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File: Sanger Sequencing Slideshare 67000 | 9780367263768 C011
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 ...

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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).
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                              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.
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                              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).
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                               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.
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                              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.
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...Courtesy of crc press taylor francis group figure sanger sequencing with fluorescent nucleotides rather than using four separate radioactive in the reactions use each labeled a different type fluorescence reduced both number top and lanes on polyacrylamide gel middle also enabled automated detection bottom reducing human error reading bands an x ray film as previously see shotgun this methodology applied to whole bacterial genomes involves first randomly dividing genome into fragments that are cloned library order they were is lost sequenced it produces overlapping sequence data joined computationally generate contiguous regions difference between comparative genomics evaluating differences similarities two sequences shows there some one not other blue vice versa red next generation principles most technologies by synthesis established method but modifications dna fragmented technology specific adapter ligated ends adapters used attach solid surface bead or flowcell primers for amplifi...

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