The main NGS method is called “sequencing by synthesis”, developed by a company: Illumina. It is commonly referred to as Illumina sequencing. This method is based on reversible chain terminators, developed at the Institut Pasteur in the 90’s43. These are marked dNTPs that can be used to elongate DNA molecules, but that have an additional molecular group that makes them terminators by default. However this terminating group can be removed once the NTP is included in a DNA molecule allowing the elongation process to continue. These dNTPs are fluorescently marked and when excited with a laser they emit light with a distinctive color. During Illumina sequencing, these reversible chain terminators are included to millions of fragments at the same time, stopping elongation. At this point all the fragments are excited with a laser and an optical system takes a picture of the emitted colors for all the fragments at once. In this image, a pixel loosely corresponds to a sequenced fragment, and its color to the most recently added dNTP. The terminating groups are then cleaved and the process can start over by incorporating a new batch of reversible terminators. By observing the successive images we can deduce the sequence of added nucleotides for each sequenced fragment and obtain all of our reads.
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