Ion Torrent next generation sequencing for accurate genotyping and detection of resistance associated variants in HCV and HIV
Poster Dec 22, 2016
Elian Rakhmanaliev, Pramila Ariyaratne, Charlie Lee, Pornpimon Nimitsuntiwong, Chortip Wathtphan, Ekawat Passomsub, Kok Siong Poon, Cui Wen Chua, Mui Joo Khoo, Zhang Rui, Wen Huang, Evelyn S. Koay, Wasun Chantratita, Gerd Michel
Background: detection of resistance-associated mutations is well established in HIV ART (as DRMs) and is increasingly used in HCV patients selected for treatment (as RAVs) with direct acting antiviral agents (DAAs). Both for DAA treatment and conventional interferon-based therapy accurate determination of HCV genotypes (GTs) is essential. Sanger sequencing has recognized limitations in sensitivity and turn around time. NGS provides excellent accuracy, speed and sensitivity enabling detection of rare mutants, HCV subtypes as well as mixed infections.
Objectives: to develop improved detection of clinically relevant viral mutations using ion torrent based NGS in an automated workflow.
Materials and methods: we have used NGS in combination with workflow automation on a newly developed platform based on the emotion 5075 system (Eppendorf, Germany) consisting of a continuous robotic process starting with sample extraction and RT-PCR followed by automated library preparation, Ion Torrent deep sequencing and direct online data analysis to determine HCV genotypes and RAVs as well as DRMs in HIV. We have employed target sequences from the HCV NS3, NS5A and NS5B regions. For HIV sequences in reverse transcriptase, protease and integrase were selected for NGS.
Results: we are reporting results from an evaluation study conducted on >200 HCV sera comparing HCV genotyping with line probe assay. Two cases of mixed GT infections were detected. Confirmation of discrepant results between NGS and line probing by Sanger sequencing indicated 100% accurate GTs by NGS whereas in several cases line probe results would have led to selection of sub-optimal therapy regimens. In an HIV pilot study (n=112 patients), comparing NGS results to TruGene sequencing the Sentosa SQ HIV Genotyping Assay detected 100% (199/199) of all mutations in the protease gene and more that 98% mutations (427/435) in the reverse transcriptase gene.
Conclusions: Given the crucial role of accurate sequencing analysis in HCV and HIV treatment management, workflow automated NGS appears as a highly reliable tool for differentiating HCV GTs and RAVs, which can help to prevent diagnostic errors potentially leading to suboptimal treatment.
Considering the pivotal role of DRMs in HIV patients under HAART the Sentosa SQ HIV Genotyping workflow appears as a valuable new tool for detecting clinically relevant HIV variants. Given its high sensitivity compared to Sanger based systems and the comparatively short turnaround time of two days the workflow offers relevant improvements in HIV DRM detection.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE