Background: HIV-1 has an extreme level of polymorphism which underpins the ability of the virus to escape HLA-restricted immune responses as well as antiretroviral drugs. Minor quasispecies in plasma may mediate drug resistance but not be detected by standard bulk sequencing methods. We have therefore developed a full-length HIV -1 pol PCR/NGS assay used to detect variants at low frequency. In addition, sequencing of longer HIV segments improves the accuracy of phylogenetic reconstructions and subtype assignment.
Method: We have designed and evaluated a complete HIV-1 full length pol PCR, next generation sequencing (NGS) and reporting pipeline that includes using nested PCR, NGS Ion Torrent S5 sequencing and Stanford HIV DB resistance algorithm for determining HIV drug resistant profiles and HIV subtypes in 41 sequences within the diverse Western Australian HIV Cohort.
Results: We successfully amplified more samples using the HIV-1 FL pol PCR method when compared to our current routine HIV-1 PCR method (95.3% vs 88.3%), including success at amplifying samples with low level viremia (<10,000cpm).
The overall average mapped reads (MR) was 312447 with a MR average length for all runs of 313.1 and the percent MR after trim was 92.5%. The read depth was 99.72%, the specificity of the mapped reads over the target region was 99.99% and the average coverage from all runs was 31849.
We obtained 100% concordance of HIV-1 subtypes in the testing cohort, and of the samples utilised in the HIV FL pol PCR/NGS evaluation, 95.0%, 97.5% and 100% of the DRM profile results obtained from Stanford HIV DB, were concordant to the current routine HIV PCR/Sanger sequencing results obtained for Protease, Reverse Transcriptase and Integrase respectively.
Conclusion: The newly developed in-house HIV-1 FL pol PCR/NGS assay has the capability to make contributions to the efficiency of HIV genotyping within the PathWest Immunology department.