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Title: Molecular Analysis of the Breast Cancer Genes BRCA1 and BRCA2 Using Amplicon-Based Massive Parallel Pyrosequencing
Authors: Michils, Geneviève ×
Hollants, Silke
Dehaspe, Luc
Van Houdt, Jeroen
Bidet, Yannick
Uhrhammer, Nancy
Bignon, Yves-Jean
Vermeesch, Joris
Cuppens Harry, Harry
Matthijs, Gert #
Issue Date: Nov-2012
Publisher: American Society for Investigative Pathology and the Association for Molecular Pathology
Series Title: The Journal of Molecular Diagnostics vol:14 issue:6 pages:623-630
Article number: 10.1016/j.jmoldx.2012.05.006
Abstract: The aim of this study was to implement the massively parallel sequencing technology for diagnostic applications. We evaluated an amplicon-based method for the analysis of the BRCA1 and BRCA2 genes on the Roche 454 GS-FLX sequencer, to identify disease-causing mutations in breast and/or ovarian cancer patients. A first evaluation relied on the analysis of DNA fragments containing known mutations. Secondly, the entire coding regions of the BRCA1 and BRCA2 genes were interrogated in more than 400 patient samples, using a multiplex PCR-based assay. Variants were filtered on the basis of their frequency (20%) and sequencing depth (>25×). Special attention was given to sequencing accuracy in homopolymers. In the initial evaluation, all known heterozygous mutations were detected. The percentage of mutant reads ranged from 22% to 62%. For the multiplex assay, 95% sensitivity and 91% specificity were obtained. In addition, we were able to reliably distinguish mutations from noise through the analysis of the raw signal intensities in homopolymers. This work presents an evaluation of the next-generation sequencing for use in diagnostics, based on a relatively high number of samples and experiments. We anticipate that the technique would further improve, and would allow reducing the costs per analysis and the turn-around time, to benefit patients who undergo BRCA molecular testing.
URI: 
ISSN: 1525-1578
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Laboratory for Molecular Diagnosis
Department of Human Genetics - miscellaneous
× corresponding author
# (joint) last author

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