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.
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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.
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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.
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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)
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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
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DOI: https://doi.org/10.1038/nprot.2011.396
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