454 Technology

Genomic DNA, PCR amplicons, BACs and cDNA can all be used as template DNA for 454 sequencing. Genomic DNA and other longer length samples are nebulised to create fragments of a more appropriate length prior to sequencing.

Adaptors are ligated to the DNA fragments for use throughout the purification, emulsion PCR amplification and sequencing steps of the process. If an amplicon library is to be sequenced, a second round of amplification may be carried out in order to attach adaptor sequences to the amplicon termini.

When the library has been purified, including the removal of any short fragments, it is attached to DNA capture beads. The library is mixed with the capture beads at a concentration that aims to ensure that, on average, no more than one strand of DNA anneals to a given bead. The beads are then emulsified to create amplification microreactors containing PCR reagents. Post-amplification, the emulsion is broken to yield a mixture where each bead is bound to millions of clonally amplified copies of one original library fragment.

Following various steps intended to purify and enrich for beads that have successfully bound library fragments, the beads are loaded into a fiber-optic slide. Each well is of sufficient size to contain one sequencing bead and additional, smaller, packing and reagent beads.

During the sequencing reaction, individual nucleotides are flowed across the wells in a fixed sequence. When a nucleotide (or more than one if there is a run of the same nucleotide) is incorporated by the polymerase, a chemiluminescent light signal is recorded by the camera. The light signal is derived from luciferase-coupled polymerisation.

This process is neatly visualised in an animation from the Wellcome Trust.