Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications.
Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications.
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DOI：
10.1039/d3cb00081h
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发表时间：
2023-11-01
影响因子：
4.1
通讯作者：
中科院分区：
文献类型：
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纳米孔直接RNA测序是一种技术，它允许对表位转录修饰进行测序，并有可能进行定量评估。在本工作中，利用pH为7的亚硫酸氢盐反应前后的纳米孔对假尿苷(Ψ)进行了测序，该纳米孔在Ψ的C1‘处产生了稳定的核糖加合物。与Ψ相比，被引用的站点以删除签名的形式产生了更大的基本呼叫错误。对pH值为7的亚硫酸氢盐反应前后的大肠杆菌rRNA和tmRNA的测序研究表明，使用化学辅助纳米孔测序在减少数据中的假阳性和假阴性方面具有明显的优势。来自大肠杆菌的rRNA有19个已知的U/C序列变体，它们给出了与Ψ相似的碱基调用特征，因此，在检查碱基调用数据时是假阳性；然而，这些位置很难与亚硫酸氢盐反应，这在纳米孔数据中很容易观察到。E.colitmRNA在一个富含嘧啶的序列环境中有一个低占有率的Ψ，被称为U代表假阴性；Ψ的部分占有率在亚硫酸氢盐反应后被揭示。在最后一项研究中，在pH为5的亚硫酸盐反应后，RNA中很容易观察到5-甲基胞苷(M5C)，在该反应中，母体C脱氨基为U，修饰部位不反应。当使用亚硫酸氢盐辅助的纳米孔直接RNA测序时，这就定位了M5C，否则观察起来很有挑战性。讨论了整体方法的优势和挑战。纳米孔直接RNA测序由伪尿嘧啶和M5C特异性亚硫酸盐处理辅助，是一种允许对表位转录修饰进行测序的技术，并有可能进行定量评估。
Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (Ψ) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose adducts at C1′ of Ψ. The adducted sites produced greater base call errors in the form of deletion signatures compared to Ψ. Sequencing studies on E. coli rRNA and tmRNA before and after the pH 7 bisulfite reaction demonstrated that using chemically-assisted nanopore sequencing has distinct advantages for minimization of false positives and false negatives in the data. The rRNA from E. coli has 19 known U/C sequence variations that give similar base call signatures as Ψ, and therefore, are false positives when inspecting base call data; however, these sites are refractory to reacting with bisulfite as is easily observed in nanopore data. The E. coli tmRNA has a low occupancy Ψ in a pyrimidine-rich sequence context that is called a U representing a false negative; partial occupancy by Ψ is revealed after the bisulfite reaction. In a final study, 5-methylcytidine (m5C) in RNA can readily be observed after the pH 5 bisulfite reaction in which the parent C deaminates to U and the modified site does not react. This locates m5C when using bisulfite-assisted nanopore direct RNA sequencing, which is otherwise challenging to observe. The advantages and challenges of the overall approach are discussed. Nanopore direct RNA sequencing assisted by pseudouridine- and m5C-specific bisulfite treatment is a technology that allows sequencing for epitranscriptomic modifications with the possibility of quantitative assessment.
DOI：
10.1093/g3journal/jkaa002
发表时间：
2021-02-09
期刊：
G3 (Bethesda, Md.)
影响因子：
--
作者：
Gifford I;Dasgupta A;Barrick JE
通讯作者：
Barrick JE
影响因子：
15
作者：
Kietrys AM;Velema WA;Kool ET
通讯作者：
Kool ET