Piroon Jenjaroenpun: Uncovering RNA modifications from single molecule native RNA sequencing using ELIGOS
Piroon Jenjaroenpun, of the University of Arkansas for Medical Sciences, described the development of ELIGOS: a software for the identification of RNA modifications in nanopore direct RNA sequencing data. He described how there are over 100 chemical modifications in RNA; whilst there are several methods of profiling these modifications, each enriches only one specific type of modification at a time. Oxford Nanopore’s direct RNA sequencing data, however, preserves all types of RNA modification via direct sequencing of native RNA strands; these can be elucidated bioinformatically from “glitches” in the signal in sequencing. Piroon and his team developed ELIGOs, Epitranscriptional Landscape Inferring from Glitches of ONT Signals, a simple workflow for capturing multiple RNA modification types. RNA samples are sequenced using both direct RNA sequencing, in which native RNA modifications are intact, and direct cDNA sequencing, in which modifications are not present. Specific base sites which are frequently called in direct RNA as insertions, deletions and substitutions, but not in cDNA, are identified via a Fisher’s exact test, to determine likely modified base sites in the native RNA sample. The team used ELIGOs to capture modifications in yeast rRNA, identifying 95% of the modified bases previously elucidated by Yang et al. (Yang (2016) PLoS One. 2016; 11(12): e0168873) using RP-HPLC and a mung bean nuclease-based assay. Piroon also demonstrated the use of ELIGOS in the identification of differential proportions of m6a modifications in the junB proto-oncogene in samples of RNA from human embryonic kidney cells, CD8-positive cells and lung adenocarcinoma epithelial cells.