-scale targeted sequencing

• Published in: Nature biotechnology 2009-11, Vol.27 (11), p.1025-1031
• Main Authors: Link, Darren R, Frazer, Kelly A, Tewhey, Ryan, Warner, Jason B, Nakano, Masakazu, Libby, Brian, Medkova, Martina, David, Patricia H, Kotsopoulos, Steve K, Samuels, Michael L, Hutchison, J Brian, Larson, Jonathan W, Topol, Eric J, Weiner, Michael P, Harismendy, Olivier, Olson, Jeff
• Format: Article
• Language: English
• Subjects: Agriculture; Base Sequence; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Deoxyribonucleic acid; Diverse techniques; DNA; DNA sequencing; Enrichment; Fundamental and applied biological sciences. Psychology; Genetic diversity; Genomics; Humans; Identification and classification; Innovations; Life Sciences; Microfluidics - methods; Molecular and cellular biology; Mutation - genetics; Nucleotide sequence; Nucleotide sequencing; Polymerase Chain Reaction - methods; Reproducibility of Results; Sequence Analysis, DNA - instrumentation; Sequence Analysis, DNA - methods
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/nbt.1583

In many sequencing applications, it is sufficient to sequence selected portions of a genome rather than the complete genome. Tewhey et al . describe an approach for massively parallel genome targeting that relies on PCR in microdroplets generated by a microfluidic device. 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 ( r 2 = 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.