RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries
RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries
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DOI：
10.1261/rna.2799511
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发表时间：
2011-09-01
期刊：
影响因子：
4.5
通讯作者：
Tuschl, Thomas
中科院分区：
文献类型：
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作者：
Hafner, Markus;Renwick, Neil;Tuschl, Thomas
小RNA cDNA文库测序是发现新RNA和分析其突变状态以及样品间表达变化的重要工具。它需要多个酶催化的步骤，包括连续的寡核苷酸接头连接到小RNA的3'和5'末端、逆转录（RT）和PCR。我们使用770种合成miRNA和45种校准寡核糖核苷酸的库评估了miRNA相对于其输入浓度的代表性的偏差，并测试了Rnl 1和Rnl 2的两种变体Rnl 2（1-249）和Rnl 2（1-249）K227 Q对3 '-接头连接的影响。与Rnll相比，使用Rnl 2变体进行衔接子连接产生了显著更少的副产物;然而，使用Rnl 2的益处仍然在很大程度上被5 '-衔接子连接步骤中的额外偏差所掩盖; RT和PCR步骤对读取频率没有显著影响。miRNA和/或miRNA/3 '-接头产物的分子内二级结构促成了这些偏差，这在限定的实验条件下是高度可重复的。我们使用合成的miRNA混合物来推导用于近似生物样品中单个miRNA的绝对水平的校正因子。最后，我们评估了5 '-末端5-nt条形码延伸对一组20个条形码化3'衔接子的影响，并观察到miRNA读段分布中的类似偏倚，从而实现了用于大规模miRNA谱分析的节省成本的多重分析。
Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.