Considerations and quality controls when analyzing cell-free tumor DNA.
Considerations and quality controls when analyzing cell-free tumor DNA.
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DOI：
10.1016/j.bdq.2018.12.003
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发表时间：
2019-03-01
影响因子：
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通讯作者：
Stahlberg, Anders
中科院分区：
文献类型：
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作者：
Johansson, Gustav;Andersson, Daniel;Stahlberg, Anders
循环无细胞肿瘤DNA （ctDNA）是一种很有前景的肿瘤生物标志物。超灵敏技术能够检测到低（< 0.1%）突变等位基因频率，这是充分利用ctDNA在癌症诊断中的潜力的先决条件。此外，整个液体活检工作流程需要仔细优化，以实现可靠的ctDNA分析。在这里，我们讨论了血浆中ctDNA检测的重要考虑因素。我们展示了如何使用简单的定量PCR分析轻松评估每个实验步骤，包括检测细胞DNA污染和PCR抑制。此外，ctDNA分析性能也被证明受到DNA片段和靶序列的影响。最后，我们表明，定量PCR是有用的估计所需的测序深度和监测整个工作流程中的DNA损失。质量控制分析的使用能够开发稳健和标准化的工作流程，从而促进ctDNA分析在临床常规中的实施。
Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.