Abstract
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, Oxford Nanopore Technologies (ONT) has released a new chemistry, a new nanopore (RNA004), and new base-calling options (m6A). Here, we present the first results on two standardized test samples: 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 an artificial RNA modification system allowing for targeted pseudouridinylation of specific RNAs in a “life-like” context. The new chemistry has significantly improved throughput and accuracy and can support 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵$ Joint senior authors