Abstract
To exploit contemporary sequencing technologies for targeted genetic analyses, we developed a hybridization enrichment strategy for DNA capture that uses PCR products as subgenomic traps. We applied this strategy to 115 kilobases of the human genome encompassing 47 genes implicated in cardiovascular disease. Massively parallel sequencing of captured subgenomic libraries interrogated 99.8% of targeted nucleotides ≥20 times (∼40,000-fold enrichment), enabling sensitive and specific detection of sequence variation and copy-number variation.
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Acknowledgements
We thank J. Aach, J. Gorham, J. Kim, J.B. Li and L. Wang for helpful discussions and advice; J. Kastelein and J.A. Kuivenhoven for HDL subject genomic DNA and for assistance in selecting HDL target genes; S. Depalma for analysis of Affymetrix custom sequencing array data; L. Merrill and D. Sonkin at Partners Center for Personalized Genetic Medicine of Harvard Medical School for Illumina sequencing and support; B. Richter, D. Gurgul, S. Roylance, J. Xu and A. Zschau at Partners Healthcare for access to high-performance computing cluster and data storage; T. Levi and B. McDonough for subject recruitment and genomic DNA isolation; S. Cox and M. Iaculli for generating HCM amplimers; and members of the Drosophila RNAi Screening Center for access to their Molecular Devices Analyst GT plate reader. G.K.H. was supported by grants from the Dutch Heart Association (NHS 2007R001) and Saal van Zwanenbergstichting. This work was supported by grants from the National Cancer Institute, the National Heart, Lung and Blood Institute, the Howard Hughes Medical Institute, and the Leducq Foundation.
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D.S.H., G.K.H., J.G.S. and C.E.S. conceived and designed the study; D.S.H. and G.K.H. constructed libraries and target concatemers; D.S.H. analyzed sequence data; O.I. and R.K. sequenced the libraries; H.L.R., D.S.H. and R.K. designed and amplified HCM sequences; D.S.H., J.G.S. and C.E.S. wrote the manuscript.
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Herman, D., Hovingh, G., Iartchouk, O. et al. Filter-based hybridization capture of subgenomes enables resequencing and copy-number detection. Nat Methods 6, 507–510 (2009). https://doi.org/10.1038/nmeth.1343
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DOI: https://doi.org/10.1038/nmeth.1343
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