High-throughput multiplex sequencing of miRNA.
High-throughput multiplex sequencing of miRNA.
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DOI：
10.1002/0471142905.hg1112s73
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发表时间：
2012-04
影响因子：
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通讯作者：
M Church, George
中科院分区：
文献类型：
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作者：
Vigneault, Francois;Ter-Ovanesyan, Dmitry;Alon, Shahar;Eminaga, Seda;C Christodoulou, Danos;Seidman, J G;Eisenberg, Eli;M Church, George
与其他microRNA （miRNA）表达谱分析方法相比，下一代测序具有许多优势，例如样品吞吐量和发现新miRNA的能力。由于当前测序平台的测序深度超过了量化mirna所需的深度，因此在一次测序运行中复用多个样本具有显著的成本优势。虽然以前的研究通过在连接步骤中向miRNA库添加条形码实现了这一目标，但最近的研究表明，这对miRNA表达数据产生了显著的偏差。然而，这种偏差可以通过在PCR步骤中对miRNA文库进行条形码来避免。在这里，我们描述了一种用户友好的PCR条形码方法，用于制备基于illumina测序的多路microRNA文库。该方法还可以防止适配器二聚体的产生，并且可以在一天内完成。
Next-Generation Sequencing offers many advantages over other methods of microRNA (miRNA) expression profiling, such as sample throughput and the capability to discover novel miRNAs. As the sequencing depth of current sequencing platforms exceeds what is necessary to quantify miRNAs, multiplexing several samples in one sequencing run offers a significant cost advantage. Although previous studies have achieved this goal by adding barcodes to miRNA libraries at the ligation step, this was recently shown to introduce significant bias into the miRNA expression data. This bias can be avoided, however, by barcoding the miRNA libraries at the PCR step instead. Here, we describe a user-friendly PCR bar-coding method of preparing multiplexed microRNA libraries for Illumina-based sequencing. The method also prevents the production of adapter dimers and can be completed in one day.