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Viruses 2014, 6(3), 1219-1236; doi:10.3390/v6031219

Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing

1,†,* , 2
2, 1
1 Center for Desert Agriculture, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia 2 Department of Plant Protection, King Saud University, Riyadh 11451, Saudi Arabia 3 School of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA Update on Address Information (12 August 2015): School of Plant Sciences, University of Arizona Tucson, AZ 85741, USA; Email:, Phone: +1-520-665-1041
* Author to whom correspondence should be addressed.
Received: 14 October 2013 / Revised: 12 February 2014 / Accepted: 20 February 2014 / Published: 12 March 2014
(This article belongs to the Section Viruses of Plants, Fungi and Protoza)
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Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.
Keywords: Illumina sequencing; geminivirus; ssDNA viruses; genome enrichment; viral genome assembly Illumina sequencing; geminivirus; ssDNA viruses; genome enrichment; viral genome assembly
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Idris, A.; Al-Saleh, M.; Piatek, M.J.; Al-Shahwan, I.; Ali, S.; Brown, J.K. Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing. Viruses 2014, 6, 1219-1236.

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