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首页> 外文期刊>BMC Genomics >Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
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Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome

机译：使用454焦磷酸测序技术研究整个登革热病毒基因组的准种的可行性

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Background Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an important role in disease severity. The study of quasispecies in dengue has been hindered because of the requirement for large amounts of cloning and sequencing, which could be overcome by 454 pyrosequencing. In this study, we attempted to demonstrate the feasibility of using 454 pyrosequencing to study genome diversity of dengue virus quasispecies by sequencing a pool of known dengue viral strains. Results Two sets of dengue DNA templates were sequenced by 454/Roche GS FLX. The total number of reads for data 1 and data 2 were 54,440 and 134,441, with average lengths of 221 and 232 bp, respectively. Reads containing ambiguous base Ns were excluded (6.00% in data 1, 7.05% in data 2). More than 99% of reads could be aligned back to the correct serotypes by BLAST. The reads covered the whole genome without any gaps, and the minimum coverage depth was 50×. Frequencies of known strains detected from each data set were highly correlated with the input ratios. We also explored criteria for filtering error reads and artifacts from true variations. Conclusions This study showed that 454 pyrosequencing, coupled with our analysis procedure, could sequence the whole genome of dengue virus with good coverage. The ratio of detected variants in the sequencing data reflected the starting ratio, proving that the proposed technique could be used to study the frequencies of variants in quasispecies.
机译：背景登革热是世界上最常见的蚊媒病毒性疾病。 RNA聚合酶在复制过程中校对不良会导致其基因组中突变的积累。这导致在病毒种群中被称为准种的基因型的多样性。准种在疾病严重程度中起重要作用。由于需要大量的克隆和测序工作，因此登革热准种的研究受到阻碍，这可以通过454焦磷酸测序来克服。在这项研究中，我们试图通过对已知的登革热病毒株进行测序来证明使用454焦磷酸测序来研究登革热病毒准种的基因组多样性的可行性。结果通过454 / Roche GS FLX对两组登革热DNA模板进行了测序。数据1和数据2的读取总数为54,440和134,441，平均长度分别为221和232 bp。排除了包含歧义碱基Ns的读取（数据1中为6.00％，数据2中为7.05％）。 BLAST可以将超过99％的读数重新排列为正确的血清型。读段覆盖了整个基因组，没有任何间隙，最小覆盖深度为50倍。从每个数据集中检测到的已知菌株的频率与输入比率高度相关。我们还探索了从真实变化中过滤错误读数和伪像的标准。结论这项研究表明454焦磷酸测序结合我们的分析程序可以对登革热病毒的整个基因组进行测序，且覆盖率高。测序数据中检测到的变异体的比率反映了起始比率，证明了所提出的技术可用于研究准物种中变异体的频率。

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