Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes

Eur J Hum Genet. 2013 Aug;21(8):864-70. doi: 10.1038/ejhg.2012.270. Epub 2012 Dec 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • BRCA1 Protein / genetics*
  • BRCA2 Protein / genetics*
  • Cost-Benefit Analysis
  • DNA, Neoplasm / chemistry
  • DNA, Neoplasm / genetics
  • Female
  • Genetic Testing / economics
  • Genetic Testing / methods
  • Hereditary Breast and Ovarian Cancer Syndrome / diagnosis*
  • Hereditary Breast and Ovarian Cancer Syndrome / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Mutation
  • Polymerase Chain Reaction
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • BRCA1 Protein
  • BRCA2 Protein
  • DNA, Neoplasm