Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.
Genome Res. 2011 Sep;21(9):1506-11. doi: 10.1101/gr.121715.111. Epub 2011 Jul 12.
Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number.
第二代测序技术逐渐成为 miRNA 检测和表达谱分析的首选方法。鉴于 miRNA 的数量相对较少，以及 DNA 测序技术的不断改进，在单个流动池道中研究多个样本的 miRNA 表达谱成为可能。多重策略需要用 DNA 条码标记每个 miRNA 文库。本文报道，通过接头连接引入的条码对 miRNA 表达谱产生显著的偏差。这种偏差比预期的泊松噪声高得多，掩盖了 miRNA 文库之间的显著表达差异。通过在文库的 PCR 扩增过程中添加条码可以消除这种偏差。多重实验中 miRNA 表达测量的准确性成为样本数量的函数。
