title: Next-generation sequencing of dried blood spot specimens: a novel approach to HIV drug-resistance surveillance.
abstract: BACKGROUND
  HIV drug-resistance (DR) surveillance in resource-limited settings can be performed using dried blood spots (DBS) because of ease of collection, transportation and storage. Analysis of pooled specimens on next-generation sequencing (NGS)-based platforms, such as the 454 pyrosequencing, is an efficient sequencing method for determining HIV DR rates. In this study, we conducted HIV DR surveillance on DBS using NGS and identified minority variants in individual patients.
  
  
  METHODS
  A total of 48 extracts of DBS from an HIV DR surveillance study in Mexico City were re-amplified using primers tagged with multiplex identifiers, pooled and pyrosequenced. Consensus sequences were generated for each specimen with mixtures identified at positions where >20% of the reads contained a variant. Individual consensus sequences were then analysed for DR mutations and compared with those derived from Sanger sequencing.
  
  
  RESULTS
  DBS analysed with tagged pooled pyrosequencing (TPP) were highly concordant with Sanger sequencing genotypes from matching plasma and DBS (99.21% and 99.51%, respectively). An exception was an M184I mutation only detected with TPP of DBS at a frequency of 20.4%. Multiple specimens had minority variant reads below the 20% mixture threshold.
  
  
  CONCLUSIONS
  TPP using DBS is an effective method for HIV DR surveillance. TPP for genotyping results in cost savings of 40% over conventional in-house methods. The effect of low-abundance DR mutations, undetectable by conventional methods, remains to be determined. This technology might be applied to any HIV specimen (plasma/serum) and can also be used for other diagnostic assays where DNA sequencing is required.
authors: National HIV & Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, ON, Canada.
authors: Hezhao
authors: H
authors: Ji
authors: Yang
authors: Y
authors: Li
authors: Morag
authors: M
authors: Graham
authors: Ben Binhua
authors: BB
authors: Liang
authors: Richard
authors: R
authors: Pilon
authors: Shari
authors: S
authors: Tyson
authors: Geoff
authors: G
authors: Peters
authors: Shaun
authors: S
authors: Tyler
authors: Harriet
authors: H
authors: Merks
authors: Silvia
authors: S
authors: Bertagnolio
authors: Luis
authors: L
authors: Soto-Ramirez
authors: Paul
authors: P
authors: Sandstrom
authors: James
authors: J
authors: Brooks
mesh_terms: D016384:Consensus Sequence; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D015658:HIV Infections / Q000175:diagnosis; D015497:HIV-1 / Q000145:classification / Q000187:drug effects* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation / Q000235:genetics; D010802:Phylogeny; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D012367:RNA, Viral; D054302:pol Gene Products, Human Immunodeficiency Virus