Journal
GENOME RESEARCH
Volume 17, Issue 8, Pages 1195-1201Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gr.6468307
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Funding
- NCRR NIH HHS [S10 RR022982, 1 S10 RR022982, S10 RR022982-01] Funding Source: Medline
- NIAID NIH HHS [R56 AI068581, R01 AI068581, AI46148, AI068581, R01 AI046148] Funding Source: Medline
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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 >= 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.
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