From Wikipedia, the free encyclopedia

454 Life Sciences
Type	Subsidiary
Founded	2000
Location	Branford, CT, USA
Industry	DNA sequencing
Products	high-throughput sequencing, sequencing machines, reagents
Revenue	$9.8 million (Q2 2006)
Website	http://www.454.com/

454 Life Sciences is a biotechnology company based in Branford, Connecticut specializing in high-throughput DNA sequencing using a novel massively parallel sequencing-by-synthesis approach. 454 has experienced rapid growth since its partnership with Roche Diagnostics and release of its GS20 sequencing machine in 2005. As of 2005, the majority of 454's revenue came from sales of sequencing machines ($5.4 million), not in-house sequencing projects ($2.3 million). [1] 454 was founded by Jonathan Rothberg, and the underlying technology was conceived while he was on paternity leave and wanted a way to sequence the genome of his new born son who had been placed in new born intensive care. For his invention, Dr. Rothberg and 454 sequencing were awarded the Wall Street Journal's Gold Medal for Innovation in 2005.

In late March, 2007, Roche Diagnostics announced an agreement to purchase 454 Life Scienes for US$154.9 million. It will remain a separate business unit, however.

Contents 1 Technology 1.1 DNA Library Preparation and emPCR 1.2 Sequencing 2 Applications 2.1 Whole Genome Assembly 2.2 Ultra Broad PCR 2.3 Ultra Deep PCR 3 Advantages and Disadvantages 4 External links 5 References

454 Sequencing is a massively-parallel sequencing-by-synthesis (SBS) system capable of sequencing roughly 20 megabases of raw DNA sequence per 4.5-hour run of their current sequencing machine, the GS20. The system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion. The DNA fixed to these beads is then amplified by PCR. Finally, each DNA-bound bead is placed into a ~44 μm well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as polymerase, ATP sulfurylase, and luciferase are also packed into the well. The PicoTiterPlate is then placed into the GS20 for sequencing.

At this stage, the four nucleotides (TAGC) are washed in series over the PicoTiterPlate. During the nucleotide flow, each of the hundreds of thousands of beads with millions of copies of DNA is sequenced in parallel. If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucleotide(s). Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. This technique is an example of pyrosequencing. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. However, the signal strength for homopolymer stretches is linear up to eight consecutive nucleotides; signal fall-off is rapid after a stretch of more than eight identical nucleotides. [2]

Genomic DNA is fractionated into smaller fragments (300-500 base pairs) that are subsequently polished (blunted). Short adaptors are then ligated onto the ends of the fragments. These adaptors provide priming sequences for both amplification and sequencing of the sample-library fragments. Adaptor B contains a 5'-biotin tag that enables immobilization of the library onto streptavidin coated beads. After nick repair, the non-biotinylated strand is released and used as a single-stranded template DNA (sstDNA) library. The sstDNA library is assessed for its quality and the optimal amount (DNA copies per bead) needed for emPCR™ is determined by titration.

The sstDNA library is immobilized onto beads. The beads containing a library fragment carry a single sstDNA molecule. The bead-bound library is emulsified with the amplification reagents in a water-in-oil mixture. Each bead is captured within its own microreactor where PCR amplification occurs. This results in bead-immobilized, clonally amplified DNA fragments.

sstDNA library beads are added to the DNA Bead Incubation Mix (containing DNA polymerase) and are layered with Enzyme Beads (containing sulfurylase and luciferase) onto the PicoTiterPlate™ device. The device is centrifuged to deposit the beads into the wells. The layer of Enzyme Beads ensures that the DNA beads remain positioned in the wells during the sequencing reaction. The bead-deposition process maximizes the number of wells that contain a single amplified library bead (avoiding more than one sstDNA library bead per well).

The loaded PicoTiterPlate device is placed into the GS20 Instrument. The fluidics sub-system flows sequencing reagents (containing buffers and nucleotides) across the wells of the plate. Nucleotides are flowed sequentially in a fixed order across the PicoTiterPlate device during a sequencing run. During the nucleotide flow, each of the hundreds of thousands of beads with millions of copies of DNA is sequenced in parallel. If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucleotide(s). Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the Instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. [3]

While 454 Sequencing can sequence any double-stranded DNA, certain applications work better than others with the technology given its penchant for extremely high throughput and relatively short read lengths. In all, there are three major types of projects.

Whole Genome Assembly (WGA) consists of projects dealing with the sequencing of the entire genome of an organism, for example, humans, dogs, mice, viruses or bacteria. Historically, the sequencing technique has focused on bacterial and viral genomes due to their lack of repetitive regions and relative ease of assembly. However, in June 2006 they launched a project with the Max Planck Institute for Evolutionary Anthropology to sequence the genome of the Neanderthal, the closest relative to humans. This has implications for the understanding of human evolution and development. At 3 billion base pairs, a complete sequence of the Neanderthal genome is expected to take two years to finish [4].

Ultra Broad PCR includes fields such as cDNA pools, small RNA, SAGE/Ditag libraries, and other amplicon pools. The high-throughput nature of 454 Sequencing allows researchers to obtain up to 200,000 100-base pair reads per run, which works well with smaller amplicons which do not have to be nebulized before sequencing or reassembled bioinformatically after sequencing.

Ultra Deep PCR is a very new field which is largely being enabled through 454 Sequencing technology. Unlike Sanger chain-termination sequencing, 454 sequencing allows mutations to be detected at extremely low levels. Thus, researchers are able to PCR amplify specific pools of cDNA or genomic segments and detect low-level mutations within those pools.

454 Sequencing runs at 20 megabases per 4.5-hour run, allowing large amounts of DNA to be sequenced at low cost compared to Sanger chain-termination methods; G-C rich content is not as much of a problem, and the lack of reliance on cloning means that unclonable segments are not skipped. Also, it is capable of detecting mutations in an amplicon pool at a low sensitivity level, which may have implications in clinical research, especially cancer and HIV. [5]

However, each read of the GS20 is only 100 base pairs long at this time, resulting in some problems when dealing with highly repetitive genomes, as repetitive regions of over 100 base pairs cannot be "bridged" and thus must be left as separate contigs. Also, the nature of the technology lends itself to problems with long homopolymer runs.

The new FLX system does 200-300 base pairs and 454 has said they expect 500 in '08.

• 454 Life Sciences Homepage

• Margulies et al. Genome Sequencing in Microfabricated High-Density Picolitre Reactions. Nature. 31 July 2005.
• Velicer et al. Comprehensive mutation identification in an evolved bacterial cooperator and its cheating ancestor. PNAS Vol. 103 pp 8107-8112. 23 May 2006.
• Dana Farber and 454 Life Sciences Annouce Breakthrough in DNA Sequencing for Cancer Research
• Max Planck Institute and 454 Life Science to Sequence Neandertal Genome
• Rohwer et al. Using Pyrosequencing to Shed Light on Deep Mine Microbial Ecology. BMC Genomics. Vol 7 pp 2164-7. 20 Mar 2006.
• Henderson et al. Dissecting Arabidopsis Thaliana DICER Function in small RNA processing, gene silencing and DNA methylation patterning. Nature Genetics. Vol 38 pp 721-25. 14 May 2006.
• Sogin et al. Microbial diversity in the deep sea and the underexplored "rare biosphere". PNAS. 31 July 2006.
• CuraGen Announces Definitive Agreement to Sell 454 Life Sciences to Roche