This workflow contains examples of using the BNW structural constraint interface to help identify biologically meaningful genetic network models for two cases:

1)    A genetic network linking genotype and phenotype

2)    A genetic network with multiple genotypes and cis- and trans-regulated genes

 

1. A genetic network linking genotype and phenotype

In this example, we will use the structural constraint interface to create a genetic network linking a genotype with intermediated phenotypes (i.e., gene expression or other cellular level traits) and a higher-order phenotype. The figure below shows a screenshot of BNW after loading the data file and selecting Go to structure learning settings and the BNW structural constraint interface. There are 5 nodes in the network, Genotype, Int1, Int2, Int3, and a Phenotype.

 

We want to assign the nodes to three tiers in this example, so we will keep the number of tiers at the default setting, and drag the nodes to the proper tiers.

In this example, we do not want the genotype to be the direct parent of the phenotype, so we made some changes to the Define interactions allowed between tiers section of the structural constraint interface. Specifically, we have unchecked two boxes that are different from the default settings: (1) we have unchecked the box that allows the node(s) in Tier1 from being the parents of the node(s) in Tier3 and (2) we have unchecked the box that allows the node(s) in Tier3 from being the children of the node(s) in Tier1. Actually, unchecking either of these boxes would have been sufficient in preventing direct interactions between Genotype and Phenotype, but, there is no harm in unchecking both boxes. There are no additional specific edges that we want to ban or require in the network, so we do not have to add any edges to the Specify additional constraint section and we can proceed with structure learning.

2. A genetic network with multiple genotypes and cis- and trans-regulated genes.

In this example, we will add restrictions to a network containing 8 nodes: 2 genotype nodes (Geno1 and Geno2), 3 cis-regulated gene expression traits (cisGene1, cisGene2, and cisGene3), 2 trans-regulated gene expression traits (transGene1 and transGene2), and a phenotype (Pheno). We have four tiers of nodes (genotypes, cis-regulated genes, trans-regulated genes, and phenotype), so we have selected 4 from the dropdown menu at the top of the page and assigned the nodes to the correct tiers. We could make a more complex system of tiers that would allow us to specify which genes are regulated by which genotypes (for example, Geno1 regulates cisGene1 and transGene1, while Geno2 regulates cisGene2, cisGene3, and transGene2), but, for this example, we will use a simpler system of 4 tiers.

We have made one change to the default setting in the Define interactions allowed between tiers section. For Tier1, which contains the genotypes, we have selected "No" for the "Are within tier interactions allowed?", as it does not make biological sense for one genotype variation to cause the variation in another genotype in this examples.

Assume that a known regulatory relationship between cisGene1 and transGene1 has been established from previous experiments. We can require that this relationship is included in the network by adding the edge list of required edges in the Specify additional constraints section.