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HIV Drug Resistance Surveillance Using Pooled Pyrosequencing

BACKGROUND: Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally...

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Autores principales: Ji, Hezhao, Massé, Nathalie, Tyler, Shaun, Liang, Ben, Li, Yang, Merks, Harriet, Graham, Morag, Sandstrom, Paul, Brooks, James
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822863/
https://www.ncbi.nlm.nih.gov/pubmed/20174661
http://dx.doi.org/10.1371/journal.pone.0009263
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author Ji, Hezhao
Massé, Nathalie
Tyler, Shaun
Liang, Ben
Li, Yang
Merks, Harriet
Graham, Morag
Sandstrom, Paul
Brooks, James
author_facet Ji, Hezhao
Massé, Nathalie
Tyler, Shaun
Liang, Ben
Li, Yang
Merks, Harriet
Graham, Morag
Sandstrom, Paul
Brooks, James
author_sort Ji, Hezhao
collection PubMed
description BACKGROUND: Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals. METHODOLOGY/PRINCIPAL FINDINGS: The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies ≥5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods. CONCLUSIONS/SIGNIFICANCE: We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required.
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spelling pubmed-28228632010-02-20 HIV Drug Resistance Surveillance Using Pooled Pyrosequencing Ji, Hezhao Massé, Nathalie Tyler, Shaun Liang, Ben Li, Yang Merks, Harriet Graham, Morag Sandstrom, Paul Brooks, James PLoS One Research Article BACKGROUND: Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals. METHODOLOGY/PRINCIPAL FINDINGS: The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies ≥5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods. CONCLUSIONS/SIGNIFICANCE: We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required. Public Library of Science 2010-02-17 /pmc/articles/PMC2822863/ /pubmed/20174661 http://dx.doi.org/10.1371/journal.pone.0009263 Text en Ji et al. http://creativecommons.org/licenses/by/4.0/ 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 properly credited.
spellingShingle Research Article
Ji, Hezhao
Massé, Nathalie
Tyler, Shaun
Liang, Ben
Li, Yang
Merks, Harriet
Graham, Morag
Sandstrom, Paul
Brooks, James
HIV Drug Resistance Surveillance Using Pooled Pyrosequencing
title HIV Drug Resistance Surveillance Using Pooled Pyrosequencing
title_full HIV Drug Resistance Surveillance Using Pooled Pyrosequencing
title_fullStr HIV Drug Resistance Surveillance Using Pooled Pyrosequencing
title_full_unstemmed HIV Drug Resistance Surveillance Using Pooled Pyrosequencing
title_short HIV Drug Resistance Surveillance Using Pooled Pyrosequencing
title_sort hiv drug resistance surveillance using pooled pyrosequencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822863/
https://www.ncbi.nlm.nih.gov/pubmed/20174661
http://dx.doi.org/10.1371/journal.pone.0009263
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