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Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome

BACKGROUND: Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an...

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Autores principales: Chin-inmanu, Kwanrutai, Suttitheptumrong, Aroonroong, Sangsrakru, Duangjai, Tangphatsornruang, Sithichoke, Tragoonrung, Somvong, Malasit, Prida, Tungpradabkul, Sumalee, Suriyaphol, Prapat
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521222/
https://www.ncbi.nlm.nih.gov/pubmed/23281804
http://dx.doi.org/10.1186/1471-2164-13-S7-S7
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author Chin-inmanu, Kwanrutai
Suttitheptumrong, Aroonroong
Sangsrakru, Duangjai
Tangphatsornruang, Sithichoke
Tragoonrung, Somvong
Malasit, Prida
Tungpradabkul, Sumalee
Suriyaphol, Prapat
author_facet Chin-inmanu, Kwanrutai
Suttitheptumrong, Aroonroong
Sangsrakru, Duangjai
Tangphatsornruang, Sithichoke
Tragoonrung, Somvong
Malasit, Prida
Tungpradabkul, Sumalee
Suriyaphol, Prapat
author_sort Chin-inmanu, Kwanrutai
collection PubMed
description BACKGROUND: Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an important role in disease severity. The study of quasispecies in dengue has been hindered because of the requirement for large amounts of cloning and sequencing, which could be overcome by 454 pyrosequencing. In this study, we attempted to demonstrate the feasibility of using 454 pyrosequencing to study genome diversity of dengue virus quasispecies by sequencing a pool of known dengue viral strains. RESULTS: Two sets of dengue DNA templates were sequenced by 454/Roche GS FLX. The total number of reads for data 1 and data 2 were 54,440 and 134,441, with average lengths of 221 and 232 bp, respectively. Reads containing ambiguous base Ns were excluded (6.00% in data 1, 7.05% in data 2). More than 99% of reads could be aligned back to the correct serotypes by BLAST. The reads covered the whole genome without any gaps, and the minimum coverage depth was 50×. Frequencies of known strains detected from each data set were highly correlated with the input ratios. We also explored criteria for filtering error reads and artifacts from true variations. CONCLUSIONS: This study showed that 454 pyrosequencing, coupled with our analysis procedure, could sequence the whole genome of dengue virus with good coverage. The ratio of detected variants in the sequencing data reflected the starting ratio, proving that the proposed technique could be used to study the frequencies of variants in quasispecies.
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spelling pubmed-35212222012-12-14 Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome Chin-inmanu, Kwanrutai Suttitheptumrong, Aroonroong Sangsrakru, Duangjai Tangphatsornruang, Sithichoke Tragoonrung, Somvong Malasit, Prida Tungpradabkul, Sumalee Suriyaphol, Prapat BMC Genomics Proceedings BACKGROUND: Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an important role in disease severity. The study of quasispecies in dengue has been hindered because of the requirement for large amounts of cloning and sequencing, which could be overcome by 454 pyrosequencing. In this study, we attempted to demonstrate the feasibility of using 454 pyrosequencing to study genome diversity of dengue virus quasispecies by sequencing a pool of known dengue viral strains. RESULTS: Two sets of dengue DNA templates were sequenced by 454/Roche GS FLX. The total number of reads for data 1 and data 2 were 54,440 and 134,441, with average lengths of 221 and 232 bp, respectively. Reads containing ambiguous base Ns were excluded (6.00% in data 1, 7.05% in data 2). More than 99% of reads could be aligned back to the correct serotypes by BLAST. The reads covered the whole genome without any gaps, and the minimum coverage depth was 50×. Frequencies of known strains detected from each data set were highly correlated with the input ratios. We also explored criteria for filtering error reads and artifacts from true variations. CONCLUSIONS: This study showed that 454 pyrosequencing, coupled with our analysis procedure, could sequence the whole genome of dengue virus with good coverage. The ratio of detected variants in the sequencing data reflected the starting ratio, proving that the proposed technique could be used to study the frequencies of variants in quasispecies. BioMed Central 2012-12-07 /pmc/articles/PMC3521222/ /pubmed/23281804 http://dx.doi.org/10.1186/1471-2164-13-S7-S7 Text en Copyright ©2012 Chin-inmanu et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Proceedings
Chin-inmanu, Kwanrutai
Suttitheptumrong, Aroonroong
Sangsrakru, Duangjai
Tangphatsornruang, Sithichoke
Tragoonrung, Somvong
Malasit, Prida
Tungpradabkul, Sumalee
Suriyaphol, Prapat
Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
title Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
title_full Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
title_fullStr Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
title_full_unstemmed Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
title_short Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
title_sort feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome
topic Proceedings
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521222/
https://www.ncbi.nlm.nih.gov/pubmed/23281804
http://dx.doi.org/10.1186/1471-2164-13-S7-S7
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