Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Microdroplet-based PCR enrichment for large-scale targeted sequencing

Nature Biotechnology volume 27, pages 1025–1031 (2009)Cite this article

A Corrigendum to this article was published on 01 February 2010

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Microdroplet PCR workflow.
Figure 2: Coverage plots of targeted sequences.
Figure 3: Normalized coverage distribution plots.
Figure 4: Intersample reproducibility of amplicon coverage.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Change history

  • 11 November 2009

    In the version of this article initially published, the email address for K.A.F. should have been kafrazer@ucsd.edu. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Levy, S. et al. The diploid genome sequence of an individual human. PLoS Biol. 5, e254 (2007).

    Article  Google Scholar 

  2. Wheeler, D.A. et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–876 (2008).

    Article  CAS  Google Scholar 

  3. Wang, J. et al. The diploid genome sequence of an Asian individual. Nature 456, 60–65 (2008).

    Article  CAS  Google Scholar 

  4. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    Article  CAS  Google Scholar 

  5. Yeager, M. et al. Comprehensive resequence analysis of a 136 kb region of human chromosome 8q24 associated with prostate and colon cancers. Hum. Genet. 124, 161–170 (2008).

    Article  CAS  Google Scholar 

  6. Ding, L. et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455, 1069–1075 (2008).

    Article  CAS  Google Scholar 

  7. McLendon, R. et al. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455, 1061–1068 (2008).

    Article  CAS  Google Scholar 

  8. Porreca, G.J. et al. Multiplex amplification of large sets of human exons. Nat. Methods 4, 931–936 (2007).

    Article  CAS  Google Scholar 

  9. Turner, E.H., Lee, C., Ng, S.B., Nickerson, D.A. & Shendure, J. Massively parallel exon capture and library-free resequencing across 16 genomes. Nat. Methods 6, 315–316 (2009).

    Article  CAS  Google Scholar 

  10. Krishnakumar, S. et al. A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proc. Natl. Acad. Sci. USA 105, 9296–9301 (2008).

    Article  CAS  Google Scholar 

  11. Albert, T.J. et al. Direct selection of human genomic loci by microarray hybridization. Nat. Methods 4, 903–905 (2007).

    Article  CAS  Google Scholar 

  12. Hodges, E. et al. Genome-wide in situ exon capture for selective resequencing. Nat. Genet. 39, 1522–1527 (2007).

    Article  CAS  Google Scholar 

  13. Okou, D.T. et al. Microarray-based genomic selection for high-throughput resequencing. Nat. Methods 4, 907–909 (2007).

    Article  CAS  Google Scholar 

  14. Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

    Article  CAS  Google Scholar 

  15. Anna, S.L., Bontoux, N. & Stone, H.A. Formation of dispersions using “flow focusing” in microchannels. Appl. Phys. Lett. 82, 364–366 (2003).

    Article  CAS  Google Scholar 

  16. Ahn, K., Agresti, J., Chong, H., Marquez, M. & Weitz, D.A. Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels. Appl. Phys. Lett. 88, 264105 (2006).

    Article  Google Scholar 

  17. Quinlan, A.R. & Marth, G.T. Primer-site SNPs mask mutations. Nat. Methods 4, 192 (2007).

    Article  CAS  Google Scholar 

  18. Harismendy, O. et al. Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biol. 10, R32 (2009).

    Article  Google Scholar 

  19. Frazer, K.A., Murray, S.S., Schork, N.J. & Topol, E.J. Human genetic variation and its contribution to complex traits. Nat. Rev. Genet. 10, 241–251 (2009).

    Article  CAS  Google Scholar 

  20. Birney, E. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).

    Article  CAS  Google Scholar 

  21. Sachidanandam, R. et al. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409, 928–933 (2001).

    Article  CAS  Google Scholar 

  22. Ng, S.B. et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461, 272–276 (2009).

    Article  CAS  Google Scholar 

  23. The International HapMap Consortium A haplotype map of the human genome. Nature 437, 1299–1320 (2005).

  24. Harismendy, O. & Frazer, K. Method for improving sequence coverage uniformity of targeted genomic intervals amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology. Biotechniques 46, 229–231 (2009).

    Article  CAS  Google Scholar 

  25. Sjoblom, T. et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006).

    Article  Google Scholar 

  26. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank X. Wang, K. Post (STSI), O. Iartchouk (Partners HealthCare Center for Personalized Genetic Medicine, K. Makowski (Agencourt Bioscience Corporation) for excellent technical assistance, N. Schork (STSI) for helpful conversations, N. Hafez (seqWise) for assistance with data analysis, and the US National Institutes of Health (CTSA grant 1U54RR025204-01; Innovative Technologies for Molecular Analysis of Cancer grant 1R21CA125693-01) and Japan Foundation for Aging and Health (MN fellowship) for support for this effort.

Author information

Author notes

  1. Masakazu Nakano, Michael P Weiner, Olivier Harismendy & Kelly A Frazer

    Present address: Present addresses: Moores UCSD Cancer Center, La Jolla, California, USA (M.N.) and Affomix, Branford, Connecticut, USA (M.P.W.).,

Authors and Affiliations

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

    Ryan Tewhey, Masakazu Nakano, Eric J Topol, Olivier Harismendy & Kelly A Frazer

  2. Division of Biological Sciences, University of California at San Diego, La Jolla, California, USA

    Ryan Tewhey

  3. RainDance Technologies, Lexington, Massachusetts, USA

    Jason B Warner, Brian Libby, Martina Medkova, Patricia H David, Steve K Kotsopoulos, Michael L Samuels, J Brian Hutchison, Jonathan W Larson, Michael P Weiner, Jeff Olson & Darren R Link

Authors
  1. Ryan Tewhey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jason B Warner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masakazu Nakano
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brian Libby
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martina Medkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patricia H David
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Steve K Kotsopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael L Samuels
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J Brian Hutchison
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jonathan W Larson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Eric J Topol
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Michael P Weiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Olivier Harismendy
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jeff Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Kelly A Frazer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.A.F., E.J.T., M.P.W., and D.R.L. conceived the project; K.A.F., O.H., J.O. and D.R.L. designed the experiments, J.W., M.N., R.T., B.L., M.M., P.D., S.K., M.S., J.B.H., J.W.L., and O.H. performed the experiments; R.T. and J.W. performed the data analysis; R.T., J.W., J.O., D.R.L. and K.A.F. wrote the manuscript.

Corresponding authors

Correspondence to Darren R Link or Kelly A Frazer.

Ethics declarations

Competing interests

J.W., B.L., M.M., P.D., S.K., M.S., J.B.H., J.W.L., J.O., M.P.W. and D.R.L. are employed by RainDance Technologies, Inc. RainDance Technologies is commercializing microdroplet PCR for targeted sequencing applications.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–5, Supplementary Tables 1–8 and Supplementary Discussion (PDF 2752 kb)

Supplementary Movie 1

Droplet merging on a microfluidic chip (MOV 137 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tewhey, R., Warner, J., Nakano, M. et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27, 1025–1031 (2009). https://doi.org/10.1038/nbt.1583

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1583

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing