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.

Incidence of chikungunya virus infections among Kenyan children with neurological disease, 2014–2018: A cohort study

“…These results support the specificity and validity of our RT-PCR assay in detection of CHIKV in CSF samples. The low success in obtaining full-length genomes could be due to several reasons (as acknowledged for other arbovirus sequencing projects (e.g., [ 36 , 37 ]): (1) low viral load (Ct values ranged 35.2–38.0); (2) template degradation (due to RNAses or freeze–thaw cycles); or (3) sequencing primer dropouts due to competition and mutations on primer binding sites. More comprehensive CHIKV genome sequencing and phylogenetic analysis of samples from different time points, disease severity, and geographic locations is planned as a future project.…”

A Serotype-Specific and Multiplex PCR Method for Whole-Genome Sequencing of Dengue Virus Directly from Clinical Samples

“…Historically, segmental reverse transcription-PCR (RT-PCR) combined with Sanger DNA sequencing of the DENV E gene (about 1,600 bp to 1,800 bp) was successfully applied ( 10 , 30 ). With the requirement of accuracy in molecular epidemiological studies, WGS of DENV has been gradually applied ( 22 , 25 , 31 – 34 ). NGS technologies have recently enabled large-scale surveillance of infectious diseases, and the power and utility of NGS are based on its massively parallel interrogation of nucleic acids.…”

Effect of prior Zika and dengue virus exposure on the severity of a subsequent dengue infection in adults

“…Details of CDS including the rationale for sample size calculation and inclusion/exclusion criteria for patient recruitment had been published previously 16 . Previous investigations with data and samples from this cohort have been helpful to design cheaper diagnostics for dengue fever 16 , to design a new pan-serotype assay for dengue full genome sequencing 17 , characterise the syndrome of post-dengue fatigue 18 , to develop a triaging system to screen patients with ultrasonography in resource limited settings for plasma leakage 19 and to model the direct costs of managing dengue patients in Sri Lanka 20 . All methods were performed in accordance with the relevant guidelines and regulations.…”