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.

Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing

Abstract

Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that 60% of target bases in the exonic 'catch', and 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overview of hybrid selection method.
Figure 2: Coverage profiles of exon targets by end sequencing and shotgun sequencing.
Figure 3: Sequence coverage along a contiguous target.
Figure 4: Normalized coverage-distribution plots.
Figure 5: Reproducibility of hybrid selection.

References

  1. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    Article  CAS  Google Scholar 

  2. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Smith, D.R. et al. Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res. 18, 1638–1642 (2008).

    Article  CAS  Google Scholar 

  5. Ley, T.J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008).

    Article  CAS  Google Scholar 

  6. Wang, J. et al. The diploid genome sequence of an Asian individual. Nature 456, 60–66 (2008).

    Article  CAS  Google Scholar 

  7. 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 

  8. Dahl, F., Gullberg, M., Stenberg, J., Landegren, U. & Nilsson, M. Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res. 33, e71 (2005).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Dahl, F. et al. Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc. Natl. Acad. Sci. USA 104, 9387–9392 (2007).

    Article  CAS  Google Scholar 

  11. Fredriksson, S. et al. Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector. Nucleic Acids Res. 35, e47 (2007).

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

    Article  CAS  Google Scholar 

  15. 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 

  16. Clamp, M. et al. Distinguishing protein-coding and noncoding genes in the human genome. Proc. Natl. Acad. Sci. USA 104, 19428–19433 (2007).

    Article  CAS  Google Scholar 

  17. Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085–2088 (1994).

    Article  CAS  Google Scholar 

  18. Hardenbol, P. et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 21, 673–678 (2003).

    Article  CAS  Google Scholar 

  19. Dohm, J.C., Lottaz, C., Borodina, T. & Himmelbauer, H. Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 36, e105 (2008).

    Article  Google Scholar 

  20. Quail, M.A. et al. A large genome center's improvements to the Illumina sequencing system. Nat. Methods 5, 1005–1010 (2008).

    Article  CAS  Google Scholar 

  21. Frazer, K.A. et al. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851–861 (2007).

    Article  CAS  Google Scholar 

  22. Lovett, M., Kere, J. & Hinton, L.M. Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc. Natl. Acad. Sci. USA 88, 9628–9632 (1991).

    Article  CAS  Google Scholar 

  23. Parimoo, S., Patanjali, S.R., Shukla, H., Chaplin, D.D. & Weissman, S.M. cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc. Natl. Acad. Sci. USA 88, 9623–9627 (1991).

    Article  CAS  Google Scholar 

  24. Bashiardes, S. et al. Direct genomic selection. Nat. Methods 2, 63–69 (2005).

    Article  CAS  Google Scholar 

  25. Jaffe, D.B. et al. Whole-genome sequence assembly for mammalian genomes: Arachne 2. Genome Res. 13, 91–96 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff of the Broad Institute Genome Sequencing Platform and Genetic Analysis Platform for generating sequencing and genotyping data. This work was supported by National Human Genome Research Institute grant HG03067-05 (to E.S.L.) and funds of the Broad Institute.

Author information

Authors and Affiliations

Authors

Contributions

A.M. and P.R. developed the wet lab protocol. J.M., W.B., T.F., C.R., S.G. and D.B.J. developed computational tools and analyzed data. E.M.L. synthesized the 200mer oligodeoxynucleotide pools. G.G. and S.F. prepared and sequenced fragment libraries. A.G., E.S.L and C.N. designed and directed the project and wrote the paper.

Corresponding author

Correspondence to Andreas Gnirke.

Ethics declarations

Competing interests

E.M.L. is employed by Agilent technologies, Inc., and Agilent reagents are used in the research presented in this article. A.G., E.S.L. and C.N. are named as inventors on a patent application on selection of nucleic acids by solution hybridization to synthetic oligonucleotide baits.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 467 kb)

Supplementary Data 1

List of 15,565 targeted exons (TXT 258 kb)

Supplementary Data 2

List of 22,000 synthetic oligodeoxynucleotides for exon capture (TXT 5629 kb)

Supplementary Data 3

List of 10,000 synthetic oligodeoxynucleotides for regional capture (TXT 2825 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gnirke, A., Melnikov, A., Maguire, J. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27, 182–189 (2009). https://doi.org/10.1038/nbt.1523

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1523

This article is cited by

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