Fig 4. Flowchart illustrating BWERF algorithm for constructing multilayered hierarchical gene regulatory network using expression data of pathway genes and regulatory genes.

A. Input for BWERF included a pathway gene expression matrix and a TFs expression matrix. B. For each pathway gene, recursively constructing of random forest model with backward elimination. C. Aggregation of the importance values of a TF to all pathway genes to produce a unified the importance value of this TF to the pathway. D. The Expectation-maximization (EM) algorithm was implemented to fit a Gaussian mixture model to the importance values. E. The most important TFs were identified and used as a layer. F. By using the new TF layer as bottom layer, we repeated all above procedure to obtain the next layer until the designated number of layer was achieved or the program was terminated due to the lack of significant TFs as input for upper layers.