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    Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing

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    Article-Viruses-Viral_meta-2014.pdf
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    Type
    Article
    Authors
    Idris, Ali
    Al-Saleh, Mohammed
    Piatek, Marek J. cc
    Al-Shahwan, Ibrahim
    Ali, Shahjahan
    Brown, Judith K.
    KAUST Department
    Bioscience Core Lab
    Biological and Environmental Sciences and Engineering (BESE) Division
    Bioscience Program
    Desert Agriculture Initiative
    Date
    2014-03-12
    Permanent link to this record
    http://hdl.handle.net/10754/325365
    
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    Abstract
    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. 2014 by the authors; licensee MDPI, Basel, Switzerland.
    Citation
    Idris A, Al-Saleh M, Piatek M, Al-Shahwan I, Ali S, et al. (2014) Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing. Viruses 6: 1219-1236. doi:10.3390/v6031219.
    Publisher
    MDPI AG
    Journal
    Viruses
    DOI
    10.3390/v6031219
    PubMed ID
    24625811
    PubMed Central ID
    PMC3970147
    ae974a485f413a2113503eed53cd6c53
    10.3390/v6031219
    Scopus Count
    Collections
    Articles; Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Bioscience Core Lab; Desert Agriculture Initiative

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