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首页> 美国卫生研究院文献>Scientific Reports >High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency
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High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency

机译：RNA文库的高通量测序分析揭示了初始文库和PCR方法对SELEX效率的影响

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摘要

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct “biased sequences” and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the “biased sequences” was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.
机译：通过指数富集（SELEX）技术进行配体的系统进化是一种功能强大且有效的适体选择程序。但是，对过程的修改可以大大提高选择效率和适体性能。例如，最近将液滴数字PCR（ddPCR）集成到SELEX选择协议中，以推定减少副产物的传播并避免由于随机文库中序列的PCR效率差异而导致的选择偏差。但是，需要对RNA适体选择过程中常规溶液PCR和ddPCR修饰的功效进行详细的平行比较，以了解对整体SELEX性能的影响。在本研究中，我们利用强大的高通量测序技术和生物信息学分析结合SELEX（HT-SELEX）来彻底研究初始文库和PCR方法在RNA适体鉴定中的作用。我们的分析表明，从两个不同的生产商处购买的初始未选择文库中存在截然不同的“有偏序列”和核苷酸组成，并且“有偏序列”的命运在选择过程中取决于靶标。我们对溶液PCR和ddPCR驱动的HT-SELEX的比较表明，PCR方法不仅影响富集序列的核苷酸组成，而且影响整体SELEX效率和适体功效。

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