Sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies. Homo sapiens

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We propose an experimental solution to avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq). Overall design: RIP-seq and short RNA seq to identify RNAs interacting with EZH2 in T98G, U87MG and K562 cells.