Nanopore-based direct sequencing of RNA transcripts with 10 different modified nucleotides reveals gaps in existing technology.
Nanopore-based direct sequencing of RNA transcripts with 10 different modified nucleotides reveals gaps in existing technology.
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DOI：
10.1093/g3journal/jkad200
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发表时间：
2023-11-01
影响因子：
2.6
通讯作者：
Cheung, Vivian G.
中科院分区：
文献类型：
--
作者：
Burdick, Joshua T.;Comai, Annelise;Bruzel, Alan;Sun, Guangxin;Dedon, Peter C.;Cheung, Vivian G.
RNA经历了复杂的转录后处理，包括核苷酸的化学修饰。由此产生的修饰核苷酸是RNA序列的组成部分，在研究RNA生物学和设计RNA疗法时必须考虑到这一点。然而，目前的“RNA测序”方法主要是对互补DNA进行测序，而不是RNA本身，这意味着RNA中存在的修改不会被捕获到测序结果中。新兴的直接RNA测序技术，如牛津纳米孔提供的技术，旨在解决这一限制。在这项研究中，我们合成并使用纳米孔技术对RNA转录本进行了测序，该转录本由标准核苷酸和10种不同浓度的不同修饰组成。结果表明，直接RNA测序仍有10%的基线错误率，尽管可以检测到一些修改，但许多修改仍然无法识别。因此，需要开发能够全面捕捉RNA的总复杂性的测序技术和分析方法。通过该项目获得的RNA序列可用于基准分析方法。
RNA undergoes complex posttranscriptional processing including chemical modifications of the nucleotides. The resultant-modified nucleotides are an integral part of RNA sequences that must be considered in studying the biology of RNA and in the design of RNA therapeutics. However, the current “RNA-sequencing” methods primarily sequence complementary DNA rather than RNA itself, which means that the modifications present in RNA are not captured in the sequencing results. Emerging direct RNA-sequencing technologies, such as those offered by Oxford Nanopore, aim to address this limitation. In this study, we synthesized and used Nanopore technology to sequence RNA transcripts consisting of canonical nucleotides and 10 different modifications in various concentrations. The results show that direct RNA sequencing still has a baseline error rate of >10%, and although some modifications can be detected, many remain unidentified. Thus, there is a need to develop sequencing technologies and analysis methods that can comprehensively capture the total complexity of RNA. The RNA sequences obtained through this project are made available for benchmarking analysis methods.
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影响因子：
14.9
作者：
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DOI：
10.15252/embj.201694885
发表时间：
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期刊：
The EMBO journal
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