Massive Parallel Amplicon Sequencing of the Breast Cancer Genes BRCA1 and BRCA2: Opportunities, Challenges, and Limitations
Massive Parallel Amplicon Sequencing of the Breast Cancer Genes BRCA1 and BRCA2: Opportunities, Challenges, and Limitations
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DOI：
10.1002/humu.21428
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发表时间：
2011-03-01
期刊：
影响因子：
3.9
通讯作者：
Claes, Kathleen
中科院分区：
文献类型：
--
作者：
De Leeneer, Kim;Hellemans, Jan;Claes, Kathleen
这项研究描述了新的大规模平行测序技术如何在乳腺癌易感基因（BRCA1和BRCA2）的诊断环境中实施。通过增加复路方法获得的覆盖均匀性来最大化吞吐量，该方法优于单路pcr的池化。我们通过分析133个不同的序列变异来评估敏感性；在大于或等于7个核苷酸的均聚物中仍未检测到3个（2%）缺失或重复，说明焦磷酸测序的局限性。此外，其他限制，如非随机测序错误，假基因扩增，和失败检测多外显子缺失被彻底描述。我们的工作流程说明了在诊断环境中对大基因进行大规模平行测序的潜力，这对于满足基因检测日益增长的期望非常重要。这种方法的实施有望大大缩短周转时间。因此，更广泛的高危妇女将能够从治疗干预和预防干预中受益。[j], 2011。(C) 2011 Wiley-Liss, Inc。
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 32:335-344, 2011. (C) 2011 Wiley-Liss, Inc.