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Abstract
New analytics of post-transcriptional RNA modifications have paved the way for a tremendous upswing of the biological and biomedical research in this field. This especially applies to methods that included RNA-Seq techniques, and which typically result in what is termed global scale modification mapping. In this process, positions inside a cell’s transcriptome are receiving a status of potential modification sites (so called modification calling), typically based on a score of some kind that issues from the particular method applied. The resulting data are thought to represent information that goes beyond what is contained in typical transcriptome data, and hence the field has taken to use the term “epitranscriptome”. Due to the high rate of newly published mapping techniques, a significant number of chemically distinct RNA modifications have become amenable to mapping, albeit with variegated accuracy and precision, depending on the nature of the technique. This review gives a brief overview of known techniques, and how they were applied to modification calling.
Highlights
Precise and reliable detection of the numerous modified nucleotides in RNA is still a highly challenging task in the epitranscriptomics field
A novel trend in the field is the development of library preparation protocols which selectively produce RNA fragments with modified nucleotides at the 50 - or 30 -end extremity, properties which can be exploited for enrichment of these fragments in RNA Seq libraries
Aldehyde reactivity at the RNA abasic site or decomposed RNA modified nucleotide was not yet explored for coupling to deep sequencing. This overview might convey the impression that RNA-Seq based detection methods can be successfully applied for mapping of almost any desired RNA modification throughout the transcriptome
Summary
Precise and reliable detection of the numerous modified nucleotides in RNA is still a highly challenging task in the epitranscriptomics field. In addition to traditional detection and quantification strategies, including various types of chromatography such as TLC, HPLC, and/or LC-MS(MS), approaches relying on deep sequencing have emerged in force, and numerous applications to large transcriptomes were reported, starting in 2012 [1,2,3,4,5], with a dynamic development during the past two–three years These methods provided an unprecedented amount of information and depth, in certain cases even allowing the analysis of low abundant RNA species. Another variant of miCLIP (PA-m6 A-Seq) used s4 U incorporation for PhotoActivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP, [24])
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