Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Nat Biotechnol. 2009 Nov;27(11):1025-31. doi: 10.1038/nbt.1583. Epub 2009 Nov 1.

Microdroplet-based PCR enrichment for large-scale targeted sequencing.

Author information

  • 1Scripps Genomic Medicine, Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, California, USA.

Erratum in

  • Nat Biotechnol. 2010 Feb;28(2):178.

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.

Comment in

PMID:
19881494
[PubMed - indexed for MEDLINE]
PMCID:
PMC2779736
Free PMC Article

Images from this publication.See all images (4)Free text

Figure 1
Figure 2
Figure 3
Figure 4
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Nature Publishing Group Icon for PubMed Central
    Loading ...
    Write to the Help Desk