After preparation of the library (and careful quantitation) and preparation of the amplified template comes the main event: the sequencing itself. While there are several methods available, the methods can be divided into three broad divisions.
The three divisions are (firstly) Pyrophosphate Release (named for the original patent by Mostafa Ronaghi and others in 1998 when he was a graduate student at the Royal Institute of Technology in Stockholm); this is the method that uses individual nucleotide flow across all templates, and then detects the signal. (Pyrosequencing – now owned by QIAGEN – detects pyrophosphate, but is not a ‘next-generation’ sequencer as it is not massively parallel; however Roche / 454 FLX used essentially the same method.) Jonathan Rothberg, who parallelized the FLX pyrosequencing method at Curagen, simply changed the detection method with Ion Torrent.
