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

In the field of virology, rapid and accurate characterization of viral genomes is vital for understanding their biology, pathogenic potential, and evolution through genomic surveillance. Oxford Nanopore Technologies (ONT) offer Direct RNA Sequencing, which is an innovative approach to obtaining comprehensive genomic information by reading RNA molecules in their native form (Garalde et al., 2019). This eliminates the need for reverse transcription (RT) and prior amplification by polymerase chain reaction (PCR), limiting sequencing biases and preserving native RNA sequences. Although Direct RNA sequencing has been shown to be effective for viral sequencing (Vacca et al., 2022), it presents certain challenges due to its requirement for high RNA inputs (300 ng of poly(A) tailed RNA or 1 ug of total RNA). Such RNA quantities can often be difficult to obtain. Additionally, a significant limitation of Direct RNA sequencing is the absence of a multiplexing protocol, necessitating the use of one RNA flow cell per sample, which results in a high cost per sample. In this study, we aimed to evaluate the feasibility of sequencing arbovirus genomic RNA with very low starting material and achieving multiplexing by sequencing several strains of two arboviruses on the same flow cell. We employed Direct RNA sequencing to sequence five arbovirus strains, including three strains of dengue virus (DENV) and two strains of chikungunya virus (CHIKV), while conducting two separate experiments. The first experiment (A) focused on using different ranges of input RNA (50, 5 and 0.5 ng) for one strain of each arbovirus, and the second experiment (B) tested the multiplexing of the five arboviruses strains on the same flow cell. For the latter, we first ensured that the percentage of genomic differences among our five arboviruses strains was sufficient to allow for multiplexing based on read mapping. We demonstrated that Direct RNA sequencing is viable even with ultra-low viral RNA input levels, achieving excellent coverage of nearly the entire genome length. This approach also enabled the effective sequencing of a custom multiplexing library for five different arboviruses strains, resulting in more than 61 X read depth and covering more than 99 % of the reference genome for both native and poly(A)-tailed RNA genomes.