Roche Genome Sequencer FLX System (GS FLX)

The Institute of Genetic Medicine at Newcastle University is equipped with the Genome Sequencer FLX System, Roche’s latest sequencing platform. Using technology established by 454 Life Sciences, the GS FLX system has the capacity to perform 400,000 sequencing reads in a single run. With potential sequencing reads of 200-300 bases, this creates a throughput approaching 100 million bases per run.

The GS FLX System offers a range of applications, including amplicon and gDNA resequencing, gene regulation and transcriptome studies, epigenetic (methylation) analysis, and de novo sequencing.

DNA sequencing using the GS FLX involves the application of revolutionary technology. The principal steps are listed below:

  1. Preparation of a DNA library:
    (a) PCR with adaptor tags (Amplicon resequencing).
    (b) Nebulisation of a purified gDNA sample followed by adaptor ligation.
  2. Annealing of DNA library to DNA capture beads.
  3. Emulsion PCR (emPCR). An emulsion is created containing microreactors which facilitate the clonal amplification of a single DNA molecule annealed to a DNA capture bead.
  4. Bead deposition. DNA beads and sequencing enzymes are loaded into a PicoTiterPlateTM.
  5. DNA sequencing. The GS FLX uses sequencing by synthesis. Each dNTP is repeatedly flowed across the growing DNA strand in the order G, T, C, A. When incorporation of the correct dNTP occurs, a cascade reaction is triggered resulting in the emission of light. This is captured by a camera.
  6. Sequence analysis. The GS FLX system features graphical software for base calling, mapping, assembly, and amplicon variation detection.

The Institute of Genetic Medicine has invested in the entire workflow required to prepare samples for GS FLX sequencing. We provide instruments for quantification of DNA (Agilent BioAnalyzer/PicoGreen Fluorometer), machinery for the preparation of emulsion PCR reactions (Qiagen TissueLyser), a particle counting facility (Beckman Coulter Counter), plus complete software packages for data analysis.

Enquiries regarding the use of the Roche GS FLX at the Institute of Genetic Medicine should be made to Dr Jonathan Coxhead, Facility Manager (j.m.coxhead@newcastle.ac.uk).