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AB™s SOLiD technology relies on ligation of tagged probes, not on synthesis. The fragments to be sequenced are ligated to specific adapter sequences, then attached to beads and clonally amplified by emulsion PCR. The beads are then attached to a glass slide. A SOLiD sequencing run consists of five rounds of ligation runs. In the first round, an initial universal sequencing primer binds to the adapter sequence. The slide is then washed with a large mixture of 8-mer probes. Each probe is labeled with one of 4 fluorophores; each fluorophore corresponds to four of 16 combinations representing the first two bases of the probe. The correct probe aligns to and is ligated to the target, and is identified by its fluorophore marker. Next, the final 3 bases, along with the fluorophore, are chemically cleaved, leaving behind a free 5â€™ phosphate. The next probe aligns, is ligated, and its marker identified. This is repeated to extend the ligated strand. This first ligation round, however, only identifies the 4 possible dinucleotides for positions 1 and 2, positions 6 and 7, etc. The ligated strand is then melted off, and the entire process repeated with a new universal sequencing primer that is offset from the first by 1 base pair. Five rounds of primer reset are required to fully query the template. Each base pair has been identified twice; thus the combination of fluorophore colors (known as the color space) identifies each base.
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