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HIV and AIDS
host immune responses such as immunoglobulin or T-cell receptor unclear whether new genotypic drug resistance interpretation systems will
rearrangements during the adaptative immune response. need to be developed for this technique. Fifth, the accuracy per
sequenced base pair is lower than with Sanger sequencing or with the
Following reverse transcription and amplification of the region of interest SOLEXA™ (Illumina
®
) or SOLiD™ (Applied Biosystems
®
) high-throughput
of the HIV-1 genome using primers with fusion adapters in the 5’-end, massively parallel sequencing systems. Sixth, accuracy is significantly
each DNA molecule is immobilised onto a 28µm DNA capture bead. The impaired in homopolymeric regions, particularly in those with more than
bead-bound amplicons are emulsified with amplification reagents in a five to six consecutive identical bases. This requires specific base-calling
water-in-oil mixture resulting in microreactors containing just one bead protocols to be established in order to analyse the output sequence data
with one unique DNA molecule. Each unique DNA molecule is amplified properly. Finally, the cost of this technology remains high, although it is
in an emulsion PCR within its own microreactor, which in principle decreasing. Techniques such as primer barcoding increase the efficiency of
excludes competing or contaminating sequences. Amplification of the reagents and hardware use.
entire fragment collection is performed in parallel; for each fragment, this
results in a copy number of several million per bead. Subsequently, the New High-throughput Massively Parallel Sequencing
emulsion PCR is broken while the amplified fragments remain bound to Systems – SOLEXA™ and SOLiD™
their specific beads. The clonally amplified fragments are enriched and The SOLEXA™ and SOLiD™ systems developed by Illumina and
loaded onto a PicoTiterPlate™ device for sequencing. The diameter of the Applied Biosystems, respectively, are at the forefront of the many
PicoTiterPlate™ wells is 44µm, which allows for only one bead per well. other high-throughput sequencing technologies currently in the
After addition of sequencing enzymes, individual nucleotides are flown in pipeline. The main characteristics of these two methods are
a fixed order across the hundreds of thousands of wells containing one summarised in Table 7. Both assays have higher accuracy per base
bead each. Addition of one (or more) nucleotide(s) complementary to the than 454 sequencing and achieve a much better treatment of
template strand results in a chemiluminescent signal recorded by a CCD homopolymeric regions. These technologies are starting to be applied
camera. The light signals are processed and transmitted to a computer, to detect minority HIV variants, but data have not yet been published.
which generates a report with the DNA sequence for each clone. Each The sensitivity threshold to detect point mutations is unknown, but it
cycle is completed in less than 10 hours. The accuracy per base is >99.5% could be at least at low as that of 454 sequencing. The main limitation
(> 99.99% if a consensus sequence is used as a reference) and the cost relative to 454 sequencing is that, at present, read lengths are 35 base
per base is less than half than by Sanger sequencing. pairs or shorter, which limits the ability to assess several resistance
mutations in single amplicons. However, these technologies – and
However, this technique has several limitations. First, it is an open many others in the pipeline – are evolving rapidly.
sequencing system that requires extensive bioinformatic support in order
to fully analyse the massive amount of genetic information generated. Conclusion
Second, the lower limit of detection of low-abundant resistant variants Resistance testing is a critical component of the clinical management of HIV
remains to be defined for this technology, although it seems to be close infection. In this article we discussed the different assays available for
to 0.5%. Third, the clinically relevant lower limit of detection of minority resistance testing in the clinic as well as the new ultrasensitive resistance
drug resistant variants remains unknown. Drug-resistant variants present assays that clinicians may have to face in the not-so-distant near future.
at significantly lower frequencies than the apparent lower detection Several technical improvements are required, however, before ultrasensitive
theshold of 454 sequencing can emerge in vivo under selective drug resistance assays find a clinical role. The most important obstacle is to
pressure. In contrast, subjects harbouring minority resistant variants can determine thresholds of minority variants that predict treatment outcomes
achieve prolonged virological suppression on triple therapy. Fourth, it is with high sensitivity and specificity. ■
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62 EUROPEAN INFECTIOUS DISEASE
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