Fig. 2 Outline of the 454 and polony sequencing process. Both systems first fragment the genomic DNA (Step 1) and then use a process of in vitro cloning followed by amplification. The 454 process is shown on the left and Polony sequencing is shown on the right. In the 454 protocol, the linkers are ligated onto the ends of the DNA (Step 2a). Polony sequencing involves circularization followed by linearization and the addition of linkers to generate two fragments with a spacer between them and linkers at the end (Step 2B). Both processes then attach the in vitro clones to beads and carry out PCR in an emulsion mixture to generate beads with many clonal copies of the target fragments (Step 3a/3b). For the sequencing step, the beads must be immobilized in a single layer to allow imaging in an environment that enables the reaction reagents to be flowed across them. In the case of 454 sequencing, a picotiter plate is used, in which most cells will contain a single bead (Step 4a). The polony method immobilizes the beads in an acrylamide matrix in a dense monolayer (Step 4b). The methods are very similar up until the point of the sequencing reaction; in the case of 454 sequencing, a DNA synthesis reaction from a single sequencing primer is carried out. Bases are flowed across the picotiter plate one at a time and incorporation is detected by the release of light (Step 5a). The polony method uses ligation to anchor primers, which can be annealed in one of four positions. In each cycle, a population of degenerate nonomers, which have been fluorescently labeled, is added to the monolayer, and only complimentary oligos will anneal and ligate to the anchor primer.