Direct RNA sequencing (RNA004) allows for improved transcriptome assessment and near real- time tracking of methylation patterns for medical applications

Direct RNA sequencing (dRNA-seq) is a nanopore-based technique to sequence RNA in its native form without enzymatic conversion steps. In general, dRNA-seq holds great promise to resolve uncharted areas of human disease diagnostics and biomarker development. However - apart from a small core community - broader usage and acceptance of the method have been impeded by several methodological hindrances, such as low throughput, low accuracy, and missing large-scale modified base-calling models. Recently, ONT has released a new chemistry, a new nanopore (RNA-004), and new base-calling options (m6A). Here, we present the first results on two standardized test samples, namely UHRR (Universal Human Reference RNA) and HEK293T. Furthermore, we demonstrate current approaches for the detection of RNA modification, most notably m6A, from human blood samples as well as in a designer organelle equipped with an internal highly specific pseudouridinylation machinery allowing for targeted pseudouridinylation of specific RNAs in a “life-like” context. We show that the new chemistry has significantly improved throughput and accuracy and can, herewith, enable real-time tracking of state-specific methylation information. From this perspective, we provide an outlook on the extent to which this RNA flow cell is suitable for bringing RNA diagnostics and the study of RNA modification disorders (RNA modopathies) into a clinical application as a routine measure.