Abstract
Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation “nanopore” technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was > 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.
Highlights
Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and combine the output to characterize a full genome
The new assay developed by us which is detailed in supplementary methods was tested on one hundred and four clinical samples (DENV1–18, DENV2–75, DENV3–7, DENV4–4)
Near full-length amplicons were visible on gel electrophoresis in 47 samples and corresponding genomes were identified from nanopore sequencing in 45 of these
Summary
Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and combine the output to characterize a full genome This process is laborious, costly and requires at least 10 primers per serotype, increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation “nanopore” technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). Genomic sequencing requires setting up infrastructure for sequencing facilities—a costly exercise beyond the economical capabilities of many low- and middle- income countries where DENV is predominantly endemic Another barrier to study viral genomics in dengue is the difficulty in generating unbiased full-length DENV genomes and successfully sequencing them as intact genomes. Doing so in a cost-effective and high throughput manner will improve the utility of a pipeline designed for this purpose
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