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

Published version
Peer-reviewed

Type

Article

Change log

Authors

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 ([Formula: see text] = 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 ([Formula: see text] = 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.

Description

Keywords

Dengue virus, Disease surveillance, Nanopore sequencing, Virus sequencing, Dengue, Dengue Virus, Genome, Viral, Humans, Indonesia, Multiplex Polymerase Chain Reaction, Nanopores, Phylogeny, Sequence Analysis, DNA

Journal Title

Virol J

Conference Name

Journal ISSN

1743-422X
1743-422X

Volume Title

17

Publisher

Springer Science and Business Media LLC
Sponsorship
Medical Research Council (MR/P017541/1)
This study was co-funded by the UK Medical Research Council and the Indonesian Science Fund (DIPI) as part of the UK-Indonesia Joint Health Research Call on Infectious Diseases (2016). SDWF was supported in part by The Alan Turing Institute via an Engineering and Physical Sciences Research Council grant.