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Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia

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Stubbs, Samuel C. B.  ORCID logo
Blacklaws, Barbara A. 
Yohan, Benediktus 
Yudhaputri, Frilasita A. 
Hayati, Rahma F. 


Abstract: Background: Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. Methods: We evaluated a method designed to address this challenge, using the ‘Primal Scheme’ multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. Results: The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested (x¯ = 99.96%, n = 18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17–99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69–99.92%). However, coding-region coverage was reduced at this depth (x¯ = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. Conclusion: The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available.



Research, Positive-strand RNA viruses, Dengue virus, Disease surveillance, Virus sequencing, Nanopore sequencing

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Virology Journal

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BioMed Central
Medical Research Council (MR/P017541/1)
Dana Ilmu Pengetahuan Indonesia (ID) (MRCUK6)
Engineering and Physical Sciences Research Council (EP/510129/1)