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

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.

Discovering and sequencing new plant viral genomes by next‐generation sequencing: description of a practical pipeline

“…Enriching viral sequences in preparations of extracted nucleic acids is thus critical. Plant DNA viruses have circular (Geminiviridae and Nanoviridae) or pseudocircular (Caulimoviridae) genomes, and rolling‐circle amplification (RCA) (Inoue‐Nagata et al ., ) is often a good choice for sample preparation (Rosario et al ., ; Idris et al ., ). The caulimoviral genome contains discontinuities in both strands but can also be amplified by RCA because replicative forms are closed circles and can serve as templates for exponential amplification reactions.…”

Reconstruction and Characterization of Full-Length Begomovirus and Alphasatellite Genomes Infecting Pepper through Metagenomics

“…When a metagenomic study is linked to a host, it is called ecogenomics [20]. In particular, rolling circle amplification (RCA) [19] as a viral genome enrichment technique, coupled with next-generation sequencing (NGS), has been used to identify geminiviruses, especially begomoviruses and their satellites in different crops [21][22][23][24][25].…”

Tomato Leaf Curl Sudan Virus (TLCSDV) causing leaf curl disease on a new host Amaranthus cruentus L.

“…The whitefly posing a serious threat to global food security and there is an urgent need to develop effective strategies to control crop loss and manage the begomoviral disease. Currently, the natural occurrence of mono-partite begomoviruses and their associated alpha and betasatellites are reported to be endemic in Eastern Hemisphere ( Briddon et al, 2001;Idris et al, 2014).…”

“…The sequence identity and phylogenetic analysis results strongly support the causative agent is a variant of Tomato leaf curl Sudan virus (ToLCSDV), a virus that has been reported earlier to cause tomato leaf curl disease in Sudan, Oman and Yemen. The association of begomovirus with leaf curl disease on various crops in Arabian Peninsula has been published in many reports (Ajlan et al, 2007;Khan et al, 2008;Idris et al, 2012;Akhtar et al, 2014;Idris et al, 2011;Khan et al, 2013a;(Ajlan et al, 2007;Khan et al, 2008;Idris et al, 2012;Akhtar et al, 2014;Idris et al, 2011;Khan et al, 2013bAl-Saleh et al, 2014Hosseinzadeh et al, 2014;Idris et al, 2014).…”