Limitations and challenges of genetic barcode quantification.
Limitations and challenges of genetic barcode quantification.
复制标题
DOI：
10.1038/srep43249
复制
发表时间：
2017-03-03
影响因子：
4.6
通讯作者：
Cornils K
中科院分区：
文献类型：
--
作者：
Thielecke L;Aranyossy T;Dahl A;Tiwari R;Roeder I;Geiger H;Fehse B;Glauche I;Cornils K
遗传条形码越来越多地用于跟踪单个细胞并定量评估它们随时间的克隆贡献。尽管条形码定量完全依赖于计数测序读数，但对该方法准确性的详细研究仍然有限。我们报告了条形码丰度与扩增和测序后产生的读取计数之间关系的系统调查，使用含有已知频率条形码的细胞混合物（“miniBulks”）。我们评估了方案修改的影响，以确定潜在的误差来源，并阐明量化方法可能存在的局限性。基于这些发现，我们设计了一种先进的条形码结构（BC32）来改进条形码调用和定量，并确保对高度稀释的条形码进行敏感检测。我们的研究结果强调了使用条形码库来获得可解释的定量数据的重要性，并强调了在可靠性和可重复性方面对任何使用的条形码库进行严格分析的必要性。
Genetic barcodes are increasingly used to track individual cells and to quantitatively assess their clonal contributions over time. Although barcode quantification relies entirely on counting sequencing reads, detailed studies about the method’s accuracy are still limited. We report on a systematic investigation of the relation between barcode abundance and resulting read counts after amplification and sequencing using cell-mixtures that contain barcodes with known frequencies (“miniBulks”). We evaluated the influence of protocol modifications to identify potential sources of error and elucidate possible limitations of the quantification approach. Based on these findings we designed an advanced barcode construct (BC32) to improved barcode calling and quantification, and to ensure a sensitive detection of even highly diluted barcodes. Our results emphasize the importance of using curated barcode libraries to obtain interpretable quantitative data and underline the need for rigorous analyses of any utilized barcode library in terms of reliability and reproducibility.