The raw FI values associated with each bead color are analyzed in a peak deconvolution step to associate the expression levels with the appropriate genes. This step is necessary because each bead color is associated with two genes rather than one. To facilitate the analysis, separate bead batches that identify each gene are mixed in a 2:1 ratio for use in the assay. To deconvolute the composite expression signal into two values and associate them with the appropriate genes, we construct a histogram of FI values. This yields a distribution that generally consists of two peaks, a larger one that designates expression of the gene for which a larger proportion of beads are present, and a smaller peak representing the other gene. Using the k-means clustering algorithm, the distribution is partitioned into two distinct clusters, such that the ratio of cluster membership is as close as possible to 2:1, and the median expression value for each cluster is then assigned as the expression value of the appropriate gene.
