Logan Mulroney from EMBL-EBI and IIT-CGS Italy presented at London Calling 2022 on “Detecting RNA modifications from nanopore ionic current signals.” They spoke about chemical and physical modifications of RNA and how they are “really dynamic.” I did not know there are “eraser” proteins that remove modifications, “writers” that add the modification, and “readers” that identify them and respond! RNA modification, Mulroney explained, can have implications in translation, decay, RNA stability, and disease states. They noted that there are ~170 known naturally occurring RNA modifications, most in tRNA. These modifications have been detected with mass spectrometry and NGS-based methods. RIP-Seq uses an antibody, Chem-Seq uses chemical modifications, and mismatch signature sequencing can be used to analyze RNA modifications. Nanopore sequencing and the direct RNA sequencing kit avoid amplification bias and provide an opportunity to sequence RNA “as in the cell.” Direct RNA nanopore sequencing takes poly(A) RNA, adds a poly(T) adaptor, and then a sequencing adaptors. Sequencing is from the 3′ to the 5′ end. RNA modifications cause disruptions in the ionic current that are detected in the traces. Nanocompore is software that detects modified RNA nucleotides and allows for biological variability and data analysis. After sequencing, there is basecalling, minimap2, q/c, and then comparison of conditions. The software clusters signals to compare modifications at different positions. Thresholds can be set to improve high probability modification sites. Mulroney shared data to review sites that were independently modified, for example. Mulroney concluded that RNA modifications can be detected by RNA nanopore sequencing and can be detected at the resolution of “near nucleotide resolution.” I find detection of RNA modifications fascinating… though I am not sure how much we could do with the system and bacteria we work with…
