超高敏突变检测的验证策略。
Validation Strategy for Ultrasensitive Mutation Detection.
机构信息
Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, MD, USA.
Department of Oncology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, MD, USA.
出版信息
Mol Diagn Ther. 2018 Oct;22(5):603-611. doi: 10.1007/s40291-018-0350-z.
BACKGROUND
Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients.
OBJECTIVE
The objective of this study was to develop a method to correct errors in low-level cell line mixes.
MATERIALS AND METHODS
We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing.
RESULTS
We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R = 0.80) and be more linear after introducing both corrections (R = 0.99).
CONCLUSIONS
The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.
背景
超敏检测低丰度 DNA 点突变是一个具有挑战性的分子生物学问题，因为几乎相同的突变型和野生型分子会发生串扰。可靠的超敏点突变检测将有助于早期发现癌症，并对癌症患者进行治疗监测。
目的
本研究旨在开发一种方法来纠正低水平细胞系混合物中的错误。
材料和方法
我们使用数字液滴 PCR（ddPCR）和下一代测序来测试样本混合物。
结果
我们引入了两种校正方法：用亲本细胞系中的基线变异等位基因频率（VAF）校正拷贝数变异；并使用单倍型计数校正细胞计数和移液中的错误。我们发现，ddPCR 在不应用任何校正的情况下，对检测低水平突变具有更好的相关性（R=0.80），在引入两种校正后，相关性更高（R=0.99）。
结论
VAF 校正比单倍型校正更显著。至关重要的是，应相互评估各种技术，并为实验室提供用于能力测试的定义质量控制样本。
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