From GenoSeq
Genome Sequencer FLX : High Throughput Sequencer
To find out more about using this technology in your research contact us, or come to the GS FLX User Group Meeting the first Wednesday of every month at 1pm in 5303 Gonda.
454 Sequencing is a Massively-parallel Pyrosequencing or Sequenicng by Synthesis.
The GS FLX system is capable of sequencing roughly 100 megabases of raw DNA per 7.5-hour run of the GSFLX instrument.
The system relies on attaching nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion. The DNA attached 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 polymerase, ATP Sulfurylase, and Luciferase are also packed into the well. The PicoTiterPlate is then placed into the sequencer for sequencing. At this stage, the four nucleotides: ACGT 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 complimentary 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 only up to eight consecutive nucleotides after which the signal falls-off rapidly.
Under optimal conditions, ~400,000 bases of an average read length of ~250 bases and can be obtained.
- Applications:
- De Novo Sequencing: Whole Genomes of Humans, Plants, Yeasts, Fungi, Bacteria, Viruses, BACS etc.
- Resequencing: SNPs, InDels, Somatic Mutations
- Transcriptome Analysis: cDNA, small non coding RNA, microRNA
- Epigenetic Changes: DNA Methylation Patterns
- Metagenomics: Analysis of Environmental DNA
- Paleogenomics : Ancient DNA
DNA Requirements:
5ug of double stranded DNA in more than 10ul volume is required. If your DNA is 500bp long or less, then about 2ug in more than 10ul volume is necessary. The DNA should be suspended in H2O or 1X TE.
Provide the DNA at a concentration > 300ng/ul and A260/280 ratio of approximately 1.8.
An image of DNA sample(s) run on a 2% agarose gel is requested to assess quality of incoming DNA. We will further assess the quality using PicoGreen Assay on Fluorimeter, Bioanalyzer and Nanodrop.
De novo sequencing requires 20X average genome coverage. Resequencing and mapping projects require 15X average coverage.
Application notes/Protocols:
- Amplicon Sequencing
- Metagenomics
- SAGE
- Methylation
- Denovo Bacteria Sequencing
- Paired End Reads
- Cloning Small RNAs for Sequencing
- RNA Virus Sequencing
- High Throughput Sequencing Services Contact Info:
- The Sequencing and Genotyping Core is located at
- 5309 Gonda Center on the UCLA campus.
- 695 Charles Young Drive South
- Los Angeles, CA 90095
- E5 on the South Campus sector of the UCLA Campus Map
- Open 9AM to 5PM Mon-Fri, excluding university holidays
- For more information about sequencing at the Core, call 310-267-2461 or contact:
- Uma Dandekar, GenoSeq Core Assistant Director
- Email: uma@ucla.edu
- Phone: 310-267-2461
- Fax: 310-794-5446