Misincorporation signatures for detecting modifications in mRNA: Not as simple as it sounds

•Next generation sequencing enables transcriptome wide detection of RNA modifications.•Analyzing misincorporation signatures facilitates detection of various modifications.•Misincorporation analysis in sequencing data is prone to diverse artifacts.•Experimental and computational artifacts lead to spurious detection of modification.•The sources of artifacts and approaches to detect and overcome them are discussed. Post-transcriptional modification on mRNA has become a field of intense interest in recent years, and next-generation sequencing based technologies are constantly emerging to detect an increasing number of modifications at a transcriptome-wide level. Some of these approaches are based on identification of misincorporation events induced by reverse transcriptase at modified sites. Although conceptually trivial, sensitive and specific identification of such events is a challenge prone to a surprising number of artifacts, which can lead to substantially inflated estimates of the abundance of diverse modifications. Here we discuss the sources of some of these artifacts and delineate approaches to overcome them.