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

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Date: 23rd October 2020 | Source: Scientific Reports

Authors: Thiruni N. Adikari, Nasir Riaz, Chathurani Sigera, Preston Leung, Braulio M. Valencia, Kirston Barton, Martin A. Smith, Rowena A. Bull, Hui Li, Fabio Luciani, Praveen Weeratunga, Tun-Linn Thein, Vanessa W. X. Lim, Yee-Sin Leo, Senaka Rajapakse, Katja Fink, Andrew R. Lloyd, Deepika Fernando, Chaturaka Rodrigo .

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.

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