Abstract
Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.
Highlights
Conventional HIV drug resistance (HIVDR) genotyping, in the context of clinical care, research and public health, is traditionally carried out using population-based Sanger sequencing techniques, which have a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20% [1,2,3,4]
We review issues and considerations for the use of next generation sequencing (NGS) for HIVDR testing from the laboratory, clinical, and implementation points of view
A recent metaanalysis and nationally representative surveys have shown that pre-treatment HIVDR to non-nucleoside reverse transcriptase inhibitors (NNRTI) has steadily increased in many low- and medium-income countries (LMIC) in the last decade, several of which still need to progress to integrase inhibitor-based antiretroviral therapy (ART) regimens as the preferred first-line option [50,51]
Summary
Conventional HIV drug resistance (HIVDR) genotyping, in the context of clinical care, research and public health, is traditionally carried out using population-based Sanger sequencing techniques, which have a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20% [1,2,3,4]. With the widespread use and decreasing costs of generation sequencing (NGS) techniques, availability of commercial kits, and development of dedicated and freely available analysis pipelines [4,10], an increasing number of laboratories around the world, especially in high-income contexts are considering or definitively moving toward these technologies for HIVDR testing [11]. NGS enables high-throughput, massively parallel sequencing of individual input templates, with the potential to improve sensitivity and quantitatively identify low-abundance variants. We believe that many of the methodological, sample-related, and analysis-associated issues raised in the manuscript are current problems being faced by experts in the field, and are of interest to more advanced groups and laboratories
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