Direct RNA Sequencing of Dengue and Chikungunya Viruses with Ultra-Low Input and Custom Multiplexing.

Accurate viral genome characterization is essential for understanding viral dynamics. Oxford Nanopore Technologies (ONT) Direct RNA Sequencing provides detailed genomic data by directly reading RNA molecules, avoiding biases from reverse transcription and PCR. However, it requires high RNA inputs and lacks a multiplexing protocol, raising costs. This study explores Direct RNA Sequencing with minimal RNA inputs and multiplexing for arboviruses. We sequenced five strains, including dengue and chikungunya viruses, to test ultra-low RNA inputs and multiplexing. Results show successful sequencing and over 99% genome coverage with ultra-low inputs, highlighting the potential of Direct RNA Sequencing in genomic surveillance and the detection of RNA modifications. This method also shows promise for future application in sequencing patient serum samples without the need for viral cultivation.