454 Life Sciences

454 Life Sciences, a Roche company, 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 acquisition by Roche Diagnostics and release of the GS20 sequencing machine in 2005, the first next-generation DNA sequencer on the market. The Genome Sequencer FLX instrument was released in 2007. In 2008, 454 Sequencing launched the GS FLX Titanium series reagents for use on the current instrument, with the ability to sequencing 400-600 million base pairs with 400-500 base pair read lengths. With its high accuracy, low cost, and long reads, many researchers have migrated away from traditional Sanger capillary sequencing instruments and toward the 454 Sequencing platform for a variety of genome projects.

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 [http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=7,211,390&OS=7,211,390&RS=7,211,390 invention] , Dr. Rothberg and 454 Life Sciences 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 Sciences for US$154.9 million. It will remain a separate business unit.

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 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. In September 2008, 454 Sequencing celebrated the 250th peer-reviewed publication using the Genome Sequencer System. The studies span a diverse group of sequencing applications including de novo whole genome sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics and RNA analysis.

Technology

454 Sequencing is a large-scale parallel pyrosequencing system capable of sequencing roughly 400-600 megabases of DNA per 10-hour run on the Genome Sequencer FLX with GS FLX Titanium series reagents. The technology is known for its unbiased sample preparation and long, highly accurate sequence reads (400-500 base pairs in length), including paired reads.Fact|date=October 2008 Software analysis tools, including an assembler, mapper and amplicon variant analyzer, are included with the system.

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. Each DNA-bound bead is placed into a ~29 μm well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as DNA polymerase, ATP sulfurylase, and luciferase are also packed into the well. The PicoTiterPlate is then placed into the GS FLX System for sequencing.

DNA library preparation and emPCR

Genomic DNA is fractionated into smaller fragments (300-800 base pairs) that are subsequently polished (made blunt at each end). 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. One adaptor (Adaptor B) contains a 5'-biotin tag for immobilization of the DNA 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.Fact|date=October 2008

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.

equencing

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 a 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 Genome Sequencer FLX Instrument. The fluidics sub-system delivers sequencing reagents (containing buffers and nucleotides) across the wells of the plate. The four DNA nucleotides are added sequentially in a fixed order across the PicoTiterPlate device during a sequencing run. During the nucleotide flow, millions of copies of DNA bound to each of the beads are sequenced in parallel. When a nucleotide complementary to the template strand is added into a well, the polymerase extends the existing DNA strand by adding nucleotide(s). Addition of one (or more) nucleotide(s) 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.Fact|date=October 2008 The signal strength is proportional to the number of nucleotides; for example, homopolymer stretches, incorporated in a single nucleotide flow generate a greater singal than single nucleotides. However, the signal strength for homopolymer stretches is linear only up to eight consecutive nucleotides after which the signal falls-off rapidly.cite journal
last = Margulies | first = Marcel | coauthors = Michael Egholm and 54 additional coauthors
date = 2005-09-15 | accessdate = 2007-12-16
title = Genome Sequencing in Open Microfabricated High Density Picoliter Reactors
journal = Nature | volume = 437 | issue = 7057 | pages = 376–380 | publisher = Nature Publishing Group
pmid = 16056220 | doi = 10.1038/nature03959 | url = http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1464427
laysummary = | laysource = | laydate = ]

Applications

454 Sequencing can sequence any double-stranded DNA and enables a variety of applications including de novo whole genome sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics and RNA analysis.

Whole Genome Sequencing ("de novo" sequencing and resequencing)

Whole Genome Sequencing (WGS) consists of projects dealing with the sequencing of the entire genome of an organism, for example, humans, dogs, mice, viruses or bacteria. 454 Sequencing technology is ideal for whole genome assembly due to it’s ultra high throughput and long single reads (500 base pairs). The ability to combine shotgun reads with paired end reads facilitates high genome coverage with minimal gaps. As a result, the 454 platform has effectively sequenced and assembled a number of complex genomes. In June 2006 they launched a project with the Max Planck Institute for Evolutionary Anthropology to sequence the genome of the Neanderthal, the extinct closest relative of 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.cite press release
title = 454 Life Sciences and Max Planck Institute to Sequence Neandertal Genome
publisher = 454 Life Sciences | date = 2006-07-20 | accessdate = 2007-12-16
url = http://www.454.com/news-events/press-releases.asp?display=detail&id=51] cite press release
title = Neandertal Genome to be Deciphered
publisher = Max Planck Society | date = 2006-07-20 | accessdate = 2007-12-16
url = http://www.mpg.de/english/illustrationsDocumentation/documentation/pressReleases/2006/pressRelease20060720/index.html] In September 2008 the complete Neanderthal mitochondrial genome was sequenced, establishing the divergence between humans and Neanderthal at 660,000 +/- 140,000 years.

In May 2007, researchers at the Baylor College of Medicine used the 454 Sequencing system to sequence and assemble the complete human diploid genome of Dr. James Watson. The feat demonstrated that generating high-quality sequence from humans, quickly and affordably, is now feasible.

Amplicon (Ultra Deep) Sequencing

Amplicon (Ultra Deep) Sequencing is a new field which is largely being enabled through 454 Sequencing technology. Unlike Sanger, 454 sequencing allows mutations to be detected at extremely low levels. Researchers are able to PCR amplify specific, targeted regions of DNA. This method is particularly useful for identifying low frequency somatic mutations in cancer samples or discovery of rare variants in HIV infected individuals. The Genome Sequencer FLX system offers dedicated analysis software, the GS Amplicon Variant Analyzer, which automatically computes the alignment of reads from amplicon-based samples against a reference sequence.

Transcriptome Sequencing

Transcriptome sequencing encompasses experiments including small RNA profiling and discovery, mRNA transcript expression analysis (full-length mRNA, expressed sequence tags (ESTs) and ditags, and allele-specific expression) and the sequencing and analysis of full-length mRNA transcripts. The transcriptome data derived from the Genome Sequencer FLX is ideally suited to detailed transcriptome investigation in the areas of novel gene discovery, gene space identification in novel genomes, assembly of full-length genes, single nucleotide polymorphism (SNP), insertion-deletion and splice-variant discovery.

Metagenomics

Metagenomics is the study of the genomic content in a complex sample. The two primary goals of this approach are to characterize the organisms present in a sample and identify what roles each organism has within a specific environment. Metagenomics samples are found nearly everywhere, including several microenvironments within the human body, soil samples, extreme environments such as deep mines and the various layers within the ocean.The Genome Sequencer FLX System enables a comprehensive view into the diversity and metabolic profile of an environmental habitat. The system’s long reads ensure the enormous specificity needed to compare sequenced reads against DNA or protein databases. Researchers often use the platform for counting environmental gene tags to analyze the relative abundance of microbial species under varying environmental conditions.

Advantages and Disadvantages

454 Sequencing with GS FLX Titanium series reagents sequence 400-600 million bases per 10-hour run, allowing large amounts of DNA to be sequenced at low cost compared to Sanger chain-termination methods. With Q20 read lengths of 400 bases (99% accuracy at the 400th base and higher for preceding bases) and significantly higher throughput, de novo assembly with 454 Sequencing is at least equivalent to Sanger assembly, while being dramatically faster and an order of magnitude less expensive. 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 highsensitivity level, which may have implications in clinical research, especially cancer and HIV.cite web
url = http://www.454.com/downloads/news-events/publications/HIV%20Resistance%20Workshop_454_final.jpg
title = Ultra-Deep Sequencing of HIV from Drug Resistant Patients
date = 2005-11-17 | accessdate = 2007-12-16
author = Jan Fredrik Simons
coauthors = Michael Egholm and 11 additional coauthors
format = JPG
publisher = 454 Life Sciences] [ name="HIVWkShop2005">cite web
url = http://www.hivandhepatitis.com/2005icr/hivdrug/docs/070805_b.html
title = Report on the XIV International HIV Drug Resistance Workshop: Parts 4-5
date = 2005-07-08 | accessdate = 2007-12-16
work = Selected Highlights from the XIV International HIV Drug Resistance Workshop | publisher = HIVandHepatitis.com]

External links

* [http://www.454.com/ 454 Life Sciences Homepage]
* [http://www.roche-applied-science.com/ Roche Applied Science Homepage]
* [http://www.researchandtesting.com/ Research and Testing Laboratory - Pyrosequencing ]
* [http://jimwatsonsequence.cshl.edu/cgi-perl/gbrowse/jwsequence/?name=Sequence:NM_005516.3 James Watson's Personal Genome Sequence]

Patents Awarded

* [http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=7,211,390&OS=7,211,390&RS=7,211,390 Method of Sequencing a Nucleic Acid]
* [http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=6,956,114&OS=6,956,114&RS=6,956,114 Sulfurylase-luciferase fusion proteins and thermostable sulfurylase]

References

A complete listing of peer-reviewed research articles can be found on the [http://www.454.com Roche/454 Sequencing website ] :
* [http://genome.cshlp.org/cgi/content/full/17/8/1195 Characterization of mutation spectra with ultra-deep pyrosequencing: Application to HIV-1 drug resistance. Genome Research.]
* [http://www.nature.com/nature/journal/v452/n7189/abs/nature06884.htm The complete genome of an individual by massively parallel DNA sequencing. Nature]
* [http://www.ncbi.nlm.nih.gov/pubmed/18704501 Comprehensive resequence analysis of a 136 kb region of human chromosome 8q24 associated with prostate and colon cancers. Human Genetics.]
* [http://content.nejm.org/cgi/content/abstract/358/10/991 A new arenavirus in a cluster of fatal transplant-associated diseases. New England Journal of Medicine.]
* [http://www.cell.com/content/article/abstract?uid=PIIS0092867408007733 A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing. Cell.]
* [http://www.sciencemag.org/cgi/content/short/281/5375/363 A Sequencing Method Based on Real-Time Pyrophosphate Release]
* [http://www.nature.com/nature/journal/v444/n7117/abs/nature05336.html Analysis of one million base pairs of Neanderthal DNA. Nature]
* [http://www.nytimes.com/2007/06/01/science/01gene.html?ex=1182139200&en=fc026710bb87a6ad&ei=5070 Genome of DNA Discoverer Is Deciphered. New York Times]
* [http://www.health-itworld.com/newsitems/2007/may/05-31-07-watson-genome Project Jim: Watson’s Personal Genome Goes Public]
* [http://www.eurekalert.org/pub_releases/2006-06/nc-dfa062006.php Dana Farber and 454 Life Sciences Announce Breakthrough in DNA Sequencing for Cancer Research]
* [http://www.pnas.org/cgi/content/abstract/0605127103v1 Sogin et al. Microbial diversity in the deep sea and the underexplored "rare biosphere". "PNAS". 31 July 2006.]
* [http://www.454.com/news-events/press-releases.asp?display=detail&id=66 CuraGen Announces Definitive Agreement to Sell 454 Life Sciences to Roche]