Abstract
Viral genetic sequencing can be used to monitor the spread of HIV drug resistance, identify appropriate antiretroviral regimes, and characterize transmission dynamics. Despite decreasing costs, next-generation sequencing (NGS) is still prohibitively costly for routine use in generalized HIV epidemics in low- and middle-income countries. Here, we present veSEQ-HIV, a high-throughput, cost-effective NGS sequencing method and computational pipeline tailored specifically to HIV, which can be performed using leftover blood drawn for routine CD4 cell count testing. This method overcomes several major technical challenges that have prevented HIV sequencing from being used routinely in public health efforts; it is fast, robust, and cost-efficient, and generates full genomic sequences of diverse strains of HIV without bias. The complete veSEQ-HIV pipeline provides viral load estimates and quantitative summaries of drug resistance mutations; it also exploits information on within-host viral diversity to construct directed transmission networks. We evaluated the method's performance using 1,620 plasma samples collected from individuals attending 10 large urban clinics in Zambia as part of the HPTN 071-2 study (PopART Phylogenetics). Whole HIV genomes were recovered from 91% of samples with a viral load of >1,000 copies/ml. The cost of the assay (30 GBP per sample) compares favorably with existing VL and HIV genotyping tests, proving an affordable option for combining HIV clinical monitoring with molecular epidemiology and drug resistance surveillance in low-income settings.
Highlights
David Bonsall and Tanya Golubchik contributed to this work, and the order in which they are listed reflects a decreasing order of managerial responsibility for the project
The veSEQ-HIV protocol was developed to obtain multiple measurements from a single assay (Fig. 1). It provides a quantitative viral load estimate across at least 5 orders of magnitude, frequency of drug resistance mutations at both consensus and minority variant levels, and accurate and unbiased genotype information that is suitable for ancestral state reconstruction and the generation of directed transmission networks
Like most high-throughput next-generation sequencing (NGS) protocols, veSEQ-HIV requires fragmentation of the virus RNA into so-called “inserts.” In previous work, we found that inserts of 350 bp or more offer useful insights into within-host phylogenetic diversity [7]; we sought to optimize the length of these inserts to be as long as possible within the limits compatible with the Illumina sequencing platform (350 to 600bp)
Summary
David Bonsall and Tanya Golubchik contributed to this work, and the order in which they are listed reflects a decreasing order of managerial responsibility for the project. We present veSEQ-HIV, a high-throughput, cost-effective NGS sequencing method and computational pipeline tailored to HIV, which can be performed using leftover blood drawn for routine CD4 cell count testing. This method overcomes several major technical challenges that have prevented HIV sequencing from being used routinely in public health efforts; it is fast, robust, and cost-efficient, and generates full genomic sequences of diverse strains of HIV without bias. The FDA approved the first NGS assay for HIV drug resistance using polspecific PCR that can sequence up to 15 samples in parallel [6]; demand remains for more high-throughput, low-cost options for use clinically and as a surveillance tool in high-prevalence settings. We describe veSEQ-HIV, a comprehensive laboratory and computational protocol developed to support clinical management and public health programs in lowincome settings
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