Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-Seq

Abstract

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 RT enzymes used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those that derive from group II self-splicing introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multi-step template switching reaction carried out by RT enzymes, in this case, by a well-characterized enzyme known as MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized and optimized the enzymatic nontemplated addition (NTA) reaction that occurs when the RT enzyme extends past the RNA 5'-terminus, and we determined the nucleotide specificity of the NTA reaction. 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 starting 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, by employing mechanistic enzymology on RT enzymes and using them to modify RNA-seq technologies, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.