The Next Generation Sequencing

Platform of Roche 454

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.

Related Links

cDNA Library Construction Service