Rocher's 454 Genome Sequencer FLX took the Sanger method and greatly simplified the preparation process. This sequencer became widely available as a commercial product in 2005. Rocher utilizes adapter-flanked fragments attached to etched fiber chips to hold primers and polymerase enzymes and start the synthesis of complementary strands. The 454 sequencer also uses emulsion PCR amplification, which replicates the strands attaching to the beads.
This ensures that the reaction can be detected at a given light intensity. The sample is then loaded onto a picoteter plate where the beads enter individual wells. Packing beads are added to wells to aid the spectrometer in reading the sample.
In the end, the 454 can analyze a large number of samples in parallel. vastly improving output compared to the original Sanger method. However, the 454 in most cases is unsuccessful in dealing with homopolymers, leading to a greater error rate.
It is also more expensive to sequence per base than some of its competitors. Illumina also relies on the chain termination method. Their current sequencer, the HiSeq 2000, integrates improvements in engineering that have given it the highest output currently on the market. Similar to the other methods, the DNA is first broken into fragments, and in this case run on a gel tray to separate them based on size. A 200-300 base pair fragment is then selected from this for further replication through PCR.
Illumina uses this automated cluster generation to distribute its fragment library to the surface of a flow cell amongst a forest of adapters. A process known as bridge amplification occurs, allowing the generation of copies of a specific molecule to be made on the surface. As each new base is added, a camera records the location of each cluster by capturing the fluorescent signal emitted by the cluster.
The combination of these images creates the sequence. Although adding and sequencing one base at a time seems extremely slow, Each flow cell analyzes approximately 150 million of these clusters, making it an extremely efficient system. Applied biosystems, however, bring something a little different to the table. The solid system is considerably flexible in that it allows for genome sequencing in different applications.
The primary steps are enzyme and sample preparation, PCR and substrate preparation, ligation, imaging, and data analysis. Enzyme and sample preparation and data analysis are the only steps that need to change based on the application desired. SOLID uses emulsion PCR amplification similar to Roche, but it distributes its fragment library onto microbeads that can vary in size and richness of slides they are on. Based on the targeted application, slides containing one, four, or eight sections can be used. Fluorescence is then emitted when each fragment is ligated onto a single strand sequence.
Data analysis is accomplished through X-ActiCol chemistry, which relies on an 8-base interrogation system with 4 different colored primers. to map out possible combinations within the sequence. The system detects single-base insertions, deletions, and SNPs with ease.