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Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR–based GS‐FLX sequencing
Author(s) -
Goossens Dirk,
Moens Lotte N.,
Nelis Eva,
Lenaerts AnSofie,
Glassee Wim,
Kalbe Andreas,
Frey Bruno,
Kopal Guido,
Jonghe Peter De,
Rijk Peter De,
DelFavero Jurgen
Publication year - 2009
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.20873
Subject(s) - biology , sanger sequencing , amplicon , genetics , multiplex , copy number variation , multiplex polymerase chain reaction , multiplex ligation dependent probe amplification , computational biology , dna sequencing , genome , massive parallel sequencing , gene , exon , polymerase chain reaction
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.