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HIV Drug Resistance Testing
Table 7: Other High-throughput Ultrasensitive Sequencing Techniques with Potential Application to Detection of Minority HIV-1 Variants
SOLiD™ (Applied Biosystems
®
) SOLEXA™ (Illumina
®
)
Principle Massively parallel sequencing of clonally amplified DNA fragments Massively parallel sequencing of DNA fragments incorporating adapters,
linked to magnetic beads, attached to a solid phase → Sequencing that are attached to a solid phase → Bridge amplification on solid phase
by sequential ligation with dye-labeled probes. → Sequencing-by-synthesis using labelled reversible terminators
Read length 35 bp 35 bp
Accuracy 99.94% (99.999% consensus accuracy @ 15x coverage) 99.99% @ 3X coverage
Sensitivity Uncertain for HIV (likely <1%) Uncertain for HIV (likely <1%)
Advantages over Bidirectional sequencing of the same amplicon feasible Accurate homopolymer sequencing
454/Roche Fewer cumulative and sequential errors High accuracy
ultra-deep sequencing Better treatment of homopolymeric zones
Disadvantages for HIV Short sequence reads, no linkage Short sequence reads, no linkage
directly quantify mutant DNA alone or to simultaneously detect and gene his3AI. Expression and reverse transcription of the TyHRT element
quantify mutant and wild-type DNA by using a pair of mutant and by the recombined HIV-1 RT results in conversion of the his3AI gene into
wild-type upstream oligonucleotides. The LigAmp assay is designed to a functional HIS3 gene. RT activity is detected as the ability to give rise
convert single-base differences into more distinctive molecules that to cells that are able to grow on medium lacking histidine. Selection in
can more easily detected and quantified. the presence of NNRTIs measures the NNRTI susceptibility of individual
RT clones. Analysis of the RT activity and NNRTI susceptibility of the
First, two oligonucleotides are hybridised to a DNA template and isolates present in large libraries of clones makes it possible to detect
ligated to one another. Each primer contains a region specific to the NNRTI-resistant RT variants that are present at low frequencies.
target gene and an M13 tail. The M13 tails permit amplification of the
ligated product in a subsequent universal quantitative realtime PCR (Q- Ultra-deep Pyrosequencing
44,45
PCR) detection reaction. The upstream oligonucleotide also contains a Ultra-deep pyrosequencing (UDS, 454 sequencing, 454 Life
region of unique foreign DNA (e.g. lacZ DNA, red) that serves as the Sciences/Roche Diagnostics) is an emulsion-based parallel
binding region for a probe in the Q-PCR reaction. The upstream primer pyrosequencing technique that provides significantly greater
is designed to match either the mutant or wild-type sequence at the 3’- throughput and lower cost per sequenced base pair than Sanger
end. When an upstream mutant primer is used, the 3’-end of the sequencing (see Figure 3). Originally designed for high-throughput
primer must match the mutant template perfectly. The same sequencing or mammal and bacterial genomes, this technique is
oligonucleotide should mispair at the 3’-end when hybridised to a wild- particularly well-suited for an in-depth analysis of a population of
type template, preventing ligation. heterogeneous genomes such as those of retroviruses.
44
Emulsion
droplets are used to separate individual DNA templates, and
Next, the ligated DNA is amplified using M13 primers and detected in a Q- amplification and sequencing are performed within the each droplet.
PCR reaction. This step is independent of the specific gene or mutation Sequences are thus generated from a large number of independent
targeted in the ligation step. A universal probe (such as LacZ) containing a
fluorophore and quencher can be used for the Q-PCR amplicons. Because
both the M13 forward primer and the lacZ probe have the same polarity as Ultra-deep sequencing is also well
the upstream ligation oligonucleotide, the lacZ probe cannot bind to the
suited to studying immune escape
ligation oligonucleotide. Binding of the probe requires ligation of the two
oligonucleotides and subsequent polymerisation of the cDNA strand in the variants and host immune responses
Q-PCR step. If no ligation occurs in the first step because of mispairing, there
such as immunoglobulin or T-cell
is no template for amplification in the Q-PCR step.
receptor rearrangements during the
The specificity of LigAmp relies on the differentiating power of a DNA
adaptative immune response.
ligase to ligate the upstream and downstream oligonucleotides only when
both hybridise to the template with no mismatches at the adjacent
terminal nucleotides. genomes, which yields a quantitative read-out of the genomic diversity.
At present, UDS allows the parallel sequencing of more than 300
The TyHRT System 000–400 000 independent HIV-1 clones at once. If lower coverage is
This is a phenotypic assay based on hybrid elements derived from the S. needed, multiple specimens can be tested in a single plate using
cerevisiae Ty1 retrotransposon in which reverse transcriptase is provided nucleotide barcoding (i.e. a sample-specific four-base code in the 5’-
by HIV-1 RT (TyHRT).
55
In the TyHRT system, amplified HIV-1 pol region end of the amplification primers). Currently, the length of sequencing
DNA from test samples is co-transformed into yeast with a plasmid reads per clone is between 300 and 400 contiguous base pairs. This
containing a TyHRT element with a deletion in the RT region. HIV-1 RT allows the genetic linkage of clinically relevant resistance mutation
DNA is introduced into the TyHRT element by homologous clusters to be studied, such as resistance mutations surrounding the
recombination. Each isolate carries a unique RT domain, and the library NNRTI-binding pocket, the integrase catalytic site, most mutations in
of isolates is representative of the RT domains present in the original viral protease and the complete V3 loop sequence of gp120. Ultra-deep
sample. The TyHRT elements carry the reverse-transcription indicator sequencing is also well suited to studying immune escape variants and
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