Direct Nanopore Sequencing of Individual Full Length tRNA Strands.
Direct Nanopore Sequencing of Individual Full Length tRNA Strands.
复制标题
单个全长tRNA链的直接纳米孔测序。
DOI：
10.1021/acsnano.1c06488
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发表时间：
2021-10-26
期刊：
影响因子：
17.1
通讯作者：
Abu-Shumays, Robin L.
中科院分区：
文献类型：
--
作者：
Thomas, Niki K.;Poodari, Vinay C.;Jain, Miten;Olsen, Hugh E.;Akeson, Mark;Abu-Shumays, Robin L.
我们描述了一种使用牛津纳米孔小基因进行tRNA直接测序的方法。主要的技术进步是定制适配器，它有助于以亚纳米精度对单个转移RNA(TRNA)分子进行端到端测序。第二个进步是针对tRNA优化的纳米孔测序流水线。我们用纯化的大肠杆菌tRNAfMet、tRNALys和tRNAPhe样品测试了这种方法。76-92%的单个比对tRNA序列读数是全长的。作为概念的证明，我们证明了纳米孔测序检测到了总E.ColiMRE600 tRNA中的所有43个预期的异构体，以及进一步定义tRNA群体的等解码子。三个纯化的tRNA和它们的合成对照之间基于比对的比较显示了系统性的核苷酸错误调用，这是已知修饰的诊断。在已知的大肠杆菌总tRNA序列的修饰附近也观察到系统性的错误调用，包括在T环中高度保守的假尿苷。这项工作突出了纳米孔直接tRNA测序的潜力以及为实现人类医疗保健应用的tRNA测序所需的改进。
We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual transfer RNA (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76–92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total E. coli MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.
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