Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR–based GS-FLX sequencing

Authors

  • Dirk Goossens,

    1. Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • Lotte N. Moens,

    1. Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • Eva Nelis,

    1. Neurogenetics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • An-Sofie Lenaerts,

    1. Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • Wim Glassee,

    1. Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • Andreas Kalbe,

    1. Roche Applied Science, Penzberg, Germany
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  • Bruno Frey,

    1. Roche Applied Science, Penzberg, Germany
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  • Guido Kopal,

    1. Roche Applied Science, Penzberg, Germany
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  • Peter De Jonghe,

    1. Neurogenetics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • Peter De Rijk,

    1. Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
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  • Jurgen Del-Favero

    Corresponding author
    1. Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
    2. University of Antwerp (UA), Antwerpen, Belgium
    • Applied Molecular Genomics Group, VIB Department of Molecular Genetics, University of Antwerp - Campus CDE, Parking P4, Building V, Room 1.14, Universiteitsplein 1, BE-2610 Antwerpen, Belgium
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  • Communicated by Mats Nilsson

Abstract

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50–500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. Hum Mutat 0,1–6, 2008. © 2008 Wiley-Liss, Inc.

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