454 Sequencing

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 and GS FLX machine in 2007. 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).[2] 454 was founded by Jonathan Rothberg, and the underlying technology is based on pyrosequencing and 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 their invention, Dr. Rothberg and 454 Life Sciences were awarded the Wall Street Journal's Gold Medal for Innovation in 2005.

In November 2006, Dr. Rothberg, Michael Egholm, and colleagues at 454 published a cover article with Svante Paabo in Nature describing the first million base pairs of the Neanderthal genome, and initiated the Neanderthal Genome Project to complete the sequence of the Neanderthal genome by 2009.

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

In May 2007, Project "Jim", a project initiated by Dr. Rothberg and 454 Life Sciences to determine the first sequence of an individual was completed. The results of the project, the complete genome sequence of James Dewey Watson, was handed to Dr. Watson at a ceremony taking place at Baylor College of Medicine.

Technology

454 Sequencing is a massively-parallel pyrosequencing system capable of sequencing roughly 100 megabases of raw DNA per 7-hour run of their current sequencing machine, the GSFLX. 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.

DNA-bound beads placed into wellsAt 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 based on sequencing-by-synthesis and is called pyrosequencing (Ronaghi et al. 1996 and 1998). 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 only up to eight consecutive nucleotides after which the signal falls-off rapidly.[3]

DNA Library Preparation and emPCR

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.