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

Autor:	Guo LT; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA., Grinko A; Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 97080 Würzburg, Germany., Olson S; Genetics and Genome Sciences, University of Connecticut Health, Farmington, Connecticut 06030, USA., Leipold AM; Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 97080 Würzburg, Germany.; University of Würzburg, Faculty of Medicine, Institute of Molecular Infection Biology (IMIB), 97070 Würzburg, Germany., Graveley B; Genetics and Genome Sciences, University of Connecticut Health, Farmington, Connecticut 06030, USA., Saliba AE; Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 97080 Würzburg, Germany.; University of Würzburg, Faculty of Medicine, Institute of Molecular Infection Biology (IMIB), 97070 Würzburg, Germany., Pyle AM; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA anna.pyle@yale.edu.; Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.; Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA.
Jazyk:	angličtina
Zdroj:	RNA (New York, N.Y.) [RNA] 2024 Oct 16; Vol. 30 (11), pp. 1495-1512. Date of Electronic Publication: 2024 Oct 16.
DOI:	10.1261/rna.080032.124
Abstrakt:	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. (© 2024 Guo et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu