title: Microdroplet-based PCR enrichment for large-scale targeted sequencing.
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 approximately 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.
authors.affiliation: Scripps Genomic Medicine, Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Tewhey
authors.forename: Jason B
authors.initials: JB
authors.lastname: Warner
authors.forename: Masakazu
authors.initials: M
authors.lastname: Nakano
authors.forename: Brian
authors.initials: B
authors.lastname: Libby
authors.forename: Martina
authors.initials: M
authors.lastname: Medkova
authors.forename: Patricia H
authors.initials: PH
authors.lastname: David
authors.forename: Steve K
authors.initials: SK
authors.lastname: Kotsopoulos
authors.forename: Michael L
authors.initials: ML
authors.lastname: Samuels
authors.forename: J Brian
authors.initials: JB
authors.lastname: Hutchison
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Larson
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Topol
authors.forename: Michael P
authors.initials: MP
authors.lastname: Weiner
authors.forename: Olivier
authors.initials: O
authors.lastname: Harismendy
authors.forename: Jeff
authors.initials: J
authors.lastname: Olson
authors.forename: Darren R
authors.initials: DR
authors.lastname: Link
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Frazer
mesh_terms: D001483:Base Sequence; D006801:Humans; D044085:Microfluidics / Q000379:methods*; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*