title: HIV drug resistance surveillance using pooled pyrosequencing.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: National HIV and Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.
authors.forename: Hezhao
authors.initials: H
authors.lastname: Ji
authors.forename: Nathalie
authors.initials: N
authors.lastname: Massé
authors.forename: Shaun
authors.initials: S
authors.lastname: Tyler
authors.forename: Ben
authors.initials: B
authors.lastname: Liang
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Harriet
authors.initials: H
authors.lastname: Merks
authors.forename: Morag
authors.initials: M
authors.lastname: Graham
authors.forename: Paul
authors.initials: P
authors.lastname: Sandstrom
authors.forename: James
authors.initials: J
authors.lastname: Brooks
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology / Q000627:therapeutic use; D001483:Base Sequence; D016000:Cluster Analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D005787:Gene Frequency; D014644:Genetic Variation; D005838:Genotype; D015658:HIV Infections / Q000097:blood / Q000188:drug therapy / Q000821:virology*; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010802:Phylogeny; D011159:Population Surveillance; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D019380:Anti-HIV Agents; D054303:HIV Reverse Transcriptase; D016333:HIV Protease