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Direct Nanopore Sequencing of Individual Full Length tRNA Strands
ACS Nano ( IF 15.8 ) Pub Date : 2021-10-07 , DOI: 10.1021/acsnano.1c06488 Niki K Thomas 1 , Vinay C Poodari 1 , Miten Jain 1 , Hugh E Olsen 1 , Mark Akeson 1 , Robin L Abu-Shumays 1
ACS Nano ( IF 15.8 ) Pub Date : 2021-10-07 , DOI: 10.1021/acsnano.1c06488 Niki K Thomas 1 , Vinay C Poodari 1 , Miten Jain 1 , Hugh E Olsen 1 , Mark Akeson 1 , Robin L Abu-Shumays 1
Affiliation
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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.
中文翻译：
单个全长 tRNA 链的直接纳米孔测序
我们描述了一种使用 Oxford Nanopore MinION 进行直接 tRNA 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。第二个进展是针对 tRNA 优化的纳米孔测序管道。我们使用纯化的大肠杆菌tRNA fMet、tRNA Lys和 tRNA Phe样品测试了该方法。76-92% 的单个比对 tRNA 序列读数是全长的。作为概念证明，我们表明纳米孔测序检测了总大肠杆菌中的所有 43 个预期同工受体MRE600 tRNA 以及进一步定义该 tRNA 种群的同源编码器。三种纯化的 tRNA 与其合成对照之间基于比对的比较揭示了系统性核苷酸错误识别，这些错误识别可用于诊断已知修饰。在大肠杆菌总 tRNA 比对的已知修饰附近也观察到系统性错误识别，包括 T 环中高度保守的假尿苷。这项工作突出了纳米孔直接 tRNA 测序的潜力以及为人类医疗保健应用实施 tRNA 测序所需的改进。
更新日期：2021-10-26
中文翻译：
单个全长 tRNA 链的直接纳米孔测序
我们描述了一种使用 Oxford Nanopore MinION 进行直接 tRNA 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。第二个进展是针对 tRNA 优化的纳米孔测序管道。我们使用纯化的大肠杆菌tRNA fMet、tRNA Lys和 tRNA Phe样品测试了该方法。76-92% 的单个比对 tRNA 序列读数是全长的。作为概念证明，我们表明纳米孔测序检测了总大肠杆菌中的所有 43 个预期同工受体MRE600 tRNA 以及进一步定义该 tRNA 种群的同源编码器。三种纯化的 tRNA 与其合成对照之间基于比对的比较揭示了系统性核苷酸错误识别，这些错误识别可用于诊断已知修饰。在大肠杆菌总 tRNA 比对的已知修饰附近也观察到系统性错误识别，包括 T 环中高度保守的假尿苷。这项工作突出了纳米孔直接 tRNA 测序的潜力以及为人类医疗保健应用实施 tRNA 测序所需的改进。
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