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Currently, the Roche/454 pyrosequencing method dominates the NGS market together with Illumina/Solexa Genome analyzer (GA). The pyrosequencing of Roche/454 is a technology to be first introduced commercially among the next-generation sequencing methods. The pyrosequencing is a massively parallel sequencing technique based on enzymatic detection of inorganic pyrophosphate release on nucleotide incorporation (Leamon et al., 2003; Ronaghi et al., 1998). This technology employed emulsion PCR for amplification of template DNA where a single DNA template is attached to a single primer-coated bead that is then amplified to form a clonal colony inside water droplets in an oil solution. The sequencing takes place in many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the incorporation of individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs (Margulies et al., 2005).

This technology provides intermediate read length and price per base compared to the conventional Sanger sequencing on one end and Illumina GA and Life Technologies SOLiD on the other (Schuster, 2008). The first version of pyrosequencing machine, called 454 Genome Sequencer (GS) 20, was released in 2004. It has been improved in the second version, 454 GS FLX, with great enhancements in terms of single-read accuracy and read length (average read length of 250 bp). The latest version of FLX series, called 454 GS FLX Titanium, generates more than 1 000 000 individual reads with improved quality of 400-500 bp in length per 10-hour instrument run (Droege and Hill, 2008; Metzker, 2010). It is currently applied to a wide variety of biological studies, such as human genetics, RNA analysis, metagenomics and ancient DNA sequencing.

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