Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Hum Mutat. 2013 Apr;34(4):629-35. doi: 10.1002/humu.22272. Epub 2013 Feb 11.
The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false-positive calls without the introduction of false-negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing-Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time- and cost-effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.
台式高通量测序仪的问世使得大多数临床诊断实验室都能够接触到这项快速发展的技术。本研究采用 Ion Torrent Personal Genome Machine，提出了一种遗传性乳腺癌和卵巢癌的分子诊断策略及其分析验证方法。该方法依赖于 BRCA1 和 BRCA2 基因的多重 PCR 扩增，结合一个变异优先级处理管道，旨在在不引入假阴性结果的情况下，最大限度地减少假阳性结果的数量。使用一组训练样本来优化整个过程，然后使用另一组样本进行验证和独立评估工作流程的性能。在相对于样本中的变异进行盲法研究，并使用常规的 Sanger 测序作为标准的情况下，该工作流程的分析灵敏度≥98.6%，置信度为 95%，特异性为 96.9%。重要的是，没有错过任何真正的变异。本研究提出了一种全面的基于高通量测序和 Sanger 测序的策略，该策略具有高分析灵敏度的检测能力，为鉴定 BRCA1 和 BRCA2 基因突变提供了一种省时、省钱的策略。
