Reducing ligation bias of small RNAs in libraries for next generation sequencing.
Reducing ligation bias of small RNAs in libraries for next generation sequencing.
复制标题
DOI：
10.1186/1758-907x-3-4
复制
发表时间：
2012-05-30
期刊：
影响因子：
--
通讯作者：
Dalmay T
中科院分区：
文献类型：
--
作者：
Sorefan K;Pais H;Hall AE;Kozomara A;Griffiths-Jones S;Moulton V;Dalmay T
核酸修饰酶的使用推动了分子生物学的快速发展。了解它们的功能对于改变或改善它们的活性很重要。然而，功能分析通常依赖于低通量实验。在这里，我们提出了一种使用下一代测序的核酸修饰酶的功能分析方法。我们证明，由RNA连接酶产生的文库的测序数据可以揭示这些酶的新的二级结构偏好，这些酶用于小RNA克隆和NGS文库制备。利用这些知识，我们证明了小RNA文库中的克隆偏倚是RNA连接酶依赖的。我们开发了一种高清晰度（HD）的协议，减少RNA连接酶依赖的克隆偏见。HD方案使读段覆盖率加倍，是定量的，并且发现了先前未鉴定的microRNA。此外，我们表明miRBase中的microRNA是主要测序平台的适配器优选的那些。小RNA的测序偏差部分影响了已经深入研究的microRNA，因此大多数以前的小RNA分析实验应该重新评估。可能会发现新的microRNA，它们是由现有的适配器选择的。目前使用的衔接子对已知microRNA的偏好表明，所有现有的小RNA（包括miRNA、siRNA和piRNA）的注释都有偏差。
The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing. We demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform. Sequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased.