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


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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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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Authors and Affiliations



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).

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