The Roche 454/GS FLX Sequencing Technology

The GS FLX sequencer supports sequencing of various different nucleic acid starting materials such as genomic DNA, PCR products, BACs and cDNA. Samples consisting of longer sequences are first sheared into a random library of 300-800 base-pair long fragments.

Adaptors essential for purification, amplification and sequencing are added to both ends of the fragments. If the sample is double stranded one strand is removed and the remaining single strandes are used in the following steps.

Aided by the adaptors individual fragments are captured on their own unique beads. A bead and the bound fragment together with a water-in-oil emulsion form a microreactor so that each fragment can be amplified without contamination via the so called emulsion PCR (emPCR). The entire fragment collection is amplified in parallel.

The emPCR amplifies each fragment several million times. After amplification the emulsion shell is broken and the clonally amplified beads are ready for loading onto the fibre-optic PicoTiterDevice for sequencing.

The PicoTiterPlate is loaded with one fragment carrying bead per well and smaller beads with the enzymes necessary for sequencing.

Sequencing is accomplished by synthesizing the complementary strands of the bead attached templates. In a number of cycles the four bases (ATGC) are sequentially washed over the PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic pyrophosphate starting a chemical cascade. This results in the generation of a light signal which is captured by a CCD camera.