Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.
Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.
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DOI：
10.1186/s12920-014-0062-0
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发表时间：
2014-11-14
影响因子：
2.7
通讯作者：
Adai AT
中科院分区：
文献类型：
--
作者：
Choudhary A;Mambo E;Sanford T;Boedigheimer M;Twomey B;Califano J;Hadd A;Oliner KS;Beaudenon S;Latham GJ;Adai AT
新一代测序 (NGS) 性能和成本的提高刺激了其在临床应用中的快速采用。我们针对 51 种成熟的癌基因和肿瘤抑制因子设计并优化了泛癌靶标富集组合，并结合了由过程控制和分析前和分析后质量控制措施提供信息的生物信息学流程。该工作流程的评估包括对完整 DNA 混合物进行测序以建立分析灵敏度和精度，利用启发式方法识别系统伪影，对完整和 FFPE 样品进行滴定研究以优化输入，以及结合正交测序策略以提高阳性预测值和变异检测。我们还使用 128 个 FFPE 样本来评估临床准确性，并纳入先前描述的定量功能指数 (QFI) 进行样本鉴定，作为详细说明完整系统性能的一部分。我们观察到，在 4 次独立测序运行中，输入量为 250 ng 时观察到的变异百分比与预期变异百分比之间的一致性相关系数为 0.99。系统变异的一个子集被证实在独立测序平台上几乎无法检测到（Wilcox 符号秩检验 p 值 <10−16），并且正交测序策略的结合将突变检测的灵敏度和阳性预测值的调和平均值提高了 41%。在一组 FFPE 肿瘤样本中，覆盖率和平台间一致性与 QFI 呈正相关，这强调了分析前样本质量控制的必要性，以降低假阳性和假阴性的风险。在单独的 FFPE 样本队列中，基于中位丰度为 7.9% 的已知突变，51 基因组的灵敏度为 78%（95% CI= 56.3, 92.5），PPV 为 100%（95% CI= 81.5, 100.0）。通过使用正交 NGS 技术对样本进行测序，灵敏度提高至 87.0% (95% CI = 66.4,97.2)，同时保持 PPV。结果凸显了综合靶向 NGS 系统中流程集成的价值，可实现发现和诊断应用，特别是在对低质量癌症样本进行测序时。本文的在线版本 (doi:10.1186/s12920-014-0062-0) 包含补充材料，可供授权用户使用。
Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures. The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance. We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10−16), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV. The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens. The online version of this article (doi:10.1186/s12920-014-0062-0) contains supplementary material, which is available to authorized users.
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影响因子：
7.3
作者：
Gao J;Aksoy BA;Dogrusoz U;Dresdner G;Gross B;Sumer SO;Sun Y;Jacobsen A;Sinha R;Larsson E;Cerami E;Sander C;Schultz N
通讯作者：
Schultz N
影响因子：
4.1
作者：
Beadling, Carol;Neff, Tanaya L.;Corless, Christopher L.
通讯作者：
Corless, Christopher L.
使用下一代 DNA 测序数据进行变异发现和基因分型的框架。
影响因子：
30.8
作者：
通讯作者：
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影响因子：
46.9
作者：
通讯作者：
--
影响因子：
14.9
作者：
Forbes SA;Bindal N;Bamford S;Cole C;Kok CY;Beare D;Jia M;Shepherd R;Leung K;Menzies A;Teague JW;Campbell PJ;Stratton MR;Futreal PA
通讯作者：
Futreal PA