TY - JOUR A2 - Cogulu, Ozgur AU - Ruiz, Anna AU - Llort, Gemma AU - Yagüe, Carmen AU - Baena, Neus AU - Viñas, Marina AU - Torra, Montse AU - Brunet, Anna AU - Seguí, Miquel A. AU - Saigí, Eugeni AU - Guitart, Miriam PY - 2014 DA - 2014/06/26 TI - Genetic Testing in Hereditary Breast and Ovarian Cancer Using Massive Parallel Sequencing SP - 542541 VL - 2014 AB - High throughput methods such as next generation sequencing are increasingly used in molecular diagnosis. The aim of this study was to develop a workflow for the detection of BRCA1 and BRCA2 mutations using massive parallel sequencing in a 454 GS Junior bench top sequencer. Our approach was first validated in a panel of 23 patients containing 62 unique variants that had been previously Sanger sequenced. Subsequently, 101 patients with familial breast and ovarian cancer were studied. BRCA1 and BRCA2 exon enrichment has been performed by PCR amplification using the BRCA MASTR kit (Multiplicom). Bioinformatic analysis of reads is performed with the AVA software v2.7 (Roche). In total, all 62 variants were detected resulting in a sensitivity of 100%. 71 false positives were called resulting in a specificity of 97.35%. All of them correspond to deletions located in homopolymeric stretches. The analysis of the homopolymers stretches of 6 bp or longer using the BRCA HP kit (Multiplicom) increased the specificity of the detection of BRCA1 and BRCA2 mutations to 99.99%. We show here that massive parallel pyrosequencing can be used as a diagnostic strategy to test for BRCA1 and BRCA2 mutations meeting very stringent sensitivity and specificity parameters replacing traditional Sanger sequencing with a lower cost. SN - 2314-6133 UR - https://doi.org/10.1155/2014/542541 DO - 10.1155/2014/542541 JF - BioMed Research International PB - Hindawi Publishing Corporation KW - ER -