title: Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes.
abstract: Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.
authors.affiliation: Hereditary Cancer Program, Catalan Institute of Oncology, L'Hospitalet de Llobregat, Barcelona, Spain.
authors.forename: Lídia
authors.initials: L
authors.lastname: Feliubadaló
authors.forename: Adriana
authors.initials: A
authors.lastname: Lopez-Doriga
authors.forename: Ester
authors.initials: E
authors.lastname: Castellsagué
authors.forename: Jesús
authors.initials: J
authors.lastname: del Valle
authors.forename: Mireia
authors.initials: M
authors.lastname: Menéndez
authors.forename: Eva
authors.initials: E
authors.lastname: Tornero
authors.forename: Eva
authors.initials: E
authors.lastname: Montes
authors.forename: Raquel
authors.initials: R
authors.lastname: Cuesta
authors.forename: Carolina
authors.initials: C
authors.lastname: Gómez
authors.forename: Olga
authors.initials: O
authors.lastname: Campos
authors.forename: Marta
authors.initials: M
authors.lastname: Pineda
authors.forename: Sara
authors.initials: S
authors.lastname: González
authors.forename: Victor
authors.initials: V
authors.lastname: Moreno
authors.forename: Joan
authors.initials: J
authors.lastname: Brunet
authors.forename: Ignacio
authors.initials: I
authors.lastname: Blanco
authors.forename: Eduard
authors.initials: E
authors.lastname: Serra
authors.forename: Gabriel
authors.initials: G
authors.lastname: Capellá
authors.forename: Conxi
authors.initials: C
authors.lastname: Lázaro
mesh_terms: D000465:Algorithms; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D003362:Cost-Benefit Analysis; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005260:Female; D005820:Genetic Testing / Q000191:economics / Q000379:methods; D061325:Hereditary Breast and Ovarian Cancer Syndrome / Q000175:diagnosis* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein; D004273:DNA, Neoplasm