A Nanopore-based method for generating complete coding region sequences of dengue virus in resource-limited settings

Dengue virus (DENV) sequencing is a vital tool for surveillance and epidemiology studies. However, the current methods employed for sequencing DENV are expensive, laborious and technically demanding, often due to intra- and inter-serotype variability. Therefore, on site DENV sequencing is not feasible in many of the areas where DENV is endemic. Surveillance in these areas can only be performed by shipping samples to well-equipped central laboratories for sequencing. However, long periods of inadequate storage and unreliable shipping conditions mean that such samples can arrive degraded, rendering sequence recovery difficult. We therefore aimed to develop an approach that is simple, portable and effective, to be used for on-site DENV sequencing in limited resource settings. To achieve this, we first used the "Primal Scheme" primer design tool to develop a simple and robust protocol for generating multiple short amplicons, covering the complete coding-region of DENV isolates. We then paired this method with the Nanopore MinION, a portable and affordable sequencing device, well-suited to minimal resource settings. The multiplex PCR method produced full-coding-region coverage of all DENV samples tested with no optimisation required, and Nanopore sequencing of the short amplicons generated consensus sequences with high accuracy (99.52 - 99.92 %). Phylogenetic analysis of the consensus sequences generated using the new method showed that they formed monophyletic clusters with those produced by the current, long-amplicon, Illumina method, thus demonstrating that the two approaches are comparable. The multiplex method's simplicity and portability compared to the current DENV sequencing approach make it well-suited for use in resource-limited, DENV-endemic regions. Deployment of the method in these regions would increase the capacity for DENV surveillance and has the potential to provide vital resolution for future DENV epidemiology studies.