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

Ali Idris, Mohammed Al-Saleh, Marek J. Piatek, Ibrahim Al-Shahwan, Shahjahan Ali, Judith K Brown

Research output: Contribution to journalArticle

31 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1219-1236
Number of pages18
JournalViruses
Volume6
Issue number3
DOIs
StatePublished - Mar 12 2014

Fingerprint

Begomovirus
Metagenomics
Satellite Viruses
Geminiviridae
Population
Satellite DNA
Plant Genome
Virus Assembly
High-Throughput Nucleotide Sequencing
Viral Structures
Single-Stranded DNA
Viral DNA
Molecular Cloning
DNA Sequence Analysis
Quality Control
Nucleic Acids
Single Nucleotide Polymorphism
Software
Costs and Cost Analysis
Polymerase Chain Reaction

Keywords

  • Geminivirus
  • Genome enrichment
  • Illumina sequencing
  • ssDNA viruses
  • Viral genome assembly

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology

Cite this

Idris, A., Al-Saleh, M., Piatek, M. J., Al-Shahwan, I., Ali, S., & Brown, J. K. (2014). Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing. Viruses, 6(3), 1219-1236. https://doi.org/10.3390/v6031219

Viral metagenomics : Analysis of begomoviruses by illumina high-throughput sequencing. / Idris, Ali; Al-Saleh, Mohammed; Piatek, Marek J.; Al-Shahwan, Ibrahim; Ali, Shahjahan; Brown, Judith K.

In: Viruses, Vol. 6, No. 3, 12.03.2014, p. 1219-1236.

Research output: Contribution to journalArticle

Idris, A, Al-Saleh, M, Piatek, MJ, Al-Shahwan, I, Ali, S & Brown, JK 2014, 'Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing', Viruses, vol. 6, no. 3, pp. 1219-1236. https://doi.org/10.3390/v6031219
Idris, Ali ; Al-Saleh, Mohammed ; Piatek, Marek J. ; Al-Shahwan, Ibrahim ; Ali, Shahjahan ; Brown, Judith K. / Viral metagenomics : Analysis of begomoviruses by illumina high-throughput sequencing. In: Viruses. 2014 ; Vol. 6, No. 3. pp. 1219-1236.
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