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Development of a Low Bias Method for Characterizing Viral Populations Using Next Generation Sequencing Technology

Willerth, Stephanie M. and Pedro, Hélder A. M. and Pachter, Lior and Humeau, Laurent M. and Arkin, Adam P. and Schaffer, David V. (2010) Development of a Low Bias Method for Characterizing Viral Populations Using Next Generation Sequencing Technology. PLOS ONE, 5 (10). Art. No. e13564. ISSN 1932-6203. PMCID PMC2962647. doi:10.1371/journal.pone.0013564.

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[img] Image (TIFF) (Figure S1. Plot of base substitution rate for the clonal sample as a function of read position. As shown in the plot, the first 6 base pairs of the read contain the highest amount of error) - Supplemental Material
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[img] Image (TIFF) (Figure S2. Alignment of the clonal and clinical samples) - Supplemental Material
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[img] MS Word (File S1. Detailed protocols for reproducing methodology.) - Supplemental Material
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[img] MS Word (File S2. Master Sequence based on Illumina sequencing of the clinical sample) - Supplemental Material
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Background: With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies. Methodology/Principal Findings: We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient's infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity. Significance: This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.

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URLURL TypeDescription CentralArticle
Pachter, Lior0000-0002-9164-6231
Additional Information: © 2010 Willerth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: May 27, 2010; Accepted: October 1, 2010; Published: October 22, 2010. This work was supported by VIRxSYS corporation and NIH R01 GM073058. H.P. is sponsored by Fundacao Para a Ciencia e a Tecnologia [FCT], Fundacao Calouste Gulbenkian, Siemens SA Portugal and was supported by FCT fellowship SFRH/BD/33204/2007. The funders (specifically the VIRxSYS corporation) provided samples for analysis and input in the experimental design of this project. The authors would like to thank Priya Shah, Katherine Hermans in the University of California-Berkeley Functional Genomics Laboratory, and Leath Tonkin of the Vincent J. Coates Genomic Sequencing Laboratory for their help. Author Contributions: Conceived and designed the experiments: SMW HAMP LP LMH APA DVS. Performed the experiments: SMW. Analyzed the data: HAMP. Contributed reagents/materials/analysis tools: HAMP LMH. Wrote the paper: SMW HAMP. Reviewed and commented on drafts of manuscaript: DVS. Revised and approved the final version of the article: LP LMH APA. Competing interests: The first author (Stephanie M. Willerth) and the fourth author (Laurent M. Humeau) had their salaries paid for by the VIRxSYS corporation. The second author (Hélder A.M. Pedro) was sponsnsored in part by Siemens SA Portugal. The funding provided by these companies did not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Funding AgencyGrant Number
NIHR01 GM073058
Fundação para a Ciência e a Tecnologia (FCT)SFRH/BD/33204/2007
Fundação Calouste GulbenkianUNSPECIFIED
Siemens SA PortugalUNSPECIFIED
Issue or Number:10
PubMed Central ID:PMC2962647
Record Number:CaltechAUTHORS:20170306-114844988
Persistent URL:
Official Citation:Willerth SM, Pedro HAM, Pachter L, Humeau LM, Arkin AP, Schaffer DV (2010) Development of a Low Bias Method for Characterizing Viral Populations Using Next Generation Sequencing Technology. PLoS ONE 5(10): e13564. doi:10.1371/journal.pone.0013564
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:74787
Deposited By: George Porter
Deposited On:06 Mar 2017 21:00
Last Modified:11 Nov 2021 05:29

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