Short-read RNA-seq yields lower estimates of A-to-I RNA editing levels than long-read cDNA sequencing.

A-to-I RNA editing, one of the most frequent RNA modifications, is essential for most aspects of RNA metabolism. However, the accuracy of quantifying A-to-I editing levels has been largely neglected, particularly when contrasting next-generation sequencing (NGS) with long-read sequencing (LRS) RNA-seq. To address it, we performed paired NGS and LRS cDNA RNA-seq of both HEK293T and U2OS cells, revealing that the A-to-I editing levels were estimated to be lower by NGS compared with LRS, a conclusion that was confirmed through full-length amplicon sequencing. Furthermore, the lower estimation of A-to-I editing levels is widespread, while we also identified this consequence using released public both same-study and cross-study NGS and LRS RNA-seq data in various human cancer cell lines. In addition, our analyses indicate that the primary factor contributing to this lower estimation is the sequencing read length difference, and a significantly positive correlation between the quantification and read length is identified by analyzing the public NGS RNA-seq data of various cell lines. We additionally found that shorter sequencing reads are more prone to aligning incorrectly to the reference genome, such as uniquely-to-multiply mapped and uniquely mapped-to-unmapped, inducing the lower estimation of A-to-I editing levels. In summary, our analyses indicate that LRS RNA-seq is preferable for accurately quantifying A-to-I editing levels.