Microdroplet-based PCR enrichment for large-scale targeted sequencing View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2009-11

AUTHORS

Ryan Tewhey, Jason B Warner, Masakazu Nakano, Brian Libby, Martina Medkova, Patricia H David, Steve K Kotsopoulos, Michael L Samuels, J Brian Hutchison, Jonathan W Larson, Eric J Topol, Michael P Weiner, Olivier Harismendy, Jeff Olson, Darren R Link, Kelly A Frazer

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. More... »

PAGES

1025-1031

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  • Journal

    TITLE

    Nature Biotechnology

    ISSUE

    11

    VOLUME

    27

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nbt.1583

    DOI

    http://dx.doi.org/10.1038/nbt.1583

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1022555745

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/19881494


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