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Roche 454 Sequencing
Roche™s platform features pyrosequencing technology, which detects the release of pyrophosphate upon incorporation of a nucleotide into the growing DNA strand. First, the library DNA fragments are tethered to a bead at a dilution factor that ensures only one fragment is bound per bead. The fragment is then amplified to cover the surface of the bead. These coated beads are each deposited into a well of picotiter plate, along with beads carrying the enzymes required for strand extension and chemiluminescence. During each round of synthesis, one of the four nucleotides is added to the well. If the nucleotide is incorporated, the resultant release of pyrophosphate generates a chemiluminescent signal proportional to the number of nucleotides incorporated. Each round cycles through A, C, G, and T to generate the sequence of the growing strand. Pyrosequencing generates longer read lengths (about 400 bp/read), making it well suited for de novo genome sequencing, splice-variant analysis or barcoding samples.
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