Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.
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.
Wang
Chunlin
C
Division of Infectious Diseases, Department of Medicine, Stanford University, Stanford, CA 94305, USA.
Mitsuya
Yumi
Y
Gharizadeh
Baback
B
Ronaghi
Mostafa
M
Shafer
Robert W
RW
D019380:Anti-HIV Agents / Q000627:therapeutic use*; D004252:DNA Mutational Analysis / Q000379:methods*; D004279:DNA, Viral / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000235:genetics; D014644:Genetic Variation; D015658:HIV Infections / Q000188:drug therapy*; D016333:HIV Protease / Q000097:blood / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000097:blood / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009154:Mutation
D019380:Anti-HIV Agents; D004279:DNA, Viral; D054303:HIV Reverse Transcriptase; D016333:HIV Protease