Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland.
SeraCare Life Sciences, Inc., Milford, Massachusetts.
J Mol Diagn. 2019 Jul;21(4):658-676. doi: 10.1016/j.jmoldx.2019.03.006. Epub 2019 May 2.
We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.
我们进行了一项多实验室评估，以确定在四种不同变异等位基因频率（VAF）下，即 2%、0.50%、0.125%和 0%，一种新型市售参考物质对于野生型 DNA 背景下 40 种癌症变异的适用性。这些变异包括单核苷酸、插入、缺失和两种结构变异，它们是根据其临床重要性和对下一代测序（NGS）方法性能的挑战选择的。碎片化 DNA 被配方模拟循环野生型和肿瘤 DNA 在合成血浆基质中的大小分布。从这些样本中提取 DNA，并通过不同的方法和多个实验室进行了表征。各种提取方法的产量存在差异，这可能是由于化学物质的差异。数字 PCR 检测用于测量 VAF，以比较来自不同 NGS 方法的结果。在不同的 NGS 方法中观察到了可比的 VAF。这项多实验室评估表明，新型参考物质是一种确定不同测量方法分析参数并确保其质量保证的合适工具。
