454 Life Sciences

454 Life Sciences

Company type	Subsidiary
Industry	Biotechnology
Founded	2000 (2000)
Founder	Jonathan Rothberg
Defunct	2013 (2013)
Fate	Discontinued by Roche
Headquarters	Branford, Connecticut , United States
Area served	Worldwide
Key people	Jonathan Rothberg, Michael Egholm
Products	High-throughput DNA sequencing technologies, Genome Sequencer FLX, GS Junior System
Parent	Roche Diagnostics (acquired 2007)

454 Life Sciences was a biotechnology firm located in Branford, Connecticut that specialized in high-throughput DNA sequencing technologies. After being acquired by Roche in 2007, the company was eventually discontinued by Roche in 2013 due to its technology losing competitive edge, although manufacturing persisted until mid-2016.

History

Originally established as 454 Corporation, a subsidiary of CuraGen, by Jonathan Rothberg, the company pioneered an affordable gene sequencing technique that won the Wall Street Journal's Gold Medal for Innovation in Biotech-Medical in 2005. The designation "454" originated from an internal project code at CuraGen.

In November 2006, Rothberg, Michael Egholm, and their team published the initial million base pairs of the Neanderthal genome in Nature, initiating the Neanderthal Genome Project with the goal of completing the entire sequence by 2009.

Roche Diagnostics purchased 454 Life Sciences in March 2007 for US$154.9 million and operated it as a distinct business division. By October 2013, Roche declared its intention to discontinue 454 and cease platform support by mid-2016.

In May 2007, 454 announced results from Project "Jim," which involved sequencing the genome of James D. Watson, renowned co-discoverer of DNA’s double helix structure.

Technology

The 454 Sequencing platform utilized a highly parallelized pyrosequencing system capable of generating roughly 400-600 megabases of DNA sequence data during a single 10-hour run on the Genome Sequencer FLX using GS FLX Titanium reagents.

This method involved attaching nebulized DNA fragments with adapters to microscopic DNA-capture beads within a water-in-oil emulsion. These beads carrying DNA were then subjected to amplification via PCR and loaded into approximately 29 μm wells on a PicoTiterPlate, an optical fiber-based chip. Prior to sequencing on the GS FLX System, enzymes such as DNA polymerase, ATP sulfurylase, and luciferase were introduced into each well.

The GS20 instrument, launched in 2005, was the first next-generation sequencing device made commercially available by 454. The introduction of GS FLX Titanium reagents in 2008 significantly enhanced the Genome Sequencer FLX's throughput to between 400 and 600 million base pairs per run with read lengths of 400-500 bases. By late 2009, the company released the GS Junior System, a compact benchtop variant of the Genome Sequencer FLX.

DNA library preparation and emPCR

Genomic DNA was fragmented into segments ranging from 300 to 800 base pairs, then end-polished to create blunt ends before ligation with short adapter sequences that provided priming sites necessary for both amplification and sequencing. One adapter contained a 5'-biotin label allowing immobilization of the DNA library onto streptavidin-coated beads. Following nick repair, the strand lacking biotin was separated and purified as single-stranded template DNA (sstDNA) library. This library underwent quality control to determine the optimal template-to-bead ratio for emPCR.

The sstDNA molecules were attached individually to beads, each bead carrying a single DNA template. These bead-bound templates were emulsified with PCR reagents within a water-in-oil mixture, creating isolated microreactors where clonal amplification occurred. The result was clonally amplified DNA fragments anchored to beads, ready for sequencing analysis.

Sequencing

Single-stranded template DNA library beads were mixed with DNA polymerase within the DNA Bead Incubation Mix and then layered alongside Enzyme Beads—which contained sulfurylase and luciferase—onto a PicoTiterPlate apparatus. Through centrifugation, the beads settled into individual wells, with the Enzyme Beads helping to immobilize the DNA beads throughout the sequencing procedure. This setup was designed to optimize the number of wells holding a single amplified library bead.

Once loaded, the PicoTiterPlate was placed into the Genome Sequencer FLX Instrument, where its fluidics system supplied sequencing reagents, including buffers and nucleotides, across the plate surface. The four DNA nucleotides were introduced sequentially in a predetermined sequence during the run. Each bead, carrying millions of identical DNA copies, underwent parallel sequencing. Upon addition of a nucleotide complementary to the template strand, polymerase extended the DNA chain, producing a light emission captured by the instrument’s CCD camera. This sequencing-by-synthesis technique, termed pyrosequencing, generated light signals proportional to nucleotide incorporation events. Homopolymer regions produced intensified signals for up to eight consecutive identical nucleotides, beyond which signal intensity dropped markedly. The resulting data were stored in standard flowgram format (SFF) files for subsequent analysis.

See also

• DNA Sequencing