Pyrosequencing

MR DNA http://www.mrdnalab.com  for next generation sequencing service

 

History

 

454 Roche FLX titanium

 

Detail major types of research performed using 454 pyrosequencing and critically review the technology and its benefits and weaknesses for these applications

1. microbial diversity/metagenomics

2. Genome sequencing

 3. epigenetics

4. small RNA

5. transcriptome

6. pick another broad category

 

 

 

454 pyrosequencing (the following was pulled from wikipedia to preserve a pretty good version  Darn things change so often )

History and Major Achievements

454 Life Sciences was founded by Jonathan Rothberg originally as 454 Corporation, a subsidiary of CuraGen Corporation. For their method for low-cost gene sequencing, 454 Life Sciences were awarded the Wall Street Journal’s Gold Medal for Innovation in the Biotech-Medical category in 2005. The name 454 was the code name by which the project was referred to at CuraGen and the numbers have no special meaning

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

In November 2006, 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

In May 2007, Project “Jim”, a project initiated by Rothberg and 454 Life Sciences to determine the first sequence of an individual was completed after sequencing the genome of James Dewey Watson, co-discoverer of the structure of DNA

http://www.mrdnalab.com pyrosequencing service microbiome metagenome genome, microbial diversity and more

454 Sequencing uses a large-scale parallel pyrosequencingsystem 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 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.

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. In 2008, 454 Sequencing launched the GS FLX Titanium series reagents for use on the Genome Sequencer FLX instrument, with the ability to sequence 400-600 million base pairs per run with 400-500 base pair read lengths. The company has said it plans to launch kits enabling sequencing read lengths of up to 1,000 bp in 2010. In late 2009, 454 Life Sciences introduced the GS Junior System, a bench top version of the Genome Sequencer FLX System.

DNA library preparation and emPCR

Genomic DNA is fractionated into smaller fragments (300-800 base pairs) and 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.

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.

 http://www.mrndalab.com  good source for microbiome 16s pyroseuquencing using 454 titanium chemistry

 

Single-stranded template DNA 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 is designed to maximize the number of wells that contain a single amplified library bead.

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. The signal strength is proportional to the number of nucleotides; for example, homopolymer stretches, incorporated in a single nucleotide flow generate a greater signal 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.. Data are stored in standard flowgram format (SFF) files for downstream analysis.

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.

 

Full genome sequencing (FGS), also referred to as whole genome sequencing (WGS), aims to sequence the entire genome of an organism, for example, humans, dogs, mice, viruses or bacteria. In June 2006, 454 Life Sciences launched a project with the Max Planck Institute for Evolutionary Anthropology to sequence the genome of the Neanderthal, the extinct closest relative of humans.[10][11]. In September 2008 the complete Neanderthal mitochondrial genome was sequenced, establishing the divergence between humans and Neanderthal at 660,000 +/- 140,000 years[12], and the full genome was published in 2010, using a combination of 454 and Illumina sequencing.[13]

 

Amplicon (ultra deep) sequencing is a new field which is largely being enabled through 454 Sequencing technology. This method is designed to allow mutations to be detected at extremely low levels, and PCR amplify specific, targeted regions of DNA. This method is used to identify low frequency somatic mutations in cancer samples or discovery of rare variants in HIV infected individuals.

 

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 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 of an environmental habitat. The system’s long reads ensure the enormous specificity needed to compare sequenced reads against DNA or protein databases. The platform is used to count environmental gene tags to analyze the relative abundance of microbial species under varying environmental conditions.