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Development of a low bias method for characterizing viral populations using next generation sequencing technology.

Willerth SM, Pedro HA, Pachter L, Humeau LM, Arkin AP, Schaffer DV - PLoS ONE (2010)

Bottom Line: 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.In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, California, United States of America.

ABSTRACT

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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Related in: MedlinePlus

Flowchart of computational methodology.Once the millions of Illumina reads were obtained, they were then aligned against the 144 HIV-1 B genomes taken from the Los Alamos National Lab database. These alignments produced regions of consensus for each of these individual genomes which were then combined for the final alignment using MAFFT, which is a multiple sequence alignment program for nucleotide sequences.
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pone-0013564-g002: Flowchart of computational methodology.Once the millions of Illumina reads were obtained, they were then aligned against the 144 HIV-1 B genomes taken from the Los Alamos National Lab database. These alignments produced regions of consensus for each of these individual genomes which were then combined for the final alignment using MAFFT, which is a multiple sequence alignment program for nucleotide sequences.

Mentions: The goal of this work is to develop a novel method for amplifying viral RNA into large quantities of DNA suitable for processing into Illumina (or other) libraries without relying on PCR and primers that assume specific sequences are present within the sample. By avoiding the use of virus-specific primers, one can reduce the bias associated with this step and potentially amplify the entire 9709 base pair HIV genome with unprecedented levels of coverage. Another challenge involved developing a novel computational method for reconstructing a “master sequence” for clinical patient samples with unknown sequences from the resulting reads obtained from the Illumina sequencing. To summarize these advances, the experimental portion of the workflow of our novel method is detailed in Figure 1, while the computational portion of the workflow is shown in Figure 2.


Development of a low bias method for characterizing viral populations using next generation sequencing technology.

Willerth SM, Pedro HA, Pachter L, Humeau LM, Arkin AP, Schaffer DV - PLoS ONE (2010)

Flowchart of computational methodology.Once the millions of Illumina reads were obtained, they were then aligned against the 144 HIV-1 B genomes taken from the Los Alamos National Lab database. These alignments produced regions of consensus for each of these individual genomes which were then combined for the final alignment using MAFFT, which is a multiple sequence alignment program for nucleotide sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2962647&req=5

pone-0013564-g002: Flowchart of computational methodology.Once the millions of Illumina reads were obtained, they were then aligned against the 144 HIV-1 B genomes taken from the Los Alamos National Lab database. These alignments produced regions of consensus for each of these individual genomes which were then combined for the final alignment using MAFFT, which is a multiple sequence alignment program for nucleotide sequences.
Mentions: The goal of this work is to develop a novel method for amplifying viral RNA into large quantities of DNA suitable for processing into Illumina (or other) libraries without relying on PCR and primers that assume specific sequences are present within the sample. By avoiding the use of virus-specific primers, one can reduce the bias associated with this step and potentially amplify the entire 9709 base pair HIV genome with unprecedented levels of coverage. Another challenge involved developing a novel computational method for reconstructing a “master sequence” for clinical patient samples with unknown sequences from the resulting reads obtained from the Illumina sequencing. To summarize these advances, the experimental portion of the workflow of our novel method is detailed in Figure 1, while the computational portion of the workflow is shown in Figure 2.

Bottom Line: 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.In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, California, United States of America.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus