Uncovering RNA and DNA Modifications from Native Sequences

Abstract

Ribonucleotides modifications to mRNA play important roles biological regulations. Over 170 types of RNA modifications have been experimentally validated. Their detection traditionally relies on specific antibody-based enrichment and analytical chemistry tools; these approaches are labor intensive and can detect only one or a few modifications at a time. This is insufficient to truly assess complete transcriptomes for sequence-specific identification and quantitation of epigenetic signals. Recently, we were the first to use third-generation Oxford Nanopore Technology (ONT) sequencing to directly sequence cellular RNA in native from, at a transcriptomic level. We determined that the method can uncover RNA modifications of any type. Based on the principle that such modifications are absent on cDNA or synthetical unmodified RNA, we conducted a study that compared sequence features of native modified RNA with unmodified RNA of the same sequence. We developed a bioinformatics tool, ELIGOS (Epitranscriptional Landscape Inferring from Glitches of ONT Signals), that successfully identified modified RNA bases from the native RNA sequences. ELIGOS accurately predicts known classes of RNA methylation sites (AUC > 0.93) in rRNAs from E. coli, yeast, and human cells, by using either unmodified in vitro transcribed RNA or our developed background-error model, which mimics the systematic error in native RNA sequences. The validity of the approach was illustrated in transcriptomes of yeast, mouse, and human cells. We further apply ELIGOS in detection of DNA adducts and for distinguishing individual alkylated DNA adducts. We analyzed a library of 16 plasmids containing site-specifically inserted O6- or N2-alkyl-deoxyguanosine lesions differing in sizes, functional group, regiochemistries, and abasic site. Based on the native DNA sequences, ELIGOS can accurately identified the location of individual DNA adducts. Moreover, individual DNA adducts were clearly distinguished from each other at the signal level. ELIGOS software is publicly available and can be used to detect possible RNA and DNA modification sites at genome-scale from native RNA/DNA sequences.