The use of CCR5 antagonists to block HIV-1 replication has accelerated the development of HIV-1 coreceptor tropism assays 
and stressed the need for novel, sensitive, and more affordable tests to increase treatment with this drug class. Although Trofile is the most commonly used phenotypic assay for HIV-1 coreceptor tropism, less sensitive genotypic tests based on HIV-1 population (Sanger) sequencing are frequently used in Europe, leading to the rapid adoption of deep sequencing technologies in genotypic HIV-1 coreceptor tropism protocols 
. Based on the need for these NGS-based genotypic assays, we have compared the ability of four NGS platforms (454™, Illumina®, PacBio®, and Ion Torrent™) to detect minority variants, and to infer the presence of non-R5 viruses within the HIV-1 population.
Next generation sequencing has been used in a multitude of biological fields, from the sequencing of whole genomes of animals, plants, and microbes, to targeted studies on polymorphisms related to various genetic disorders and cancer, most of them based on Illumina® 
and 454™ 
platforms and more recently using PacBio® 
and Ion Torrent™ 
systems. To date all published HIV-related studies have used the 454™ platform, due in part to being one of the first NGS systems to provide longer read lengths 
. As expected each deep sequencing platform differs in terms of the chemistry, read length, yield, error rate, turn-around time, and overall cost 
. Here, we sequenced the HIV-1 V3 region from the same RNA aliquots obtained from 12 patients and showed that all four NGS platforms had similar substitution and insertion rates (ranging from 1.3 to 1.8 and 1.7 to 2.4 mean #/read, respectively), while Illumina® had the fewest deletions per read (0.01 versus a range of 0.2–0.5 mean #/read for the other three platforms). This is consistent with the reduced number of indels reported for Illumina® when compared with 454™ during the genome sequencing of Gallus gallus
or influenza virus 
and the sequencing of a strain of Escherichia coli
using 454™ and Ion Torrent™ 
We observed differences in the number of V3 sequences and mean read length among the NGS platforms, which were due to both the size of the PCR product selected for sequencing and intrinsic characteristics of the sequencing method. Short amplicons (337 nt) containing the V3 region were sequenced using 454™ and PacBio®; however, library preparation and shotgun sequencing was performed on the entire PCR-amplified env
gene (2,302 nt) using Illumina® and Ion Torrent™. In addition, the 454™ sequencing was performed in-house using barcoded sequencing primers while for Illumina®, PacBio®, and Ion Torrent™ the amplicons were sent for sequencing at the respective company. Despite these differences, all NGS platforms were able to detect the same higher frequency variants but showed slightly variations in the detection of low frequency variants (<0.5%), which had limited implications for HIV-1 tropism. It is important to note that based on HIV-1 clonal sequencing the error rate for in-house 454™ sequencing assays has been calculated to be between 0.1% and 0.5% 
; therefore, we only used variants present at ≥1% of the viral population for diversity and HIV-1 tropism analyses.
Multiple studies have compared the efficacy of phenotypic and genotypic HIV-1 tropism assays to detect non-R5 variants 
. In general, population-based sequencing tests are less sensitive and less specific than phenotypic assays 
, although a few studies have shown significant concordance and predictive values 
. More sensitive deep sequencing methods for HIV-1 coreceptor tropism assays resulted in the detection of minor variants, which correlated well with both phenotypic assays 
and virological response to maraviroc 
. Here we have shown that all four NGS platforms provide equal and sensitive detection of minority non-R5 viruses in 12 patients, with minor differences depending on the bioinformatic algorithm used to infer HIV-1 tropism. However, it is important to stress that maraviroc was combined with at least two other antiretroviral drugs and increased viral loads could be due to several factors including poor or selective drug adherence and resistance to other drugs while maintaining partial maraviroc suppression. Nevertheless, all four NGS platforms showed similar concordance with virologic response at week 12 (ranging from 75% to 80% depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). Despite the limited number of samples, these results are comparable to previous studies where deep sequencing had a good concordance with phenotypic HIV1- tropism tests (82% to 87%) 
and matched Trofile™ in predicting the success of maraviroc-based antiretroviral regimens 
In conclusion, this is the first study comparing the ability of the four current leaders in deep sequencing (454™, Illumina®, PacBio®, and Ion Torrent™) to detect minority variants, and to infer the presence of non-R5 viruses, within the HIV-1 population. Despite minor differences in error rates and profiles (types of errors), all four NGS platforms successfully detected the same unique viral variants present at high frequencies, which are the sequences relevant for the clinical determination of HIV-1 coreceptor tropism. Further studies with larger number of patients and the latest chemistry and software for each NGS system will be needed to corroborate our findings; however, despite intrinsic parameters to each NGS platform (e.g., read length, error rates, cost per run, and turn-around time) we suspect that any of the current NGS platforms will be effective in a genotypic test to predict HIV-1 coreceptor usage.