An overlay of three channels of fluorescence. In practice, there are four fluorescent channels, one corresponding to each base. Only three channels and the corresponding overlay are shown here for clarity. NIS-Elements Basic Research 3.0 (Nikon Instruments Inc, Melville, N.Y) software was used to generate this image and analyze the data. Pixel values are taken from beads in each channel to ascertain sequencing accuracy. The pixel values of brightness in each channel are used as a gauge of nucleotide identity. The pixel values of the brightest channel for a given bead and the values of other channels, provide the signal to noise ratio for comparison.