Nonetheless, Illumina/Solexa terminators are reversible, permitting polymerization to proceed even after fluorophore detection. In this method, DNA fragments are immobilized on a flow cell surface and bridge PCR is used for amplification (34). The DNA sequencing is initiated with addition of the sequencing primer, DNA polymerase, and four reversible dye terminators. Fluorescence Wortmannin DNA-PK is recorded after incorporation by a four-channel fluorescent scanner (33). The newest sequencers using this technology can generate sequence reads that are about 100 bp long. The HeliScope System by Helicos Biosciences is a single molecule sequencing platform that also utilizes the sequencing-by-synthesis principle. In the HeliScope platform, single DNA molecules are sequenced directly, obviating the need for a previous clonal PCR amplification step.
The sample DNA is fragmented and polyadenylated at the 3�� end, with the final adenine being labeled with Cy3 (34). The poly-A template molecules are captured by hybridization to poly-T oligonucleotides immobilized on a flow cell surface. Because templates are fluorescently labeled, imaging can identify the array coordinates, where a sequencing read is expected. The label is then cleaved and sequencing proceeds by adding the DNA polymerase and each of four Cy5-labeled nucleotides to the flow cell (29). Fluorescent imaging detects incorporation into the individual strands. After chemical cleavage of the label, the cycle is repeated for the next nucleotide. Pacific Biosciences is another company that has developed another single molecule sequencing platform, the SMRT technology (35).
Table 1 depicts many of the features of the most currently used NGS technologies. For a more detailed description of the NGS technologies and chemistries, the reader is referred to some comprehensive reviews (29, 31, 34, 36). Table 1 Next-generation sequencing technologies Advantages and limitations of pyrosequencing One of the greatest advantages of the massive parallel pyrosequencing approach over the Sanger sequencing method is that hundreds of thousands of sequence reads can be obtained in a single run, generating sequence information data that are orders of magnitude larger (37). Moreover, the cost per base is much lower for pyrosequencing when compared with the Sanger method. This has also helped to widely expand the utilization of DNA-sequencing approaches.
Another advantage of the pyrosequencing technique is that it also avoids the biases inherent to the cloning procedure. In the pyrosequencing method, sequences from different samples can be identified in the same run using the barcoding multiplex approach, in which unique sequences are incorporated into the primers and barcoded amplicons are generated38. This multiplex approach increases efficiency and throughput, and reduces Anacetrapib costs (10, 39).