Roche 454
Roche 454 sequencing system is the first commercial platforms for the next generation sequencing technology. Its main principle of sequencing is illustrated as follows. 
a. Preparation of DNA Library
DNA Library construction in 454 sequencing system is different from that of Illumina. It uses spray method to break DNA samples into small fragments of 300-800bp, and adds different adapters at both ends. Otherwise, use primers for amplification after DNA denaturation, clone into specific vectors, and finally constructing single stranded DNA library.
b. Emulsion PCR
Of course, DNA amplification process of Roche 454 system is different from that of Illumina. These single stranded DNAs would be fixed by 28um beads which are buried in emulsion.
The biggest feature of emulsion PCR is the formation of a large number of independent reaction space for DNA amplification. The key technology is to separate different beats using the characteristics of emulsion. The basic process is as follows. Before sample DNA amplification, aqueous solution with all components of PCR reaction will be infused into the surface of mineral oil with high-speed rotation, and it forms numerous small water droplets wrapped by mineral oil. One small droplet forms an independent PCR reaction space. Ideally, each small drop of water contains only one DNA template and one bead.
On the surface of beads, which are wrapped by small water droplets, there are complementary oligos to match those adapters, so the single stranded DNA can specifically bind to the beads. At the same time, incubation system contains PCR reagents to ensure that each small DNA fragment fixed on the bead can be the unique template for amplification. Moreover, PCR products can be also combined with magnetic beads. After the reaction accomplishment, emulsion system can be destroyed and target DNAs would be accumulated. Finally, each small fragment will be amplified about 1 million times, so as to achieve the amount level required by the sequencing process. 
c. Pyrosequencing
A polymerase and single strand DNA binding protein are needed to process beads with DNAs before sequencing. Then these beads are put on PTP plate. This plate has many special nanopores of 44um diameters. Each nanopore can only accommodate one bead, which can fix the position of each bead through this method, in order to be convenient for sequencing.
The method used in this sequencing process is pyrosequencing. Put a smaller bead into the nanopore, and start the sequencing reaction. DNA sequencing reaction is based on the single stranded DNAs which have been amplified and fixed. If one dNTP can pair with the template DNA, the pyrophosphate group will be released after synthesis. The released pyrophosphate group reacts with ATP sulfuric acid chemical enzymes to produce ATP. CO-oxidation of ATP and luciferase makes the fluorescein molecule triggered and fluoresce, and the CCD camera on the other side of the PTP board records the signal of fluorescent. Finally the results are processed by computer software. Because each kind of dNTP produces unique fluorescence color in the reaction, DNA sequence can be measured according to the fluorescence colors. After the reaction, ATP are degraded by diphosphatase, leading to fluorescence quenching, so that sequencing reaction goes into the next cycle.
For 454 sequencing technology, each reaction is carried out in independent nanopore of PTP board, thus it greatly reduces mutual interference and sequencing bias. The biggest advantage of 454 technology is to get longer sequencing read length. Currently, average read length of 454 technology is up to 400bp. 454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
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