A hybridization-based enrichment strategy to increase the accuracy of next generation sequencing in phylogenetic analysis of dengue viruses in Sri Lanka

• Introduction: Sri Lanka has experienced confirmed dengue outbreaks since the 1960s although severe dengue disease (DHF/DSS) didn’t appear until 1989. Since then, cyclical outbreaks associated with severe disease have occurred throughout the island. The most recent epidemic began in 2009 with the apparent introduction of a new genotype of DENV-1. To better understand the mechanisms underlying the persistence of this ongoing epidemic, a longitudinal study was conducted in hospitals in the Colombo district from April 2012 to March 2014. In order to glean as much information as possible about the viral genetics from this large cohort, we developed a novel Next Generation Sequencing (NGS) platform that can function without any a priori knowledge of the target dengue genome.Methods: The principle problem encountered when employing NGS directly on patient samples is the high ratio of host to viral RNA. To compensate for this, we developed a hybridization-based enrichment strategy consisting of DENV-specific 120nt, biotinylated oligodeoxynucleotides to capture DENV genomic material from an NGS library prepared directly from patient sera.Results: The strategy developed here allowed us to enrich DENV genomic material over 5000 fold relative to unenriched material. Full genome data and phylogenetic analysis indicate that the DENV-1 are predominantly genotype 1 although a smaller number of genotype 5 isolates was also identified.Conclusion: The platform developed for this study has the inherent ability to capture all four serotypes of DENV and can significantly increase the virus to host RNA ratio. The principle driver of the current dengue epidemic in Sri Lanka is the same DENV-1 genotype that has been in circulation since 2009.This research was funded by the Singapore Infectious Disease Initiative (SIDI/2013/012) and the European Union 7th Framework Programme through ‘DengueTools’. (www.dengue-tools.net).Disclosure: Nothing to disclose.

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MEDICIN OCH HÄLSOVETENSKAP  -- Hälsovetenskap -- Folkhälsovetenskap, global hälsa, socialmedicin och epidemiologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Health Sciences -- Public Health, Global Health, Social Medicine and Epidemiology (hsv//eng)

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