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

Nature Biotechnology volume 27, pages 182–189 (2009)Cite this article

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.

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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.

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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.

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Author notes

  1. William Brockman

    Present address: Present address: Google, Inc., 5 Cambridge Center, Cambridge, Massachusetts 02142, USA.,

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Andreas Gnirke, Alexandre Melnikov, Jared Maguire, Peter Rogov, William Brockman, Timothy Fennell, Georgia Giannoukos, Sheila Fisher, Carsten Russ, Stacey Gabriel, David B Jaffe, Eric S Lander & Chad Nusbaum

  2. Agilent Technologies Inc., 5301 Stevens Creek Blvd., Santa Clara, 95051, California, USA

    Emily M LeProust

  3. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, 02139, Massachusetts, USA

    Eric S Lander

  4. Department of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston, 02115, Massachusetts, USA

    Eric S Lander

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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.

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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)

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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

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