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Next-generation systems also analyze fragments of DNA but step up the process by multiplying the number of sequencing reactions that occur on each piece of template DNA (see figure), yielding vastly more data. Solexa produces shorter DNA sequence reads than traditional methods of sequencing, but many more of them—up to 50 million sequences in a single two-day run. 454 and SOLiD use different techniques for sequencing but likewise produce large amounts of data.
HHMI has purchased 15 of the $500,000 Solexa sequencers for its investigators, and its investment in next-generation sequencing equipment is already paying dividends.
Joe DeRisi, an HHMI investigator researching malaria and emerging viral diseases at the University of California, San Francisco, gives the example of a fragment of a novel virus discovered in his lab a couple of years ago.
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A: Next-generation DNA sequencers are designed to read sequences from a very large number of individual DNA molecules within a mixed sample. However, not even the new techniques are sensitive enough to truly read a sequence from a single molecule of DNA. Instead, individual DNA strands must be immobilized and amplified in a fixed location to form a group of many identical strands that can provide enough signal for a sequence to be read. As a first step, DNA mixtures are prepared by fragmenting the DNA to be sequenced into small sizes (~100-500 bases); then, defined snippets of DNA are added to the two ends of each resulting fragment.
B: In the Solexa sequencing system (Illumina, Inc.) two kinds of amplification primers—short pieces of DNA—are fixed to a glass surface, much like a microscope slide. Then, the mixture of template DNA fragments is added to the slide, where individual molecules bind at random positions to the surface-bound primers. Multiple cycles of amplification yield double-stranded DNAs, which are created by bridging between the two types of surface-bound primers. Because each molecule of DNA can reach only so far, this approach tends to create a “forest” of fragments at a given spot on the slide. By controlling the number of input DNA molecules, the density of the amplified spots can be set as desired.
C: The Solexa sequencing strategy uses modified nucleotides: each base bears a different fluorescent group that also blocks further nucleotide addition. All four nucleotide bases are flowed simultaneously over the surface, and each template can incorporate only a single nucleotide. A laser activates each fluorescent group in turn and optics collect images of the surface. The pattern of colored light emission can be used to read the sequence directly from each spot (see magnification at left). Once the fluorescent group and blocking group are cleaved from the growing DNA strand, the next base can be read by precisely the same strategy.
D: Though it may seem complex, the process works remarkably well. The Solexa platform permits short sequence reads, with reliable reports of 50 nucleotides being obtained from each template. Moreover, the system can read tens of millions of DNA sequences simultaneously.
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“This virus was very different than anything that had ever been published before,” he says. “A very talented postdoc threw everything but the kitchen sink at this project, trying to sequence a complete copy of the virus' genome and was unable to do so.”
The DeRisi team was stumped. Last year, they began working with the Solexa sequencer and decided to revisit the problem. They attempted to sequence the whole pool of DNA from which the virus fragment was isolated.
“After a single run, we had recovered the entire genome in one go. In two days we were able to accomplish in totality what we couldn't in over a year of hard trying,” says DeRisi, whose team is preparing to publish their discovery.
DeRisi points out that the new technologies are not a replacement for traditional sequencing methods, which are ideal for sequencing a very specific piece of DNA. But because of the ability of the new technologies to tackle large projects or complex mixtures of DNA, he and other HHMI researchers have great expectations.
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