ABSTRACT
One of the early NGS technologies, 454 (later acquired by Roche), achieved high-throughput sequencing by further developing a method called pyrosequencing. This method is based on the detection of the pyrophosphate released after each nucleotide incorporation in the new DNA strand synthesis [66]. In this technology, each of the four different nucleotides is added into the sequencing reaction in a fixed order one at a time. If complementary, the corresponding nucleotide (or more than one, if there is a homopolymer on the template) is ligated to the new strand, and as part of the ligation reaction a pyrophosphate is released as a side product. An enzyme called ATP sulfurylase converts this pyrophosphate to ATP, which in turn is used to convert luciferin to oxyluciferin by luciferase. The generated oxyluciferin emits light, and the amount of light emitted is generally proportional to the number of nucleotides incorporated. By detecting light emission after each cycle of nucleotide addition, the sequence on each DNA template is deduced. High throughput is achieved when massive numbers of DNA templates are sequenced in this fashion simultaneously. Using the 454/ Roche technology, sequence reads of 400 to 500 bp in length are generated. A number of widely used NGS technologies, including Illumina reversible dye-terminator sequencing, Ion Torrent semiconductor sequencing, and Pacific Biosciences single-molecule real-time sequencing, are all based on the sequencing-by-synthesis principle. The specifics of these methods will be detailed in Section 4.3.
