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Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.
- Source :
-
Genome research [Genome Res] 2007 Aug; Vol. 17 (8), pp. 1195-201. Date of Electronic Publication: 2007 Jun 28. - Publication Year :
- 2007
-
Abstract
- The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.
- Subjects :
- DNA, Viral metabolism
Drug Resistance, Viral genetics
Genetic Variation
HIV Protease blood
HIV Reverse Transcriptase blood
HIV-1 enzymology
Humans
Mutation
Anti-HIV Agents therapeutic use
DNA Mutational Analysis methods
HIV Infections drug therapy
HIV Protease genetics
HIV Reverse Transcriptase genetics
HIV-1 genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1088-9051
- Volume :
- 17
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Genome research
- Publication Type :
- Academic Journal
- Accession number :
- 17600086
- Full Text :
- https://doi.org/10.1101/gr.6468307