Artifacts and biases of the reverse transcription reaction in RNA-sequencing

RNA sequencing has spurred a great number of research areas in recent years. Most protocols rely on converting RNA into a more stable complementary DNA copy during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process.

In this Review, Ghent University researchers confront the reader with intra- and inter-sample biases, and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader’s despair, the researchers also provide solutions to most issues and inform on good RNA sequencing practices. Their hope the reader can use this Review to his advantage, thereby contributing to scientifically sound RNA studies.

Overview of reverse transcription artifacts

Graphical overview of the five discussed priming artifacts. The reverse transcriptases are shown as yellow proteins (note the difference between yellow and orange reverse transcriptases). This figure was created using BioRender.