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

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-...

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Autores principales: Idris, Ali, Al-Saleh, Mohammed, Piatek, Marek J., Al-Shahwan, Ibrahim, Ali, Shahjahan, Brown, Judith K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970147/
https://www.ncbi.nlm.nih.gov/pubmed/24625811
http://dx.doi.org/10.3390/v6031219
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author Idris, Ali
Al-Saleh, Mohammed
Piatek, Marek J.
Al-Shahwan, Ibrahim
Ali, Shahjahan
Brown, Judith K.
author_facet Idris, Ali
Al-Saleh, Mohammed
Piatek, Marek J.
Al-Shahwan, Ibrahim
Ali, Shahjahan
Brown, Judith K.
author_sort Idris, Ali
collection PubMed
description 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.
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spelling pubmed-39701472014-03-31 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. Viruses Article 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. MDPI 2014-03-12 /pmc/articles/PMC3970147/ /pubmed/24625811 http://dx.doi.org/10.3390/v6031219 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
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
title Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing
title_full Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing
title_fullStr Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing
title_full_unstemmed Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing
title_short Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing
title_sort viral metagenomics: analysis of begomoviruses by illumina high-throughput sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970147/
https://www.ncbi.nlm.nih.gov/pubmed/24625811
http://dx.doi.org/10.3390/v6031219
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