Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance

Genome Res. 2007 Aug;17(8):1195-201. doi: 10.1101/gr.6468307. Epub 2007 Jun 28.

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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Anti-HIV Agents / therapeutic use*
  • DNA Mutational Analysis / methods*
  • DNA, Viral / metabolism
  • Drug Resistance, Viral / genetics
  • Genetic Variation
  • HIV Infections / drug therapy*
  • HIV Protease / blood
  • HIV Protease / genetics*
  • HIV Reverse Transcriptase / blood
  • HIV Reverse Transcriptase / genetics*
  • HIV-1 / enzymology
  • HIV-1 / genetics*
  • Humans
  • Mutation

Substances

  • Anti-HIV Agents
  • DNA, Viral
  • HIV Reverse Transcriptase
  • HIV Protease