2.1 Main callback methods

The first step is to build the server port, which will be required in all remote procedure calls. By default the constructor RedPort should set all details:

library(RedeR)

## ***This is RedeR 1.30.0! For a quick start, please type 'vignette('RedeR')'.
##    Supporting information is available at Genome Biology 13:R29, 2012,
##    (doi:10.1186/gb-2012-13-4-r29).

rdp <- RedPort() 

Next, invoke RedeR using the method calld:

calld(rdp)

Within an active interface, then the method addGraph can easily send R graphs to the application. For example, the following chunk adds an igraph object (Fig. 2):

library (igraph)
g1 <- graph.lattice(c(5,5,5))
addGraph( rdp, g1, layout.kamada.kawai(g1) )

Figure 2. A toy example added to RedeR by the addGraph function.

Conversely, RedeR graphs can be transferred to R and wrapped in igraph objects:

g2 <- getGraph(rdp)
resetd(rdp)

The interface accepts additional graph attributes, as for example edge direction, edge width, edge weight, node shape, node size, node color etc. In order to pass these extensible features the attributes must be provided in a valid syntax (see getGraph and addGraph specification for additional details).

Another strategy is to wrap graphs into containers and then send it to the Java application. Next, the subgraphs g3and g4 are assigned to different nested structures (Fig. 3).

g3 <- barabasi.game(10)
g4 <- barabasi.game(10)
V(g3)$name<-paste("sn",1:10,sep="")
V(g4)$name<-paste("sm",1:10,sep="")
addGraph(rdp, g3, isNest =TRUE, gcoord=c(25,25), gscale=50)
addGraph(rdp, g4, isNest =TRUE, gcoord=c(75,75), gscale=50)

Figure 3. Nested graphs in RedeR using the command addGraph.

In this case, the subgraphs can be handled apart from each other. For example, the following chunk selects all nodes assigned to the container “N0” and then gets back the subgraph (the selection step can also be done interactively).

selectNodes(rdp,"N0")
g5 <- getGraph(rdp, status= "selected")
resetd(rdp)

As a suggestion, try some RedeR features in the Java side (e.g. open samples s2 or s3 in the main panel and enjoy the dynamic layout options!).

2.2 Interactive work

The next chunk generates a scale-free graph according to the Barabasi-Albert model (Csardi and Nepusz 2006) and sends the graph to RedeR without any layout information.

g6 <- barabasi.game(500)
addGraph(rdp, g6, zoom=20)

Then using the “relax” options available in the app you can tune the graph layout as presented in Figure 4.

relax(rdp,p2=400,p5=30,ps=T)

Figure 4. Scale-free graph according to the Barabasi-Albert model (Csardi and Nepusz 2006).

In Figure 5 the same graph is used to exemplify the community structure mapped by the edge-betweenness algorithm (available on both the Java interface and the ** igraph ** package). In Figure 6 these communities are nested to hidden containers, which are objects of the same class of nodes but with additional behaviors. You can build these containers either using R or Java functions (see options available in the clustering main menu and in the shortcuts of the nested objects, i.e., right-click a container).

Figure 5. Subgraphs detected based on edge betweenness.

Figure 6. Subnetworks within hidden containers.

3.1 Subgraphs

Start the app (i.e. run the ‘calld’ method), and then get the indicated data frame and interactome:

data(ER.limma)
data(hs.inter)
dt <- ER.limma
gi <- hs.inter

Extract and set attributes to a subgraph represented by genes differentially expressed at a given time point (i.e. logFC from t3-t0 contrast):

gt3  <- subg(g=gi, dat=dt[dt$degenes.t3!=0,], refcol=1)

## ...note: not all genes found in the network!

gt3  <- att.setv(g=gt3, from="Symbol", to="nodeAlias")
gt3  <- att.setv(g=gt3, from="logFC.t3", to="nodeColor", breaks=seq(-2,2,0.4), pal=2)

Note that some genes will not be found in the interactome, and that’s okay. Extract another subgraph and set its attributes (i.e. logFC from t6-t0 contrast):

gt6  <- subg(g=gi, dat=dt[dt$degenes.t6!=0,], refcol=1)

## ...note: not all genes found in the network!

gt6  <- att.setv(g=gt6, from="Symbol", to="nodeAlias")
gt6  <- att.setv(g=gt6, from="logFC.t6", to="nodeColor", breaks=seq(-2,2,0.4), pal=2)

Extract another subgraph and set its attributes (i.e. logFC from t12-t0 contrast):

gt12 <- subg(g=gi, dat=dt[dt$degenes.t12!=0,], refcol=1)

## ...note: not all genes found in the network!

gt12 <- att.setv(g=gt12, from="Symbol", to="nodeAlias")
gt12 <- att.setv(g=gt12, from="logFC.t12", to="nodeColor", breaks=seq(-2,2,0.4), pal=2)

Now add all subgraphs to the app (Fig.7):

addGraph(rdp, gt3, gcoord=c(10,25), gscale=20, isNest=TRUE, theme='tm1', zoom=30)
addGraph(rdp, gt6, gcoord=c(20,70), gscale=50, isNest=TRUE, theme='tm1', zoom=30)
addGraph(rdp, gt12, gcoord=c(70,55), gscale=80, isNest=TRUE, theme='tm1', zoom=30)

… and nest overlapping subgraphs (i.e. indicating overlapping time series!):

nestNodes(rdp, nodes=V(gt3)$name, parent="N1", theme='tm2')
nestNodes(rdp, nodes=V(gt6)$name, parent="N2", theme='tm2')
nestNodes(rdp, nodes=V(gt3)$name, parent="N4", theme='tm3')

To simplify the graph, the mergeOutEdges method assigns edges to containers:

mergeOutEdges(rdp)

Relax the network:

relax(rdp,50,400)

Add color legend (other types are available):

scl <- gt3$legNodeColor$scale
leg <- gt3$legNodeColor$legend 
addLegend.color(rdp, colvec=scl, labvec=leg, title="node color (logFC)")

Select a gene:

selectNodes(rdp,"RET")

Reset graph:

resetd(rdp)

Figure 7. Nested subnetworks. This graph shows genes differentially expressed in estrogen-treated MCF-7 cells at 3, 6 and 12 h (relative to 0 h). The insets correspond to the overlap between consecutive time points (adapted from Castro et al. (2012))

3.2 Nested networks and clustering

Get the indiated data frame and igraph object:

data(ER.deg)
dt <- ER.deg$dat
sg <- ER.deg$ceg

Map the data frame to the graph:

sg <- att.mapv(sg, dat=dt, refcol=1)

Set attributes to the graph (i.e. gene symbols and two available numeric data):

sg <- att.setv(sg, from="Symbol", to="nodeAlias")
sg <- att.setv(sg, from="logFC.t3", to="nodeColor", breaks=seq(-1,1,0.2), pal=2)    
sg <- att.setv(sg, from="ERbdist", to="nodeSize", nquant=10, isrev=TRUE, xlim=c(5,40,1))

Add graph to the app (Fig.8):

addGraph(rdp,sg)

Compute a hierarchical clustering using standard R functions:

hc <- hclust(dist(get.adjacency(sg, attr="weight")))

Map the hclust object onto the network and return corresponding ids (pvclust objects are also compatible):

nestID <- nesthc(rdp,hc, cutlevel=3, nmemb=5, cex=0.3, labels=V(sg)$nodeAlias)

…at this point nested objects from the network should appear mapped onto a dendrogram. Different levels of the nested structure can be set by the nesthc method. Additionally, clustering stability can be assessed by the pvclust package, which is already compatible with the interface.

Assign edges to containers:

mergeOutEdges(rdp,nlev=2)

Relax the network:

relax(rdp)

Add color and size legends:

scl <- sg$legNodeColor$scale
leg <- sg$legNodeColor$legend
addLegend.color(rdp, colvec=scl, labvec=leg, title="diff. gene expression (logFC)")

scl <- sg$legNodeSize$scale
leg <- sg$legNodeSize$legend
addLegend.size(rdp, sizevec=scl, labvec=leg, title="bd site distance (kb)")

Reset graph:

resetd(rdp)

Figure 8. Hierarchical networks. This graph is obtained by superimposing a dendrogram onto the corresponding co-expression gene network (adapted from Castro et al. (2012)).

4.1 R package

The RedeR package is freely available from the Bioconductor at https://bioconductor.org/packages/RedeR/.

4.2 Java application

The RedeR jar file is already included in the R package and, as usual, to run Java applications your system should have a copy of the JRE (Java Runtime Environment, version >= 6).