Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification
Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification
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DOI：
10.1186/s13059-019-1691-6
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发表时间：
2019-04-29
期刊：
影响因子：
12.3
通讯作者：
Beckman, Kenneth B.
中科院分区：
文献类型：
--
作者：
Gohl, Daryl M.;Magli, Alessandro;Beckman, Kenneth B.
定量DNA序列标签工程结构，如质粒，转座子，或其他转基因基础上的许多功能基因组学测量。通常，这种测量依赖于PCR，然后是下一代测序。然而，PCR扩增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。将定义的质粒池的测量值与液滴数字PCR数据进行比较，表明重新计数是高度准确和可重复的。我们使用重新计数来对不同Illumina测序仪的分子长度引起的聚类偏差提供新的见解，并说明对下一代测序数据解释和数据生成经济学的影响。
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.