Next generation sequencing demonstrates minor variant HIV drug resistance mutations

Fisher, Randall (2016-03)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

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.

AFRIKAANSE OPSOMMING: die tevoorskyning van MIV middelweerstandigheid (MIVMW) in terapie-naïewe en behandelde pasiënte. Hierdie middel-weerstandige virusstamme word in die virus reservoir opgeneem en kan voortbestaan as minderheid-variante wanneer dit oorgroei word deur wilde tipe variante, in die afwesigheid van voldoende geneesmiddel-druk. Alhoewel minderheid-middelweerstandige variante terapie faling mag voorspel, kan dit nie geredelik met PKR en Sanger nukleinsuurbasispaaropeenvolgingbepaling (NSOB) aangetoon word wanneer dit minder as 20% van die viruspopulasie uitmaak nie. Nuwe generasie NSOB (NGB) laat die aantoning van minderheid middel weerstandige variante toe en die ondersoek van gepaartgaande MIVMW mutasies wanneer die lees-lengte voldoende is. Verskillende NGB platforms, beskikbaar in Suid Afrika, bied kostebesparing per monster wanneer daar voldoende monsters saamgepoel word. Nietemin vereis geteikende NGB die verryking van die templaat deur PKR wat kan lei tot PKR-geïnduseerde ewekansige foute of inleier-seleksie-sydigheid en is meer uitgeproke wanneer lang fusie-inleiers gebruik word vir NGB amplikon analise. Metodes: Aangesien data beperk is oor minderheids-MIVMW variante in babas wat geïnfekteer is, ten spyte van die Wes-Kaap Voorkoming van Moeder-na-Kind MIV Oordrag (VMNKO) behandelingsplan en in volwassenes met virologiese faling sonder major protease inhibitor (PI)-weerstandigheid mutasies, op ‘n PI-gebaseerde middelkombinasie, het ons NGB in hierdie populasies uitgevoer. In die eerste deursnit studie het ons monsters geneem van minderheidsvariante van sewe volwassenes wat faling van PI-gebaseerde terapie gehad het weens swak behandelingsgetrouheid. Monsters is verryk vir NGB met ‘n geneste fusie-inleier PKR en minderheidsvariante is geïdentifiseer op die 454 Life Sciences, FLX Titanium platform (Connecticut, VSA). In die tweede deursnitstudie het ons monsters van virus variante van 15 VMNKO –gefaalde babas geneem. Geneste PKR amplikons is grootte-gefragmenteer en ligeer aan platform-spesifieke NGB-aanpassers. Die NGB biblioteek is in parallel op die Ion Torren Personal Genome Machine (PGM) (Life Technologies, California, USA) en die Illumina MiSeq (California, VSA) getoets en bevestig deur klonale NSOB. In die derde studie, het ons gepoog om die herproefneming fout wat voorkom wanneer fusie-inleiers gebruik word the karakteriseer en te korrigeer deur PKR reaksies “af te sper” in emulsie. Ons het die 454 NGB opbrengs vergelyk tussen “oop” PKR en geëmulsifiseerde PKR reaksies vir templaat verryking en vir die effek van variërende inleier templaat mispassings wanneer die populasie-steekproef opgemaak word deur plasmiedmengsels. Bevindings: In die eerste studie het NGB die aantoning van MIVMW mutasies in vyf van sewe pasiënte verbeter, alhoewel geen meerderheid-variante met PI-weerstandigheid geïdentifiseer is nie. Ons het tot die gevolgtrekking gekom dat beperkte of afwisselende middel-druk gelei het tot onvoldoende seleksie om meerderheid middel-weerstandheid variante te laat verskyn. In die tweede studie het NGB, uitgevoer op die PGM en MiSeq, middel weerstandigheid-aantoning in 15 VMNKO –gefaalde babas, verbeter. Alhoewel amplikon NSOB met fusie-inleiers die effektiefste gebruik van NGB dekking bewerkstellig, is dit geneig tot PKR herproefneming foute, afhangend van die graad van templaat-inleier mispassing. In studie drie, was emulsie PKR instaat om hierdie fout te verminder maar nie te korrigeer nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98389
This item appears in the following collections: