University of Minnesota Genomics Center, Minneapolis, MN, 55455, USA.
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
Genome Biol. 2019 Apr 29;20(1):85. doi: 10.1186/s13059-019-1691-6.
Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.
从工程构建体（如质粒、转座子或其他转基因）中定量 DNA 序列标签是许多功能基因组学测量的基础。通常，此类测量依赖于聚合酶链式反应（PCR），然后是下一代测序。然而，PCR 扩增可能会引入显著的定量误差。我们描述了一种新的无 PCR 直接计数方法 REcount。通过将定义的质粒池的测量值与液滴数字 PCR 数据进行比较，证明了 REcount 具有高度的准确性和可重复性。我们使用 REcount 来深入了解不同 Illumina 测序仪中由于分子长度引起的聚类偏差，并说明其对下一代测序数据解释和数据生成经济性的影响。
