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

群体遗传学：使用分子计数对 DNA 进行绝对和相对定量

• 批准号: 710191
• 金额: $ 12.33万
• 依托单位: POPULATION GENETICS TECHNOLOGIES LTD
• 依托单位国家: 英国
• 项目类别: GRD Proof of Concept
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=710191
• 关键词: Population; Genetics; Absolute; Relative; Quantification

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.

尽管有许多不同的技术可用于测量 DNA 量，但每种技术在灵敏度、选择性、动态范围、便利性、稳健性和成本方面都存在权衡。由于固有的统计优势，数字方法（数字 PCR [dPCR]、NanoString、单分子测序）正在逐渐取代模拟替代方法（定量 PCR [qPCR]、微阵列）。群体遗传学 (PG) 和其他机构最近描述了基于下一代测序 (NGS) 的新数字计数方法，称为“CounterStamping”。感兴趣样本中的 DNA 分子用来自大量反序列的 DNA 反序列进行标记。计数器及其附着的标记分子的测序可以提供有关不同分子种类的相对丰度的信息并估计绝对分子数。由此生成的数据可以对已知和先前未识别的遗传变异进行量化，这与通常只能量化已知变异的现有方法不同。尽管有显着的好处，但计数方法的使用受到阻碍，因为与 qPCR/dPCR 相比，现有方案复杂且不方便。该提案旨在开发概念验证，以简化基于 PCR 的绝对和相对定量计数。与目前的方法相比，PG 提出的方法更快、更容易，并且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桌面测序仪的日益普及，使得计数 PCR 可能对现有的 qPCR 和 dPCR 技术产生颠覆性影响。此外，所提出的技术将推动疾病监测、伴随诊断和分子诊断等领域的新应用，这些领域需要更多的多重分析和精确的测量。