Massive parallel amplicon sequencing of the breast cancer genes BRCA1&2 : opportunities, challenges and limitations.

Kim de Leeneer (1) , Jan Hellemans (1) , Joachim de Schrijver (2) , Machteld Baetens (1) , Bruce Poppe (1) , Wim van Criekinge (2) , Anne Depaepe (1) , Paul Coucke (1) , Kathleen B.M. Claes (1)

1 Center for Medical Genetics [Ghent]
2 UGENT - Universiteit Gent = Ghent University

This study describes how the new massive parallel sequencing technology can be implemented in a diagnostic setting by proof of concept studies for the breast cancer susceptibility genes (BRCA1&2). Throughput was maximized by increasing uniformity in coverage. This was obtained by a multiplex approach, which outperformed pooling of singleplex PCRs. We evaluated the sensitivity by analysis of 133 distinct sequence variants; 3 (2%) deletions or duplications in homopolymers of ≥7 nucleotides remained undetected, illustrating a limitation of pyrosequencing. Furthermore, other limitations like non random sequencing errors, pseudogene amplification and failure to detect of multi exon deletions are thoroughly described. Our workflow certainly has the potential for high throughput analysis of large genes in diagnostic settings, 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, so a wider spectrum of at risk women will be able to benefit from therapeutic interventions for which knowledge of the DNA sequence is essential.

Mots clés: Life Sciences

Kim de Leeneer, Jan Hellemans, Joachim de Schrijver, Machteld Baetens, Bruce Poppe, et al.. Massive parallel amplicon sequencing of the breast cancer genes BRCA1&2 : opportunities, challenges and limitations.. Human Mutation, 2011, 32 (3), pp.335. ⟨10.1002/humu.21428⟩. ⟨hal-00613912⟩