Stubbs, Samuel CB; Blacklaws, Barbara A; Yohan, Benediktus; Yudhaputri, Frilasita A; Hayati, Rahma F; Schwem, Brian; Salvaña, Edsel M; Destura, Raul V; Lester, James S; Myint, Khin S; +2 more... Sasmono, R Tedjo; Frost, Simon DW; (2020) Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia. VIROLOGY JOURNAL, 17 (1). 24-. ISSN 1743-422X DOI: https://doi.org/10.1186/s12985-020-1294-6
Permanent Identifier
Use this Digital Object Identifier when citing or linking to this resource.
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.
Item Type | Article |
---|---|
Faculty and Department |
Faculty of Epidemiology and Population Health > Dept of Infectious Disease Epidemiology (-2023) Faculty of Epidemiology and Population Health > Dept of Infectious Disease Epidemiology & Dynamics (2023-) |
PubMed ID | 32054488 |
Elements ID | 144844 |
Download
Filename: Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia.pdf
Licence: Creative Commons: Attribution 3.0
Download