Introduction We propose to perform near full-length deep sequencing of HIV-1 genomes from approximately 100 newly-diagnosed cases in San Francisco in the previous year (2016), using stored specimens from the clinical genotyping service that serves the majority of HIV cases in San Francisco.
Methods Stored, frozen blood plasma from approximately100 newly-diagnosed cases in San Francisco over the previous year will be analysed. Four amplification products covering most of the HIV-1 genome will be derived by RT-PCR adapted from existing protocols. These protocols have been shown to be successful for the near full length analysis of approximately 85% of specimens having viral load greater than 10 000 copies per ml, which represent the majority of newly diagnosed people in San Francisco. Briefly, four overlapping regions spanning all reading frames of the HIV genome will be amplified in a published one-step RT-PCR strategy. Pooled amplicons will be purified and sequenced on an Illumina HiSeq instrument operated by GENEWIZ.
Results This work is ongoing and we have generated approximately half of the near full length HIV genomes for this project. This data and techniques will be presented as part of the results.
Conclusion Deep sequencing provides better resolution for characterising the duration of infection. Viral diversity increases over time on average and can be estimated from the frequency of sequence ambiguities in population sequences. However, sequence ambiguities can occur due to dual infection, which can occur early in infection, and make the virus population sequence appear older.
Support This project is funded entirely by an RO1 grant from the National Institutes of Health, USA