Single molecule, near full-length genome sequencing of dengue virus

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.

Figure 1

Success rate of near-full genome amplification assay stratified by the Cq value of the quantitative PCR per sample. Please see Supplementary Tables 1 and 2 for corresponding viral loads.

Figure 2

Maximum likelihood phylogenetic tree (consensus of 1000 bootstrap replicates with 99% bootstrap support) showing clustering of DENV2 sequences of this study (red) with Cosmopolitan genotype. Caption: Each color indicates a different genotype of DENV2; Orange: cosmopolitan, Green: American, Blue: Asian American, Pink: Asian I, Black: Asian II.

Figure 3

Combined maximum likelihood (consensus of 1000 bootstrap replicates with 99% bootstrap support) of Illumina and nanopore consensuses demonstrating the clustering of each nanopore consensus with its Illumina counterpart (denoted with a _I against the sample number).

Figure 4

Comparison of Illumina and ONT generated consensuses each with a > 100 coverage throughout the genome with a maximum likelihood phylogenetic tree.

Figure 5

The number of minor variants (< 5% of abundance) reconstructed with Nano-Q tool changes linearly with the total number of eligible reads for the pipeline. However, as most minor variants have an abundance < 1%, detection of more variants (if more reads were available), is unlikely to impact the frequency estimation of major variants.

Figure 6

Maximum likelihood phylogenetic tree (consensus of 1000 bootstrap replicates with 99% bootstrap support) of 57 near-full genome within host variants isolated from 14 subjects with DENV2 infection demonstrating clustering of variants by the host. Variants from each host is in the same color. In three subjects, only one variant was found. The relative abundance of the variant is given as a percentage next to variant name (e.g. 0.46 refers to a 46% abundance).