title: Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.
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.
authors.affiliation: Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: Goossens
authors.forename: Lotte N
authors.initials: LN
authors.lastname: Moens
authors.forename: Eva
authors.initials: E
authors.lastname: Nelis
authors.forename: An-Sofie
authors.initials: AS
authors.lastname: Lenaerts
authors.forename: Wim
authors.initials: W
authors.lastname: Glassee
authors.forename: Andreas
authors.initials: A
authors.lastname: Kalbe
authors.forename: Bruno
authors.initials: B
authors.lastname: Frey
authors.forename: Guido
authors.initials: G
authors.lastname: Kopal
authors.forename: Peter
authors.initials: P
authors.lastname: De Jonghe
authors.forename: Peter
authors.initials: P
authors.lastname: De Rijk
authors.forename: Jurgen
authors.initials: J
authors.lastname: Del-Favero
mesh_terms: D002607:Charcot-Marie-Tooth Disease / Q000175:diagnosis / Q000235:genetics; D017630:Connexins / Q000235:genetics; D051767:Early Growth Response Protein 2 / Q000235:genetics; D020558:GTP Phosphohydrolases; D018628:Gene Dosage; D020022:Genetic Predisposition to Disease / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics; D009154:Mutation; D018993:Myelin P0 Protein / Q000235:genetics; D009185:Myelin Proteins / Q000235:genetics; D016900:Neurofilament Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D000097002:Gap Junction beta-1 Protein
chemical_list: D017630:Connexins; D051767:Early Growth Response Protein 2; D020558:GTP Phosphohydrolases; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D018993:Myelin P0 Protein; D009185:Myelin Proteins; D016900:Neurofilament Proteins; D000097002:Gap Junction beta-1 Protein; C497326:EGR2 protein, human; C073687:PMP22 protein, human; C044882:neurofilament protein L; C470018:MFN2 protein, human