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.

Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

Abstract

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3–5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5–6 d for array-based enrichment and 3–4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Workflow of the protocol for highly multiplexed enrichment.
Figure 2: Mapping and enrichment statistics for a multiplexed microarray-based enrichment experiment with 20 rat samples and a design size of 1.4 Mb.
Figure 3: Mapping and enrichment statistics for a multiplexed microarray-based enrichment experiment with 96 human samples and a design size of 0.4 Mb.
Figure 4: Mapping and enrichment statistics for a multiplexed solution-based enrichment experiment with 23 human samples and design size of 3 Mb (human exome on the X chromosome).
Figure 5: Mapping and enrichment statistics for a multiplexed solution-based enrichment experiment with four human samples and design size of 50 Mb (human whole exome).
Figure 6: Effect of multiplexing level of up to 96 bar-coded samples on allelic competition.

References

  1. 1

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Li, Y. et al. Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants. Nat. Genet. 42, 969–972 (2010).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Metzker, M.L. Sequencing technologies—the next generation. Nat. Rev. Genet. 11, 31–46 (2010).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Vissers, L.E. et al. A de novo paradigm for mental retardation. Nat. Genet. 42, 1109–1112 (2010).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Igartua, C. et al. Targeted enrichment of specific regions in the human genome by array hybridization. Curr. Protoc. Hum. Genet. 66, 18.3.1–18.3.14 (2010).

    Article  Google Scholar 

  6. 6

    Durbin, R.M. et al. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

    CAS  Article  PubMed  Google Scholar 

  7. 7

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

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Hodges, E. et al. Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nat. Protoc. 4, 960–974 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9

    Nijman, I.J. et al. Mutation discovery by targeted genomic enrichment of multiplexed bar-coded samples. Nat. Methods 7, 913–915 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Harbour, J.W. et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 330, 1410–1413 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Varela, I. et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539–542 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Krawitz, P.M. et al. Identity-by-descent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome. Nat. Genet. 42, 827–829 (2010).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Gilissen, C. et al. Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am. J. Hum. Genet. 87, 418–423 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Musunuru, K. et al. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl. J. Med. 363, 2220–2227 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Bilguvar, K. et al. Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations. Nature 467, 207–210 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Ng, S.B. et al. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat. Genet. 42, 790–793 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Cummings, N. et al. Combining target enrichment with barcode multiplexing for high throughput SNP discovery. BMC Genomics 11, 641 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Teer, J.K. et al. Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res. 20, 1420–1431 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Harakalova, M. et al. Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations. J. Cardiovasc. Transl. Res. 4, 271–280 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Fisher, S. et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol. 12, R1 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Mokry, M. et al. Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichment of short-fragment sequencing libraries. Nucleic Acids. Res. 38, e116 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Bainbridge, M.N. et al. Whole exome capture in solution with 3 Gbp of data. Genome Biol. 11, R62 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  24. 24

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

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Johansson, H. et al. Targeted resequencing of candidate genes using selector probes. Nucleic Acids. Res. 39, e8 (2011).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Lee, H. et al. Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing. BMC Genomics 10, 646 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank I. Wortel and E. Slob for testing the protocol. B. Hrdlickova was supported by The Rector's grant MUNI/E0136/2009 provided by Masaryk University, Czech Republic.

Author information

Affiliations

Authors

Contributions

All authors contributed extensively to protocol development and to the preparation of the manuscript. M.H. and M.M. created the protocol and wrote the manuscript. M.H., M.M., B.H., I.R., K.D., H.V. and E.D. performed the experiments and optimized experimental steps. I.R., N.L. and E.D. performed the multiplexed sequencing runs. I.J.N. wrote custom scripts for the array-based probe design. M.V. and I.J.N. performed the bar code splitting, data mapping and distribution analysis. W.P.K. and E.C. supervised the experiments and the development of the protocol.

Corresponding author

Correspondence to Edwin Cuppen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Graphical example of a manual for the level of multiplexing based on the size of design. (PDF 25 kb)

Supplementary Table 1

Oligonucleotide sequences (PDF 44 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Harakalova, M., Mokry, M., Hrdlickova, B. et al. Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing. Nat Protoc 6, 1870–1886 (2011). https://doi.org/10.1038/nprot.2011.396

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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