Feasibility Study of Using Massively Parallel Pyrosequencing in Detecting Variation in Dengue Virus Genome

摘要

@@BackgroundDengue is a positive single-stranded RNA virus which causes Dengue hemorrhagic fever. Due to the lack of 'proof reading' featare in RNA polymerasc, a collection of genetically-different but closely related viral population occurs during viral replication in a host; this is referred to as "quasispecies" [1]. Quasispecies are believed to play an important role in the survival and evolution of dengue viruses as well as in the pathogenesis of disease. The study of Dengue quasispecies has been hampered technically by the sequencing technology. Most of the past investigations were carfled out by culturing the viruses or by RT-PCR amplification followed by cloning. Culture of the viruses is known to allow adaptations and selections to occur, putting artifacts into the studies.Massively parallel pyrosequeneing is a new high throughput sequencing system using emPCR instead of cloning technique This allows us to comprehensively study the quasispecies directly from patients' sera/plasma without cloning or culture process. In this feasibility study, we tested whether Roche GS-FLX sequencer is suitable for studying quasispecies in Dengue virus. Two main questions were asked -whether we could cover the whole viral genome and whether we could detect variations at different allele frequencies.