Tsutomu Suzuki from the University of Tokyo in Japan presented at London Calling 2023 a fifteen-minute recorded session about “Nanopore sequencing of tRNA modifications.” Suzuki is a biochemist in the field of RNA biology. They began describing how RNA modification enriches the diversity of RNA. Suzuki noted that tRNA modifications have been studied for a long time! There are also numerous known modifications of tRNA: tRNAs are “heavily decorated” according to Suzuki. tRNA modifications are important because they may regulate elongation speed and protein production. There are several approaches using Illumina-seq to learn about tRNA modifications. However, most of the information about tRNA modifications is eliminated during the library production. Nanopore sequencing is able to directly sequence RNA, thus helping identify modifications. Suzuki and colleagues extract total tRNAs from target specimens and profile them using Nanopore sequencing. However, Guppy and current basecallers have trouble interpreting signals from tRNA. Thus, Ueda and Suzuki created a new algorithm. For this project, Escherichia coli tRNA modifications were mapped. Suzuki shared a visual of how available basec allers have limitations when it comes to tRNA. The E. coli system is amenable to studying tRNA modifications with Nanopore sequencing because there are several deletion strains that are important. In the Suzuki lab, tRNAs were purified and used for adapter ligation and sequencing. The signals produced were used for a deep learning model for tRNAs. All highly pure tRNAs were mixed and then the sample was divided for Illumina and Nanopore sequencing. tRNA abundance varied. Normalization is important, and then E. coli tRNA profiling by nanopore seq and classification were conducted. Next, Suzuki and team developed a “detector” for unique modifications. Suzuki’s group is continuing to investigate and ways to detect modifications and train models.
