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https://doi.org/10.1038/s41598-020-75374-1
Title: | Single molecule, near full-length genome sequencing of dengue virus | Authors: | Adikari, T.N. Riaz, N. Sigera, C. Leung, P. Valencia, B.M. Barton, K. Smith, M.A. Bull, R.A. Li, H. Luciani, F. Weeratunga, P. Thein, T.-L. Lim, V.W.X. Leo, Y.-S. Rajapakse, S. Fink, K. Lloyd, A.R. Fernando, D. Rodrigo, C. |
Issue Date: | 2020 | Publisher: | Nature Research | Citation: | Adikari, T.N., Riaz, N., Sigera, C., Leung, P., Valencia, B.M., Barton, K., Smith, M.A., Bull, R.A., Li, H., Luciani, F., Weeratunga, P., Thein, T.-L., Lim, V.W.X., Leo, Y.-S., Rajapakse, S., Fink, K., Lloyd, A.R., Fernando, D., Rodrigo, C. (2020). Single molecule, near full-length genome sequencing of dengue virus. Scientific Reports 10 (1) : 18196. ScholarBank@NUS Repository. https://doi.org/10.1038/s41598-020-75374-1 | Rights: | Attribution 4.0 International | 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. © 2020, The Author(s). | Source Title: | Scientific Reports | URI: | https://scholarbank.nus.edu.sg/handle/10635/199312 | ISSN: | 20452322 | DOI: | 10.1038/s41598-020-75374-1 | Rights: | Attribution 4.0 International |
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