Doctoral Degrees (Medical Physiology)

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Browsing Doctoral Degrees (Medical Physiology) by Author "Fisher, Randall"

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    Next generation sequencing demonstrates minor variant HIV drug resistance mutations
    (Stellenbosch : Stellenbosch University, 2016-03) Fisher, Randall; Van Zyl, Gert; Engelbrecht, Susan; Preiser, Wolfgang; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
    ENGLISH ABSTRACT: Introduction: The South African public sector antiretroviral therapy (ART) roll-out has been associated with emergence of HIV drug resistance (HIVDR) in therapy-naïve and treated patients. These drug-resistant viral strains are archived in viral reservoirs and may persist as minority variants when outgrown by fitter wild-type strains, in the absence of sufficient drug pressure. Although minority drug resistant variants may predict failure, they are not readily detectable with PCR and Sanger sequencing when constituting less than 20% of the viral population. Next generation sequencing (NGS) allows the detection of minor drug resistant variants and investigation of linkage of HIVDR mutations when sufficient read-length is obtained. Various NGS platforms, available in South Africa, offer per-sample cost reductions when sufficient numbers of samples are pooled. However, targeted resequencing on NGS platforms requires template enrichment by PCR that can result in PCR-induced error and template re-sampling error. Resampling error is a result of random error or a primer selection bias and is more pronounced when using long fusion primers for NGS amplicon sequencing. Methods: As data were limited on minor HIVDR variants in infants who became infected despite the Western Cape Prevention of Mother-to-Child HIV Transmission (PMTCT) regimen and in adults with virological failure without major protease inhibitor (PI)-resistance mutations on a PI-based regimen, we performed NGS in these populations. In the first cross-sectional study we sample minority variants from seven adults who failed PI-based therapy due to poor adherence. Samples were enriched for NGS with a nested fusion primer PCR and minor variants were identified on the 454 Life Sciences, FLX Titanium platform (Connecticut, USA). In the second cross-sectional study we sampled viral species from 15 PMTCT-failed infants. Nested PCR amplicons were size-fragmented and ligated to platform-specific sequencing adaptors. The sequence library was sequenced in parallel on the Ion Torrent Personal Genome Machine (PGM) (Life Technologies, California, USA) and the Illumina MiSeq (California, USA) and was validated by clonal sequencing. In study three, we attempted to characterise resampling error when using fusion primers and correct it by partitioning PCR reactions in emulsion. We compared the 454 NGS sequence yields when using “open” or emulsified PCR for template enrichment and investigated the effect of varying degrees of primer-template mismatches when the sampled population consisted of mixtures of plasmids. Findings: In the first study, NGS improved the drug resistance mutation detection in five of the seven patients although no majority variants with PI-resistance were identified. We concluded that limited or intermittent drug pressure resulted in insufficient selection for major drug-resistant variants to emerge. In study two, NGS conducted on the PGM and MiSeq improved the drug resistance detection in 15 PMTCT-failed infants. Although amplicon sequencing using fusion primers allows the most efficient use of NGS coverage, it is prone to PCR resampling error depending on the degree of template-to-primer mismatches. In study three, emulsion PCR was able to reduce but not correct this resampling error.