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Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing

Abstract

We describe an approach for targeted genome resequencing, called oligonucleotide-selective sequencing (OS-Seq), in which we modify the immobilized lawn of oligonucleotide primers of a next-generation DNA sequencer to function as both a capture and sequencing substrate. We apply OS-Seq to resequence the exons of either 10 or 344 cancer genes from human DNA samples. In our assessment of capture performance, >87% of the captured sequence originated from the intended target region with sequencing coverage falling within a tenfold range for a majority of all targets. Single nucleotide variants (SNVs) called from OS-Seq data agreed with >95% of variants obtained from whole-genome sequencing of the same individual. We also demonstrate mutation discovery from a colorectal cancer tumor sample matched with normal tissue. Overall, we show the robust performance and utility of OS-Seq for the resequencing analysis of human germline and cancer genomes.

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Figure 1: Overview of OS-Seq.
Figure 2: Targeted sequencing coverage profile along the KRAS gene from the OS-Seq-366 assay.
Figure 3: Coverage assessment of OS-Seq.

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Sequence Read Archive

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    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).

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

    Natsoulis, G. et al. A flexible approach for highly multiplexed candidate gene targeted resequencing. PLoS ONE 6, e21088 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Mamanova, L. et al. Target-enrichment strategies for next-generation sequencing. Nat. Methods 7, 111–118 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Korn, J.M. et al. Integrated genotype calling and association analysis of SNPs, common copy number polymorphisms and rare CNVs. Nat. Genet. 40, 1253–1260 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Gonzalez, G., Uribe, J.C., Armstrong, B., McDonough, W. & Berens, M.E. GeneRanker: an online system for predicting gene-disease associations for translational research. Summit on Translat. Bioinforma. 2008, 26–30 (2008).

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Pruitt, K.D. et al. The consensus coding sequence (CCDS) project: Identifying a common protein-coding gene set for the human and mouse genomes. Genome Res. 19, 1316–1323 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Jurka, J. et al. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet. Genome Res. 110, 462–467 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Fedurco, M., Romieu, A., Williams, S., Lawrence, I. & Turcatti, G. BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies. Nucleic Acids Res. 34, e22 (2006).

    Article  Google Scholar 

  17. 17

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health grants K08CA96879 (H.P.J.), DK56339 (H.P.J.), P01HG000205 (J.D.B., J.M.B. and H.P.J.), RC2HG005570 (G.N., J.M.B. and H.P.J.), R21CA140089 (G.N., J.M.B. and H.P.J.), the Sigrid Jusélius Foundation Fellowship (S.M.), the Academy of Finland Grant (S.M.), Doris Duke Clinical Foundation Clinical Scientist Development Award (H.P.J.), the Howard Hughes Medical Foundation Early Career Grant (H.P.J.), the Reddere Foundation Award (H.P.J.), the Liu Bie Ju Cha and Family Fellowship in Cancer (H.P.J.), and the Wang Family Foundation Research Grant (G.N. and H.P.J.).

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The project was conceived and experiments planned by S.M., J.D.B. and H.P.J. S.M. and J.D.B. carried out all experiments. J.D.B., S.M., J.M.B., G.N. and H.P.J. performed data analysis. J.D.B., S.M., J.M.B. and H.P.J. wrote the manuscript, and all authors reviewed it. All aspects of the study were supervised by H.P.J.

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Correspondence to Hanlee P Ji.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Methods and Supplementary Figures 1–8 (PDF 890 kb)

Supplementary Table 4 (PDF 1057 kb)

Supplementary Table 5 (PDF 48 kb)

Supplementary Data Files

OS-Seq programs (ZIP 5 kb)

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Myllykangas, S., Buenrostro, J., Natsoulis, G. et al. Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing. Nat Biotechnol 29, 1024–1027 (2011). https://doi.org/10.1038/nbt.1996

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