Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA.
Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA.
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DOI：
10.1016/j.jmoldx.2020.01.012
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发表时间：
2020-02
期刊：
影响因子：
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通讯作者：
M. Gulley;Sandra H. Elmore;G. Gupta;Sunil Kumar;Matthew Egleston;Ian J. Hoskins;A. Garnett
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文献类型：
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作者：
M. Gulley;Sandra H. Elmore;G. Gupta;Sunil Kumar;Matthew Egleston;Ian J. Hoskins;A. Garnett
大规模平行测序的一个问题是，体细胞突变和病毒载量通常被量化为相对于野生型人类DNA的分数，但野生型水平随着不同的生物学和分析前干扰而变化。设计了一种新的策略，在对加标DNA的读段计数进行标准化后，以每mL血浆的拷贝数定量靶分析物。在文库制备之前，将五种合成DNA（称为EndoGenus spikes）添加到血浆中（修改的ArcherDX Liquidescent 28）。通过归一化至EndoGenus加标读数的部分回收率，每种疾病标志物的数值以拷贝/mL为单位进行报告。为了显示该系统的运行情况，对具有23个工程化突变的40个模拟血浆和21个天然血浆进行了重复测定。回收了所有5个EndoGenus加标物的读数（平均值，在模拟和天然血浆中分别为313和376拷贝/mL）。标准化读数计数以实现峰值的比例回收有助于控制多步方案中的变量，将重复检测中突变的CV从34%降至22%，将病毒载量的CV从25%降至7%。总之，EndoGenus系统可用于评估整个测试系统的效率和精确定量靶分子。该系统可能有利于监测疾病负担的患者，同时还可以跟踪新兴的亚克隆。
A problematic aspect of massive parallel sequencing is that somatic mutations and viral loads are typically quantified as a fraction relative to wild-type human DNA, yet wild-type levels vary with diverse biologic and preanalytic interferences. A novel strategy was devised to quantify target analytes in copies per mL of plasma after normalizing for read counts of spiked DNAs. Five synthetic DNAs (called EndoGenus spikes) were added to plasma before library preparation (modified ArcherDX LiquidPlex 28). By normalizing to the fractional recovery of EndoGenus spike reads, numerical values for each disease marker were reportable in units of copies per mL. To show how well this system operates, replicate assays were performed on 40 mock plasmas having 23 engineered mutations and on 21 natural plasmas. Reads for all five EndoGenus spikes were recovered (means, 313 and 376 copies/mL in mock and natural plasmas, respectively). Normalizing read counts for the proportional recovery of spikes helped control for variables in the multistep protocol, reducing the CV in replicate tests from 34% to 22% for mutations and from 25% to 7% for viral loads. In conclusion, the EndoGenus system is useful for evaluating efficiency of the total test system and for precisely quantifying target molecules. This system may benefit patients being monitored for disease burden while also tracking emerging subclones.