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Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing
Genetic analyses play a central role in infectious disease research. Massively parallelized “mechanical cloning” and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have,...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728572/ https://www.ncbi.nlm.nih.gov/pubmed/26751471 http://dx.doi.org/10.3390/v8010012 |
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author | Vrancken, Bram Trovão, Nídia Sequeira Baele, Guy van Wijngaerden, Eric Vandamme, Anne-Mieke van Laethem, Kristel Lemey, Philippe |
author_facet | Vrancken, Bram Trovão, Nídia Sequeira Baele, Guy van Wijngaerden, Eric Vandamme, Anne-Mieke van Laethem, Kristel Lemey, Philippe |
author_sort | Vrancken, Bram |
collection | PubMed |
description | Genetic analyses play a central role in infectious disease research. Massively parallelized “mechanical cloning” and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely limited to small genomic regions. The growing need to monitor multiple genome regions for drug resistance testing, as well as the obvious benefit for studying evolutionary and epidemic processes makes complete genome sequencing an important goal in viral research. In addition, a major drawback for NGS applications to RNA viruses is the need for large quantities of input DNA. Here, we use a generic overlapping amplicon-based near full-genome amplification protocol to compare low-input enzymatic fragmentation (Nextera™) with conventional mechanical shearing for Roche 454 sequencing. We find that the fragmentation method has only a modest impact on the characterization of the population composition and that for reliable results, the variation introduced at all steps of the procedure—from nucleic acid extraction to sequencing—should be taken into account, a finding that is also relevant for NGS technologies that are now more commonly used. Furthermore, by applying our protocol to deep sequence a number of pre-therapy plasma and PBMC samples, we illustrate the potential benefits of a near complete genome sequencing approach in routine genotyping. |
format | Online Article Text |
id | pubmed-4728572 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-47285722016-02-08 Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing Vrancken, Bram Trovão, Nídia Sequeira Baele, Guy van Wijngaerden, Eric Vandamme, Anne-Mieke van Laethem, Kristel Lemey, Philippe Viruses Article Genetic analyses play a central role in infectious disease research. Massively parallelized “mechanical cloning” and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely limited to small genomic regions. The growing need to monitor multiple genome regions for drug resistance testing, as well as the obvious benefit for studying evolutionary and epidemic processes makes complete genome sequencing an important goal in viral research. In addition, a major drawback for NGS applications to RNA viruses is the need for large quantities of input DNA. Here, we use a generic overlapping amplicon-based near full-genome amplification protocol to compare low-input enzymatic fragmentation (Nextera™) with conventional mechanical shearing for Roche 454 sequencing. We find that the fragmentation method has only a modest impact on the characterization of the population composition and that for reliable results, the variation introduced at all steps of the procedure—from nucleic acid extraction to sequencing—should be taken into account, a finding that is also relevant for NGS technologies that are now more commonly used. Furthermore, by applying our protocol to deep sequence a number of pre-therapy plasma and PBMC samples, we illustrate the potential benefits of a near complete genome sequencing approach in routine genotyping. MDPI 2016-01-07 /pmc/articles/PMC4728572/ /pubmed/26751471 http://dx.doi.org/10.3390/v8010012 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Vrancken, Bram Trovão, Nídia Sequeira Baele, Guy van Wijngaerden, Eric Vandamme, Anne-Mieke van Laethem, Kristel Lemey, Philippe Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing |
title | Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing |
title_full | Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing |
title_fullStr | Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing |
title_full_unstemmed | Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing |
title_short | Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing |
title_sort | quantifying next generation sequencing sample pre-processing bias in hiv-1 complete genome sequencing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728572/ https://www.ncbi.nlm.nih.gov/pubmed/26751471 http://dx.doi.org/10.3390/v8010012 |
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