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.
Key words: Illumina Miseq sequencing, geminivirus, ssDNA viruses, viral sequence enrichment, de novo genome assembly, rolling cycle amplification (RCA).
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