Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain

Abstract Sequencingof a bulk polymerase chain reaction (PCR) product to identify drug resistance mutationsinforms antiretroviral therapy selection but has limited sensitivity for minority variants. Alternatively, deep sequencingis capable of detecting minority variants but is subject to sequencingerrors and PCR resamplingdue to low input templates. We screened for resistance mutationsamong 184 HIV-1-infected, therapy-naive subjects using the 454 sequencingplatform to sequencetwo ampliconsspanning HIV-1 reverse transcriptase codons 34–245. Samples from 19 subjects were also analyzed using the MiSeq sequencingplatform for comparison. Errors and PCR resamplingwere addressed by tagging each HIV-1 RNA template copy (i.e., cDNA) with a unique sequencetag (Primer ID), allowing a consensus sequenceto be constructed for each original template from resampled sequences. In control reactions, Primer ID reduced 454 and MiSeq errors from 71 to 2.6 and from 24 to 1.2 errors/10,000 nucleotides, respectively....