Population Genetics: Absolute and Relative Quantification of DNA Using Molecular Counting

尽管有许多不同的技术可用于测量DNA的量，但每种技术在灵敏度、选择性、动态范围、方便性、鲁棒性和成本方面都有折衷。由于固有的统计学优势，数字方法（数字PCR [dPCR]、NanoString、单分子测序）正逐渐取代模拟方法（定量PCR [qPCR]、微阵列）。群体遗传学（PG）和其他人最近描述了基于下一代测序（NGS）的新数字计数方法，称为“计数器”。感兴趣的样品中的DNA分子用来自大量计数序列库的DNA计数序列标记。计数器及其标记分子的测序可以提供不同分子种类的相对丰度信息，并估计分子的绝对数量。由此产生的数据允许量化已知和以前未识别的遗传变异，不像现有的方法，通常只能量化已知的变异。尽管有显着的好处，计数方法的使用受到阻碍，因为现有的协议是复杂和不方便相比，qPCR/dPCR。该提案旨在开发概念验证，以简化和简化基于PCR的绝对和相对量化计数。PG提出的方法更快，更容易，与现有方法相比，减少了污染的可能性。PG还建议开发算法来纠正计数器序列中的错误，以提高该方法的准确性。NGS成本的持续降低以及台式测序仪的日益普及，使得PCR计数对现有的qPCR和dPCR技术具有潜在的破坏性。此外，拟议的技术将推动疾病监测，伴随诊断和分子诊断的新应用，其中需要更大的多路复用和精确的测量。

Although many different techniques are available to measure amounts of DNA, each has tradeoffswith respect to sensitivity, selectivity, dynamic range, convenience, robustness and cost.Digital methods (digital PCR [dPCR], NanoString, single-molecule sequencing) are graduallysuperseding analog alternatives (quantitative PCR [qPCR], microarrays) because of inherentstatistical advantages. Population Genetics (PG), and others, have recently described newdigital counting methods based on next-generation sequencing (NGS) termed‘CounterStamping’. DNA molecules in a sample of interest are labelled with a DNA countersequence from a large repertoire of counter sequences. Sequencing of the counter and itsattached labelled molecule can provide information on the relative abundance of differentmolecular species and estimate the absolute molecule number. The generated data therebyallow quantification of both known and previously unidentified genetic variation, unlikeexisting methods that can generally only quantify known variation.Despite significant benefits, use of counting methods is hampered because existing protocolsare complicated and inconvenient compared to qPCR/dPCR. This proposal is to develop proofof concept to streamline and simplify PCR-based counting for absolute and relativequantification. PG’s proposed method is quicker, easier and has reduced possibility ofcontamination compared to current approaches. PG also proposes to develop algorithms tocorrect errors in counter sequences to improve the accuracy of the method. Continuingreductions in NGS costs, and increased adoption of desktop sequencers, make counting PCRpotentially disruptive to existing qPCR and dPCR technologies. Further, the proposedtechnology will drive new applications in disease monitoring, companion diagnostics andmolecular diagnostics where greater multiplexing and precise measurements are required.