Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 97080 Würzburg, Germany.
RNA. 2024 Oct 16;30(11):1495-1512. doi: 10.1261/rna.080032.124.
End-to-end RNA-sequencing methods that capture 5'-sequence content without cumbersome library manipulations are of great interest, particularly for analysis of long RNAs. While template-switching methods have been developed for RNA sequencing by distributive short-read RTs, such as the MMLV RTs used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those derived from group II introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multistep template-switching reaction carried out by RTs, in this case, by MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized the nucleotide specificity of nontemplated addition (NTA) reaction that occurs when the RT extends past the RNA 5'-terminus. We then evaluated the binding specificity of specialized template-switching oligonucleotides, optimizing their sequences and chemical properties to guide efficient template-switching reaction. Having dissected and optimized these individual steps, we then unified them into a procedure for performing RNA sequencing with MarathonRT enzymes, using a well-characterized RNA reference set. The resulting reads span a six-log range in transcript concentration and accurately represent the input RNA identities in both length and composition. We also performed RNA-seq from total human RNA and poly(A)-enriched RNA, with short- and long-read sequencing demonstrating that MarathonRT enhances the discovery of unseen RNA molecules by conventional RT. Altogether, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.
无需繁琐的文库操作即可捕获 5'序列内容的端到端 RNA 测序方法非常有吸引力，特别是对于长 RNA 的分析。虽然已经开发了用于通过分布式短读 RT（如 SMART-Seq 方法中使用的 MMLV RT）进行 RNA 测序的模板转换方法，但它们尚未适应利用超延伸 RT（如衍生自 II 组内含子的 RT）的优势。为了促进这种转变，我们剖析了指导 RT 进行多步模板转换反应的酶特异性和效率的各个过程，在这种情况下，是通过 MarathonRT 进行的。值得注意的是，这是同类研究中的第一项，适用于任何 RT。首先，我们表征了当 RT 延伸超过 RNA 5'-末端时发生的非模板添加（NTA）反应的核苷酸特异性。然后，我们评估了专门的模板转换寡核苷酸的结合特异性，优化了它们的序列和化学性质以指导有效的模板转换反应。在剖析和优化这些单独的步骤之后，我们将它们统一到使用经过充分表征的 RNA 参考集进行 MarathonRT 酶 RNA 测序的程序中。生成的读数跨越转录物浓度的六个对数范围，并准确代表输入 RNA 在长度和组成上的身份。我们还从总人 RNA 和 poly(A)-富集 RNA 进行了 RNA-seq，短读和长读测序表明 MarathonRT 通过常规 RT 增强了对未见 RNA 分子的发现。总之，我们已经生成了一种新的快速、准确的方法，用于对包含长 RNA 转录物混合物的复杂 RNA 文库进行测序。
