title: Short Communication: Analysis of Minor Populations of Human Immunodeficiency Virus by Primer Identification and Insertion-Deletion and Carry Forward Correction Pipelines.
abstract: Accurate analysis of minor populations of drug-resistant HIV requires analysis of a sufficient number of viral templates. We assessed the effect of experimental conditions on the analysis of HIV pol 454 pyrosequences generated from plasma using (1) the "Insertion-deletion (indel) and Carry Forward Correction" (ICC) pipeline, which clusters sequence reads using a nonsubstitution approach and can correct for indels and carry forward errors, and (2) the "Primer Identification (ID)" method, which facilitates construction of a consensus sequence to correct for sequencing errors and allelic skewing. The Primer ID and ICC methods produced similar estimates of viral diversity, but differed in the number of sequence variants generated. Sequence preparation for ICC was comparably simple, but was limited by an inability to assess the number of templates analyzed and allelic skewing. The more costly Primer ID method corrected for allelic skewing and provided the number of viral templates analyzed, which revealed that amplifiable HIV templates varied across specimens and did not correlate with clinical viral load. This latter observation highlights the value of the Primer ID method, which by determining the number of templates amplified, enables more accurate assessment of minority species in the virus population, which may be relevant to prescribing effective antiretroviral therapy.
authors.affiliation: 1 Center for Global Infectious Disease Research, Seattle Children's Research Institute , Seattle, Washington.
authors.forename: Paul
authors.initials: P
authors.lastname: Hughes
authors.affiliation: 2 Department of Microbiology, University of Washington , Seattle, Washington.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Deng
authors.affiliation: 1 Center for Global Infectious Disease Research, Seattle Children's Research Institute , Seattle, Washington.|3 Department of Pediatrics, University of Washington , Seattle, Washington.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Olson
authors.affiliation: 4 Department of Laboratory Medicine, University of Washington , Seattle, Washington.|5 Department of Medicine, University of Washington , Seattle, Washington.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Coombs
authors.affiliation: 4 Department of Laboratory Medicine, University of Washington , Seattle, Washington.|5 Department of Medicine, University of Washington , Seattle, Washington.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Chung
authors.affiliation: 1 Center for Global Infectious Disease Research, Seattle Children's Research Institute , Seattle, Washington.|3 Department of Pediatrics, University of Washington , Seattle, Washington.|4 Department of Laboratory Medicine, University of Washington , Seattle, Washington.|6 Department of Global Health, University of Washington , Seattle, Washington.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Frenkel
mesh_terms: D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D014644:Genetic Variation; D005828:Genetics, Population; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006678:HIV / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D015658:HIV Infections / Q000821:virology*; D006801:Humans; D016254:Mutagenesis, Insertional; D009154:Mutation; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D017931:DNA Primers