Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism

Abstract

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

Figure 1. Schema summarizing the strategy followed in this study to compare the use of the four next-generation sequencing platforms (454™, Illumina®, PacBio®, and Ion Torrent™) to determine HIV-1 coreceptor tropism (see text for full details).

Figure 2. Comparison of data processing across NGS platforms.

Number of sequencing errors, substitutions, deletions, and insertions (per read) for the NGS platforms: 454™, Illumina®, PacBio®, and Ion Torrent™. The mean and interquartile range (IQR) are indicated for each sample. Whiskers indicate 1.5 times the IQR as is the default value in the R-statistical package .

Figure 3. Comparison of the frequency of variants across NGS platforms.

The heights of the bars represent the combined frequency of V3 variants detected by the NGS platforms 454™, Illumina®, PacBio®, and Ion Torrent™ prior to filtering. The colors within each bar denote the proportional contribution made by each platform after normalization based on coverage. Insets show low frequency variants up to a maximum of 20 unique sequences.

Figure 4. Comparison of the clustering of variants across platforms.

The ten most common nucleotide V3 sequences from samples 10–65, 10–69, and 10–73 -obtained with each of the four NGS platforms (454™, Illumina®, PacBio®, and Ion Torrent™)- were aligned against the respective population (sanger) sequence from the respective patient using Clustal X 2.0 . For each patient, every unique variant is identified by the NGS platform used and the number of sequences (frequency) obtained, e.g., 454#1290. For each position only those nucleotides that differ from the population sequence are depicted. Dashes indicated the same nucleotide as the population sequence while gaps introduced to maintain the alignment are indicated by dots. Relative clustering of the data from the NGS platforms was inferred by neighbor-joining, phylogenetic analyses determined using MEGA 5.05 and displayed in a circle with topology only to facilitate their interpretation. Bootstrap resampling (1,000 data sets) of the multiple alignments tested the statistical robustness of the trees, with percentage values above 60% indicated by an asterisk. The size of the circles in the phylogenetic trees correlates with the frequency of the unique sequence determined by each NGS platform (color) in the logarithmic scale. The black box denotes the population (sanger) sequence for each sample.

Figure 5. Comparison of the clustering of variants across platforms.

The ten most common nucleotide V3 sequences from samples 10–75, 10–80, and 10–91 -obtained with each of the four NGS platforms (454™, Illumina®, PacBio®, and Ion Torrent™)- were aligned against the respective population (sanger) sequence and analyzed as described in the Figure 4 legend.

Figure 6. Comparison of the clustering of variants across platforms.

The ten most common nucleotide V3 sequences from samples 10–105, 10–133, and 10–137 -obtained with each of the four NGS platforms (454™, Illumina®, PacBio®, and Ion Torrent™)- were aligned against the respective population (sanger) sequence and analyzed as described in the Figure 4 legend.

Figure 7. Comparison of the clustering of variants across platforms.

The ten most common nucleotide V3 sequences from samples 10–172, 10–176, and 10–180 -obtained with each of the four NGS platforms (454™, Illumina®, PacBio®, and Ion Torrent™)- were aligned against the respective population (sanger) sequence and analyzed as described in the Figure 4 legend.

Figure 8. HIV-1 coreceptor tropism determination using deep sequencing.

(A) HIV-1 tropism determined at baseline using Trofile™ (Monogram Biosciences) ; R5, CCR5-tropic virus; D/M, dual mixed. (B) Virologic response at week 12 of a maraviroc-based antiretroviral regimen. Y or N corresponds to plasma viral load below or not 400 copies/ml at week 12, respectively. E.S., end of study (patient did no enter the study following the detection of non-R5 variants at baseline using Trofile™). (C) Quantification of non-R5 variants detected by deep sequencing as predicted using four HIV-1 tropism algorithms, i.e., 11/24/25 rule , Geno2Pheno 3.5% FPR , , , Geno2Pheno 10% FPR, and Web PSSM using the subtype B x4r5 matrix . Dotted line represents the ≥2% suggested cutoff for the minimal amount of non-R5 sequences to be present in the viral population in order to classify a given virus as non-R5 , .