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首页> 外文期刊>African Journal of Biotechnology >The rolling circle amplification and next generation sequencing approaches reveal genome wide diversity of Kenyan cassava mosaic geminivirus
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The rolling circle amplification and next generation sequencing approaches reveal genome wide diversity of Kenyan cassava mosaic geminivirus

机译：滚环扩增和下一代测序方法揭示了肯尼亚木薯花叶双生病毒的全基因组多样性

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Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family, Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep sequencing using Illumina Miseq and bioinformatics to assess population diversity of begomoviruses in naturally infected cassava. The approach is suitable for detecting rare members in a population in begomoviral populations in situation where mixtures of isolates, strains, and multiple species occur. The main objectives were to increase the sensitivity of detection of next generation sequencing by enriching it using rolling circle amplification then determination of the diversity of the cassava mosaic geminivirus. This was done by total nucleic acids isolated from symptomatic, field cassava infected plants, then using rolling circle amplification to multiply the less abundant viral sequences. Enriched and non-enriched virus-libraries were subjected to deep sequencing using Illumina Miseq. Using bioinformatic CLC Genomics 5.5.1 software programs the quality assessment of reads and contig assembly of viral sequences. This was done through de novo and reference-guided assembly. The identity and diversities of the begomoviral sequences were compared with sequences in Sanger sequencing of viral components deposited in the NCBI Gene Bank. In this study we have demonstrated that RCA increases the chances of detecting the virus by approximately 10 to 1000 fold and wide genome diversity of cassava mosaic geminivirus in various cassava growing zones in Kenya were detected. In conclusion, this approach described herein is simple and will enhance the exploration of begomovirus diversities from cassava infected plants, irrespective of their viral abundance. This will make it possible for routine screening of field samples as the cost of deep sequencing NGS is decreasing and the advances of bioinformatic software development become enhanced. This is the first report of the RCA-Illumina-NGS approach to explore cassava infected with begomoviruses under field conditions and their diversities.
机译：滚环扩增是富集单链DNA植物begomovirus基因组（属，Begomovirus;家族，Geminiviridae）的简单方法。这是一种创新的方法，利用了在环扩增中使用的噬菌体phi29 DNA聚合酶的稳健性，以及使用Illumina Miseq和生物信息学进行的深度测序来评估自然感染木薯中begomovirus病毒的种群多样性。该方法适用于在分离株，菌株和多种物种混合出现的情况下，在前胎种群中检测种群中的稀有成员。主要目标是通过使用滚环扩增技术来丰富下一代测序的检测灵敏度，并确定木薯花叶双子病毒的多样性，从而提高检测下一代测序的灵敏度。这是通过从有症状的，木薯感染的有症状植物中分离出的总核酸来完成的，然后使用滚环扩增法来扩增不那么丰富的病毒序列。使用Illumina Miseq对富集和非富集的病毒库进行深度测序。使用生物信息学的CLC Genomics 5.5.1软件可以对病毒序列的读段和重叠群进行质量评估。这是通过从头和参考指南组装完成的。将bemovoviral序列的同一性和多样性与NCBI基因库中病毒成分的Sanger测序中的序列进行了比较。在这项研究中，我们证明了RCA将检测病毒的机会增加了约10到1000倍，并且在肯尼亚的各种木薯种植区中检测到了木薯花叶双生病毒的广泛基因组多样性。总之，本文所述的方法是简单的，并且将增强从木薯感染的植物中探索begomovirus多样性的途径，无论其病毒丰度如何。随着深度测序NGS成本的降低以及生物信息软件开发的进步，这将使常规筛查样品成为可能。这是RCA-Illumina-NGS方法的首次报道，该方法用于在野外条件及其多样性下探索被begomovirus感染的木薯。

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