Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

Archer, John; Weber, Jan; Henry, Kenneth R.; Winner, Dane; Gibson, Richard; Lee, Lawrence; Paxinos, Ellen E.; Arts, Eric J.; Robertson, David L.; Mimms, Larry; Quiñones‐Mateu, Miguel E.

doi:10.1371/journal.pone.0049602

Cited by 78 publications

(70 citation statements)

References 70 publications

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“…Using a fixed cutoff can retain a large proportion of offspring sequences, introducing skewing from rerepresentation of templates with the abundant Primer ID reads as the apparent templates of offspring Primer IDs. Second, previous studies used the Roche 454 platform, which has a much lower throughput compared to the MiSeq platform used in the present study (21,24,25), which, along with the Ion Torrent platform, has a high error rate at homopolymer runs (26,27) that is not a feature of MiSeq platform. When the number of raw sequence reads per template is not sufficient (due either to low capacity or too much multiplexing), the peak of the Primer ID read distribution will be shifted toward the low-abundance error end, making template recovery significantly less than optimal (Fig.…”

Section: Discussionmentioning

confidence: 90%

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Zhou

Jones

Mieczkowski

et al. 2015

J Virol

123

167

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Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/ sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. IMPORTANCEAlthough next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set. Studies of viral population diversity are increasingly using nextgeneration sequencing (NGS) technologies to extend the depth of population sampling. Key aspects of understanding within-host viral population diversity are knowing the true depth of template/genome sampling and documenting the accuracy of the sequencing method to validate the detection of rare variants. Current approaches using NGS in viral population studies usually require a preceding PCR amplification step. Thus, PCR errors, including nucleotide misincorporation ...

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Section: Discussionmentioning

confidence: 90%

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Zhou

Jones

Mieczkowski

et al. 2015

J Virol

123

167

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“…Another study required 2 to 6 cDNA reactions and 5 to 35 separate PCRs to generate libraries for full-genome sequencing on three different NGS platforms (13). The speed of Nextera XT, the tremendous sequence depth, an error rate of 0.1%, and the current read lengths (v3 ϭ 600 nt) obtained on the ubiquitous Illumina MiSeq are also preferable to the time-consuming library amplification steps, lower throughput, and higher error rates associated with other platforms (11,36). We estimate the cost of the m2000 extraction, HIV-SMART library prep, and MiSeq sequencing reagent was approximately $165 per genome for the multiplex run of 23 libraries.…”

Section: Discussionmentioning

confidence: 99%

“…The utility of next-generation sequencing (NGS) has been demonstrated for a variety of applications related to HIV patient monitoring, including identification of drug resistance mutations (6)(7)(8)(9)(10) and prediction of coreceptor tropism (11,12). Examples generally entail sequencing only of pol or env amplicons, respectively, to a depth necessary to identify low-level variants.…”

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confidence: 99%

A Pan-HIV Strategy for Complete Genome Sequencing

et al. 2016

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bMolecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5= end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ϳ2,000؋ depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of <4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

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“…Most of those studies used 454 sequencing, which was limited by homopolymer errors and relative low throughput compared to the Illumina or IonTorrent platform (25). In these studies, the presence of X4 variants was defined by the detection of Ͼ2% of sequences with FPR values of Ͻ3.5% in the viral population, as seen in the MERIT and MOTIVATE cohort studies (12,13,49).…”

Section: Discussionmentioning

confidence: 99%

“…EPD PCR is time-consuming and labor-intensive, making it challenging to analyze more than a few dozen viral genomes per sample (20)(21)(22), which limits the depth of sampling of viral genomes when assessing the complexity of the viral population. Recently, viral population studies have started to incorporate deep-sequencing or next-generation sequencing (NGS) technologies that have the capacity to greatly extend the depth of sampling (12,(23)(24)(25)(26)(27)(28). However, conventional approaches using NGS in HIV-1 population studies have serious limitations in representing an accurate sampling of the original viral population.…”

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confidence: 99%

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

Zhou

Bednar

Sturdevant

et al. 2016

J Virol

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HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of <2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated. IMPORTANCEPrimer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants by using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting that X4 and R5 variants are genetically isolated and may be replicating in different cell types or that X4/R5 recombinants have reduced fitness. H uman immunodeficiency virus type 1 (HIV-1) requires the CD4 receptor and a coreceptor to infect host cells. Entry is mediated by the viral envelope protein (Env), which is processed to give two associated subunits, gp120 and gp41, that assemble into a trimeric structure embedded in the viral envelope/membrane. The binding of gp120 to CD4 triggers a structural change that exposes its variable region 3 (V3) loop, which is part of the coreceptor domain (1, 2). The majority of transmitted/founder (T/F) viruses and viruses isolated from the clinically latent stage in HIV-infected subjects use CCR5 as the coreceptor, making the virus CCR5 tropic (or an R5 virus). The virus can evolve to use CXCR4 (CXCR4 tropic [or an X4 virus]), and viruses that have switched core...

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Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

Cited by 78 publications

References 70 publications

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

A Pan-HIV Strategy for Complete Genome Sequencing

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

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