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The Keck Center provides an ultra-high-throughput sequencing option using the Roche 454 GS-FLX, a next-generation genome sequencing system utilizing pyrosequencing technology. This supports the sequencing of samples from a wide variety of starting materials, including genomic DNA (large eukaryotic genomes, fungi, bacteria, viruses), metagenomes (environmental samples), PCR amplicons, amplified tag fragments (microRNA, ChiP-derived, methlyation enriched, captured sequences, SAGE, etc.), BAC clones or pools, and cDNA. The Keck Center can prepare cDNA libraries (primary or normalized) for pyrosequencing at a much lower price than for Sanger sequencing, since no cloning is required for pyrosequencing.
Sample requirements include a minimum of 5 ug of DNA in 100 ul TE with an image on an agarose gel containing a mass ladder to access quantity and quality. Samples that are larger than 500 bp, such as genomic DNA, cDNA and BACs, are sheared into small fragments ranging from 300 to 800 bp, followed by adaptor (A and B) ligation of each fragment. Small fragment that are less than 500 bp, such as PCR amplicons, can be amplified using fusion primers containing genome sequencing adaptors. These adaptors will then be used for capturing, purification, amplification, and sequencing steps. One fragment, immobilized on one DNA capture bead in its own oil droplet microreactor is amplified during emulsion PCR, resulting in several million copies of each fragment per bead. Beads carrying clonally amplified DNA fragments are loaded onto a PicoTiterPlate for sequencing. The fluidic subsystem of the Genome Sequencer FLX Instrument controls the flow of individual nucleotides in a fixed order across hundreds of thousands of wells containing one bead each. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera of the Genome Sequencer FLX Instrument.
Under optimal sequencing conditions, the Roche 454 GS-FLX system produces ~400,000 reads with an average read length of ~250 bases per read from a 7.5 hour instruments run. The Keck center offer many sequencing options depending on the type and size of project as well as other variables. Various bioinformatics options are available for data analysis.
Please contact the Keck Center to arrange a meeting or to discuss your project with Dr. Ryan Kim (wrkim@uiuc.edu) or Dr. Alvaro Hernandez (aghernan@uiuc.edu) at 217-244-3480.
Pricing
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High-Throughput Sequencing and Genotyping Unit
Ryan Kim, Ph.D - Director
340 Edward R. Madigan Laboratory, 1201 W. Gregory Drive, Urbana, IL 61801
Phone: (217) 244-3480 FAX: (217) 265-5066
Email: wrkim@uiuc.edu
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