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Introduction to Roche 454 Sequencing

Roche was one of the first companies that commercialized the next generation sequencing (NGS) technology. Roche 454 technology is based on pyrosequencing. Roche was long time considered as the gold standard in next generation sequencing. Due to the high sequencing chemistry costs and the relative low maximal throughput the per nucleotide sequencing cost is higher compared to other NGS technologies.

How does Roche pyrosequencing works?

Pyrosequencing is based on light signals that are generated during the incorporation of fluorescence labelled nucleotides into a growing DNA strand in the DNA polymerase reaction. Each nucleotide (G, A, T, and C) is labelled with a fluorescent marker.
During DNA library and template preparation individual DNA fragments are bound to beads. In order to enhance the light signals the DNA of each fragment/ bead complex is clonally amplified in a single oil droplet by emulsions PCR. Prior sequencing reaction the bead/template complexes are separated into thousands of individual wells on a PicoTiter plate. During sequential flooding of the plate with the different nucleotide enzyme reaction solutions only light signals arise if the corresponding nucleotides are incorporated in the DNA strand. The series of light signals are translated into sequence information leading to sequencing reads for millions of individual wells.
The following video demonstrates in more detail the method of the 454 pyrosequencing.

Roche 454 sequencing time, read length and throughput Roche GS Junior

The sequencing time depends on the desired read length, the sequencing system and the chemistry used. For maximal read length the sequencing time is between 18-23 hours.
The maximal read length is about 1000 nucleotides, the average read length is between 700 to 800 nucleotides.
The read number per sequencing run is between 100.000 (GS Junior) and 1million (GS FLX+).

Roche sequencing systems and its applications Roche GS FLX

Roche provides two sequencing systems, the GS Junior and the GS FLX+.
In general this sequencing technology can be applied in a number of different fields. This technology is especially applicable were long read information coupled with high accuracy is needed. This could be quasi species investigations or sequencing of complex genomic regions.
For sequencing projects with very large data output it need to be considered that the throughput of the 454 technology is limited compared to other NGS technologies. 
For more information about the systems and its applications visit the 454 website.

In 2014, Roche acquired Genia Technologies in order to strengthen their NGS pipeline. Genia is currently developing a single-molecule, semi conductivity based DNA sequencing platform, using nanopore technology.

Furthermore, Roche started investments in Stratos Genomics, another new NGS technology provider that develops an improved sequencing technology based on nanopore technology. 

Both technologies aim to improve sequencing accuracy of the existing nanopore technology.