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Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2 : opportunities, challenges, and limitations
Author(s) -
De Leeneer Kim,
Hellemans Jan,
De Schrijver Joachim,
Baetens Machteld,
Poppe Bruce,
Van Criekinge Wim,
De Paepe Anne,
Coucke Paul,
Claes Kathleen
Publication year - 2011
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21428
Subject(s) - biology , amplicon , pyrosequencing , pseudogene , pooling , multiplex , computational biology , genetics , dna sequencing , massive parallel sequencing , gene , polymerase chain reaction , genome , computer science , artificial intelligence
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. Hum Mutat 31:1–10, 2011. © 2011 Wiley‐Liss, Inc.