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

Sequencing of a bulk polymerase chain reaction (PCR) product to identify drug resistance mutations informs antiretroviral therapy selection but has limited sensitivity for minority variants. Alternatively, deep sequencing is capable of detecting minority variants but is subject to sequencing errors and PCR resampling due to low input templates. We screened for resistance mutations among 184 HIV-1-infected, therapy-naive subjects using the 454 sequencing platform to sequence two amplicons spanning HIV-1 reverse transcriptase codons 34-245. Samples from 19 subjects were also analyzed using the MiSeq sequencing platform for comparison. Errors and PCR resampling were addressed by tagging each HIV-1 RNA template copy (i.e., cDNA) with a unique sequence tag (Primer ID), allowing a consensus sequence to 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. MiSeq also allowed accurate sequencing of codon 65, an important drug resistance position embedded in a homopolymeric run that is poorly resolved by the 454 platform. Excluding homopolymeric positions, 14% of subjects had evidence of ≥1 resistance mutation among Primer ID consensus sequences, compared to 2.7% by bulk population sequencing. When calls were restricted to mutations that appeared twice among consensus sequence populations, 6% of subjects had detectable resistance mutations. The use of Primer ID revealed 5-15% template utilization on average, limiting the depth of deep sequencing sampling and revealing sampling variation due to low template utilization. Primer ID addresses important limitations of deep sequencing and produces less biased estimates of low-level resistance mutations in the viral population.

Epidemiology and evolution of rotaviruses and noroviruses from an archival WHO Global Study in Children (1976-79) with implications for vaccine design.

Prompted by the discovery of new gastrointestinal viruses, the NIH, NIAID and WHO investigated the etiology of acute diarrhea that occurred from 1976-1979 in a global cohort of infants and young children. Rotaviruses were found to be major pathogens worldwide, whereas the Norwalk virus could not be detected using a radioimmunoassay. The aim of this study is to re-evaluate the role and diversity of rotaviruses and noroviruses in the original cohort using more sensitive current technologies. Stools collected from Asia, Africa, and South America (n = 485) were evaluated for viral genotypes by RT-PCR and sequencing. Rotaviruses were detected in 28.9% and noroviruses in 9.7% of the specimens, with G1 rotaviruses and GII noroviruses accounting for the majority of each respective virus. Various strains in this study predated the currently assigned dates of discovery for their particular genotype, and in addition, two noroviruses (KL45 and T091) could not be assigned to current genotypes. Phylogenetic analyses demonstrated a relative constancy in circulating rotavirus genotypes over time, with several genotypes from this study becoming established in the current repertoire of viral species. Similarly, GII noroviruses have maintained dominance, with GII.4 noroviruses continuing as a predominant genotype over time. Taken together, the complex molecular epidemiology of rotaviruses and noroviruses circulating in the 1970's is consistent with current patterns, an important consideration in the design of multivalent vaccines to control these viruses.