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Sequencing Chemistry
454 Sequencing, based on sequencing-by-synthesis (Figure 1), is the power behind the performance of the Genome Sequencer FLX System and GS Junior System.
- Nucleotides are flowed sequentially in a fixed order across the PicoTiterPlate device during a sequencing run.
- During the nucleotide flow, hundreds of thousands of beads each carrying millions of copies of a unique single-stranded DNA molecule are sequenced in parallel.
- If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existing DNA strand by adding nucelotide(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.
- The signal strength is proportional to the number of nucleotides incorporated in a single nucelotide flow.
Figure 1: Sequencing reaction of the Genome Sequencer System. Millions of copies of a single clonal fragment are contained on each DNA Capture Bead.
Image Processing
The Genome Sequencer System software tracks the location of DNA carrying beads on a XY axis. Each bead corresponds to a XY-coordinate on a series of images. The signal intensity per nucleotide flow is recorded for each bead over time and is plotted to generate a flowgram. Each 10-hour sequencing run with the GS FLX Titanium series will typically produce over 1,000,000 flowgrams, one flowgram per read, while each 10-hour sequencing run with the GS Junior Titanium series will produce approximately 100,000 flowgrams (Figure 2).
Figure 2: Genome Sequencer System Image processing overview.