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Filter-based hybridization capture of subgenomes enables resequencing and copy-number detection

Nature Methods volume 6, pages 507–510 (2009)Cite this article

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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Figure 1
Figure 2: Captured subgenomic library uniformity and SNP detection for HCM and HDL subgenomes.
Figure 3: Detection of CNV.

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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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Author notes

  1. J G Seidman and Christine E Seidman: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Daniel S Herman, G Kees Hovingh, Raju Kucherlapati, J G Seidman & Christine E Seidman

  2. Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

    G Kees Hovingh

  3. Partners Healthcare Center for Personalized Genetic Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Oleg Iartchouk, Heidi L Rehm, Raju Kucherlapati, J G Seidman & Christine E Seidman

  4. Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Heidi L Rehm

  5. Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Christine E Seidman

Authors
  1. Daniel S Herman
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  2. G Kees Hovingh
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  3. Oleg Iartchouk
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  4. Heidi L Rehm
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  6. J G Seidman
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  7. Christine E Seidman
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Contributions

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.

Corresponding author

Correspondence to Christine E Seidman.

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Supplementary Figures 1–9, Supplementary Tables 1–7 and Supplementary Methods (PDF 5040 kb)

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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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