Massively parallel pyrosequencing in HIV research

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Abstract
The new massively parallel sequencing methods are so astonishing that one wonders whether space aliens are secretly behind them. One technician, running a single instrument, can obtain up to approximately 1 billion bases of DNA sequence in a few days. Here we describe the new sequencing methods, briefly present a few applications in HIV research, and then speculate on future directions. Several methods for massively parallel pyrosequencing have recently been commercialized. As an example, consider the use of the 454 Life Sciences pyrosequencing method for metagenomic analysis of woolly mammoth DNA [1,2]. DNA from a mammoth carcass was purified and fragmented, and DNA linkers were ligated to the free ends. DNAs were then denatured and strands annealed to beads conjugated with oligonucleotides complementary to the linker sequences. This step is carried out with very low DNA concentrations so that on average only one strand binds to each bead. Bead-bound DNA is then PCR amplified in an oil–water emulsion, where each water droplet in the emulsion contains on average a single bead. The amplified DNA strands anneal to the beads, yielding beads with many copies of homogeneous PCR products. Pools of up to 400 000 beads are then distributed in a picotiter plate and further manipulations carried out in a custom fluidics station (Fig. 1). A polymerase is used to extend a DNA chain from a bound primer on each strand. The four nucleoside triphosphates are sequentially flowed over the picotiter plate. With each incorporation event, pyrophosphate is liberated into solution (hence ‘pyrosequencing’). An enzyme system is present in the aqueous phase that directs incorporation of pyrophosphate into ATP, which in turn activates purified luciferase, also present in the aqueous phase, to produce a flash of light. Each flash from each well is quantified by a charge coupled device camera and the signals detected and stored in a computer. Sequential application of the four nucleotides (nts) allows DNA sequences of approximately 100 nt to be built up several hundred thousand fold at a time. Using this method a detailed comparison of the mammoth and elephant genomes can be carried out (yes, there is also an elephant genome project). With the improved 454 technology released recently, it is possible to generate reads of approximately 260 nt on approximately 400 000 beads per run, yielding a whopping 100 million bases of DNA sequence in a day or two. An illustrated description of the method can be found at http://www.454.com/enabling-technology/index.asp. A second pyrosequencing technology, commercialized by Solexa/Illumina (San Diego, California, USA), yields shorter sequence reads, only approximately 35 bp, but a single run yields up approximately 1 billion bases of DNA sequence [3,4] (see www.illumina.com/downloads/SS_DNAsequencing.pdf). Table 1 compares the Sanger, 454/Roche (Branford, Connecticut, USA), and Solexa/Illumina methods. A variety of additional technologies are also under development [5]. The pyrosequencing methods are well suited to addressing questions on the dynamics of HIV quasi-species in response to selective pressures. HIV reverse transcriptase is very error prone, making roughly one base pair substitution mutation per round of replication [6]. The viral populations in infected individuals are also very large, with some 1010 virions produced and destroyed per day [7–9]. After infection, this activity quickly results in the formation of a diverse pool, or quasispecies, in which most viral sequences differ from all others.