Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations

Hum Mutat. 2011 Mar;32(3):335-44. doi: 10.1002/humu.21428. Epub 2011 Feb 8.


This study describes how the new massive parallel sequencing technology can be implemented in a diagnostic setting for the breast cancer susceptibility genes (BRCA1 and BRCA2). The throughput was maximized by increasing uniformity in coverage, obtained by a multiplex approach, which outperformed pooling of singleplex PCRs. We evaluated the sensitivity by analysis of 133 distinct sequence variants; three (2%) deletions or duplications in homopolymers of greater than or equal to seven nucleotides remained undetected, illustrating a limitation of pyrosequencing. Furthermore, other limitations like nonrandom sequencing errors, pseudogene amplification, and failure to detect multiexon deletions are thoroughly described. Our workflow illustrates the potential of massive parallel sequencing of large genes in a diagnostic setting, which is of great importance to meet the increasing expectations of genetic testing. Implementation of this approach will hopefully lead to a strong reduction in turnaround times. As a consequence a wider spectrum of at risk women will be able to benefit from therapeutic interventions and prophylactic interventions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • BRCA1 Protein / genetics*
  • BRCA2 Protein / genetics*
  • Base Sequence
  • Breast Neoplasms / genetics*
  • DNA Mutational Analysis / methods
  • Female
  • Genes, BRCA1*
  • Genes, BRCA2*
  • Genetic Predisposition to Disease
  • Genetic Testing / methods
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Screening Assays / methods
  • Humans
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods


  • BRCA1 Protein
  • BRCA1 protein, human
  • BRCA2 Protein
  • BRCA2 protein, human