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Courtesy of CRC Press/Taylor & Francis Group Figure 11.2 Sanger sequencing with fluorescent nucleotides. Rather than using four separate radioactive nucleotides in the sequencing reactions, use of nucleotides each labeled with a different type of fluorescence reduced both the number of reactions (top) and the number of lanes on the polyacrylamide gel (middle). Fluorescence also enabled automated detection (bottom), reducing human error in reading bands on an x-ray film as previously (see Figure 11.1). 011x002 Courtesy of CRC Press/Taylor & Francis Group Figure 11.3 Shotgun sequencing. This methodology, applied to sequencing whole-bacterial genomes, involves first randomly dividing the genome into fragments that are cloned into a library. The order to the fragments as they were in the genome is lost. As the library is sequenced, it produces overlapping sequence data that is joined computationally to generate contiguous sequence data. 011x003 Courtesy of CRC Press/Taylor & Francis Group Figure 11.4 Regions of difference between bacterial genomes. Comparative genomics, evaluating differences and similarities between two genome sequences (top and bottom), shows that there are some regions in one genome that are not in the other (blue), and vice versa (red). 011x004 Courtesy of CRC Press/Taylor & Francis Group Figure 11.5 Next-generation sequencing principles. Most next-generation sequencing technologies use sequencing by synthesis, as established in the Sanger sequencing method, but with modifications. DNA is fragmented and sequencing technology specific adapter sequences are ligated to the ends of the fragments. The adapters are used to attach the fragments to a solid surface (bead or flowcell), as primers for amplification of the fragments, and to initiate sequencing of the fragments. As nucleotide bases are incorporated into the DNA, the identity of each incorporated base is determined via fluorescence or changes in pH within the reaction vessel. 011x005 Courtesy of CRC Press/Taylor & Francis Group Figure 11.6 Repetitive regions complicate assembly. If a genome contains, for example, six repetitive regions that are identical to one another (A, B, C, D, E, and F), this can confound attempts to assemble sequencing fragments. The fragment shown could belong in any of the six regions of the genome. 011x006
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