The 454 pyrosequencing technology was developed by 454 Life Sciences as a new, sealable, highly parallel two step DNA sequencing system with significantly greater throughput than the Sanger sequencing systems. It is based on the "sequencing by synthesis" principle which involves utilizing single strand DNA, to be sequenced, and sequencing its complementary strand with enzymatic action. It is a system integrated with PCR amplification of numerous DNA fragments linked to high throughput parallel pyrosequencing in picolitre-sized wells. The "sequencing by synthesis" principle also relies on the detection of pyrophosphate (PPi) released on nucleotide incorporation, generating a light signal, rather than chain termination with dideoxynucleotides.
HOW IT WORKS
454 pyrosequencing is a highly parallel two step approach. The DNA is firstly prepared by cutting the DNA into blunt ends and attaching oligonucleotides adaptors to both ends of the cut DNA molecules. The fragments are then individually attached to a bead, which is then amplified via PCR in droplets of an oil-water emulsion, generating multiple copies of the same DNA sequence on each bead. The beads are then captured in picotitre wells on a fabricated substrate and then pyrosequenced.
The sequencing occurs in five steps, using the Genome Sequencer FLX system for trascriptome sequencing. Firstly, the template, a single stranded PCR amplicon, is hybridized to a sequencing primer and then incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin. The first dNTP is then added and if complementary, it is bound to the DNA strand by DNA polymerase and this is accompanied by the release of a PPi. The PPi is converted to ATP by ATP sulfyrelase in the presence of APS and then, a luciferase catalysed reaction generates visible light proportional to the amount of the generated ATP. Apyrase continuously degrades unincoporated nucleotides and ATP. After the degradation process, another nucleotide is added. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the pyrogram trace.
THERE ARE SOME DISADVANTAGES
The Newbler software assembles the read data in "flow space" rather than "nucleotide space", in other words, it considers the light intensity measurements and not predicted read sequences. The assembler is designed to correct the systematic errors associated with pyrosequencing such as homopolymer related errors. The quality of 454 sequencing reads and the resulting assembly is not well characterised. Another limitation is the short read length from 454 systems, in the neighbourhood of 300-500 nucleotides. Given the short length of the reads, 454's Newbler assembler is expected to perform poorly with repeat regions. However, this problem may be solved by a strategy which combines scaffold information from traditional clone end-sequencing with 454 data.
The strengths of pyrosequencing technology include great flexibly in primer placement, the assays are mutation tolerant, the data is fully quantitative, and sequence information is fully obtainable. The technology is highly sensitive compared to traditional sequencing methods with 99.9% accuracy up to the 200 base and 99% at the 400 base. The system sequences 400-600 million bp in 10 hours thus lower cost as large amounts of DNA can be sequenced at the given time as compared to the Sanger method which will take days to sequence the same amount of DNA. This technology together with other next generation sequencing technologies, have had a lot of impact on genomic research as a large output of data becomes available at a shorter period of time leading to a break through in biological fields such as biotechnology, systematic and forensic sciences and also in the medical field.
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