Next Generation MUT-MAP, a High-Sensitivity High-Throughput Microfluidics Chip-Based Mutation Analysis Panel
Next Generation MUT-MAP, a High-Sensitivity High-Throughput Microfluidics Chip-Based Mutation Analysis Panel
复制标题
DOI：
10.1371/journal.pone.0090761
复制
发表时间：
2014-03-21
期刊：
影响因子：
3.7
通讯作者：
Raja, Rajiv
中科院分区：
文献类型：
--
作者：
Schleifman, Erica B.;Tam, Rachel;Raja, Rajiv
对肿瘤组织进行分子分析以检测变化，如致癌突变，在确定肿瘤学治疗方案方面起着至关重要的作用。因此，越来越需要一种稳健的高通量技术来检测致癌热点突变。尽管商业检测可用于检测单个基因中的遗传改变，但通常仅可从患者获得有限量的组织，需要多路复用以允许使用低DNA输入同时检测许多基因中的突变。尽管下一代测序（NGS）平台为此提供了强大的工具，但它们面临着高成本、大量DNA输入要求、复杂的数据分析和长周转时间等挑战，限制了它们在临床环境中的使用。我们报告了下一代突变多分析物面板（MUT-MAP）的开发，这是一种高通量微流体面板，用于使用等位基因特异性PCR（ASPCR）和Taqman技术检测11个治疗相关基因（AKT 1，BRAF，EGFR，FGFR 3，FLT 3，HRAS，KIT，KRAS，MET，NRAS和PIK 3CA）的120个体细胞突变。该突变组仅需要来自新鲜冷冻的2ng高质量DNA或来自福尔马林固定石蜡包埋（FFPE）组织的100 ng DNA。已实施包括自动数据分析过程在内的突变调用，以每天运行88个样本。使用质粒对该平台的验证在所有新添加的测定中显示出稳健的信号和低交叉反应性，并且发现细胞系样品中的突变调用与癌症体细胞突变目录（COSMIC）数据库一致，允许将我们的平台与桑格测序进行直接比较。当与在FFPE稀释实验中在Ion Torrent平台上运行的SuraSeq 500面板相比时，与NGS的高度相关性显示测定灵敏度低至0.45%。这种多重突变面板是个性化医疗和癌症药物开发中高通量生物标志物发现的宝贵工具。
Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multianalyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (ASPCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development.