- 1 Overview
- 2 Network reconstruction
- 3 Calculation of centrality measures
- 4 Assessment of the association of centrality measures
- 5 Identification of the most
`influential`

network nodes - 6 Identification of the most important network spreaders
- 7 Identification of the most important network hubs
- 8 Ranking the influence of nodes on the topology of a network based on the
`SIRIR`

model - 9 Experimental data-based classification and ranking of top candidate features
- 10 Computational manipulation of cells
- 11 References

`influential`

is an R package mainly for the identification of the most influential nodes in a network as well as the classification and ranking of top candidate features. The `influential`

package contains several functions that could be categorized into five groups according to their purpose:

- Network reconstruction
- Calculation of centrality measures
- Assessment of the association of centrality measures
- Identification of the most
`influential`

network nodes - Experimental data-based classification and ranking of features

The sections below introduce these five categories. However, if you wish not going through all of the functions and their applications, you may skip to any of the novel methods proposed by the `influential`

, including:

`library(influential)`

Three functions have been obtained from the `igraph`

^{1} R package for the reconstruction of networks.

In the data frame the first and second columns should be composed of source and target nodes.

A sample appropriate data frame is brought below:

lncRNA | Coexpressed.Gene |
---|---|

ADAMTS9-AS2 | A2M |

ADAMTS9-AS2 | ABCA6 |

ADAMTS9-AS2 | ABCA8 |

ADAMTS9-AS2 | ABCA9 |

ADAMTS9-AS2 | ABI3BP |

ADAMTS9-AS2 | AC093110.3 |

This is a co-expression dataset obtained from a paper by Salavaty **et al**.^{2}

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(d=MyData) My_graph
```

If you look at the class of `My_graph`

you should see that it has an `igraph`

class:

```
class(My_graph)
#> [1] "igraph"
```

A sample appropriate adjacency matrix is brought below:

LINC00891 | LINC00968 | LINC00987 | LINC01506 | MAFG-AS1 | MIR497HG | |
---|---|---|---|---|---|---|

LINC00891 | 0 | 1 | 1 | 0 | 0 | 0 |

LINC00968 | 0 | 0 | 1 | 0 | 0 | 0 |

LINC00987 | 0 | 1 | 0 | 0 | 0 | 0 |

LINC01506 | 0 | 0 | 0 | 0 | 0 | 0 |

MAFG-AS1 | 0 | 0 | 0 | 0 | 0 | 0 |

MIR497HG | 0 | 1 | 1 | 0 | 0 | 0 |

- Note that the matrix has the same number of rows and columns.

```
# Preparing the data
<- coexpression.adjacency
MyData
# Reconstructing the graph
<- graph_from_adjacency_matrix(MyData) My_graph
```

A sample appropriate incidence matrix is brought below:

Gene_1 | Gene_2 | Gene_3 | Gene_4 | Gene_5 | |
---|---|---|---|---|---|

cell_1 | 0 | 1 | 1 | 0 | 1 |

cell_2 | 1 | 1 | 1 | 0 | 0 |

cell_3 | 1 | 1 | 1 | 0 | 0 |

cell_4 | 0 | 0 | 0 | 1 | 0 |

```
# Reconstructing the graph
<- graph_from_adjacency_matrix(MyData) My_graph
```

SIF is the common output format of the Cytoscape software.

```
# Reconstructing the graph
<- sif2igraph(Path = "Sample_SIF.sif")
My_graph
class(My_graph)
#> [1] "igraph"
```

To calculate the centrality of nodes within a network several different options are available. The following sections describe how to obtain the names of network nodes and use different functions to calculate the centrality of nodes within a network. Although several centrality functions are provided, we recommend the IVI for the identification of the most `influential`

nodes within a network.

By the way, the results of all of the following centrality functions could be conveniently illustrated using the centrality-based network visualization function.

Network vertices (nodes) are required in order to calculate their centrality measures. Thus, before calculation of network centrality measures we need to obtain the name of required network vertices. To this end, we use the `V`

function, which is obtained from the `igraph`

package. However, you may provide a character vector of the name of your desired nodes manually.

- Note in many of the centrality index functions the entire network nodes are assessed if no vector of desired vertices is provided.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
My_graph_vertices
head(My_graph_vertices)
#> + 6/794 vertices, named, from 775cff6:
#> [1] ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
```

Degree centrality is the most commonly used local centrality measure which could be calculated via the `degree`

function obtained from the `igraph`

package.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating degree centrality
<- degree(My_graph, v = GraphVertices, normalized = FALSE)
My_graph_degree
head(My_graph_degree)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 172 121 168 26 189 176
```

Degree centrality could be also calculated for *directed* graphs via specifying the `mode`

parameter.

Betweenness centrality, like degree centrality, is one of the most commonly used centrality measures but is representative of the global centrality of a node. This centrality metric could also be calculated using a function obtained from the `igraph`

package.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating betweenness centrality
<- betweenness(My_graph, v = GraphVertices,
My_graph_betweenness directed = FALSE, normalized = FALSE)
head(My_graph_betweenness)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 21719.857 28185.199 26946.625 2940.467 33333.369 21830.511
```

Betweenness centrality could be also calculated for *directed* and/or *weighted* graphs via specifying the `directed`

and `weights`

parameters, respectively.

Neighborhood connectivity is one of the other important centrality measures that reflect the semi-local centrality of a node. This centrality measure was first represented in a Science paper^{3} in 2002 and is for the first time calculable in R environment via the `influential`

package.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating neighborhood connectivity
<- neighborhood.connectivity(graph = My_graph,
neighrhood.co vertices = GraphVertices,
mode = "all")
head(neighrhood.co)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 11.290698 4.983471 7.970238 3.000000 15.153439 13.465909
```

Neighborhood connectivity could be also calculated for *directed* graphs via specifying the `mode`

parameter.

H-index is H-index is another semi-local centrality measure that was inspired from its application in assessing the impact of researchers and is for the first time calculable in R environment via the `influential`

package.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating H-index
<- h_index(graph = My_graph,
h.index vertices = GraphVertices,
mode = "all")
head(h.index)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 11 9 11 2 12 12
```

H-index could be also calculated for *directed* graphs via specifying the `mode`

parameter.

Local H-index (LH-index) is a semi-local centrality measure and an improved version of H-index centrality that leverages the H-index to the second order neighbors of a node and is for the first time calculable in R environment via the `influential`

package.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating Local H-index
<- lh_index(graph = My_graph,
lh.index vertices = GraphVertices,
mode = "all")
head(lh.index)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 1165 446 994 34 1289 1265
```

Local H-index could be also calculated for *directed* graphs via specifying the `mode`

parameter.

Collective Influence (CI) is a global centrality measure that calculates the product of the reduced degree (degree - 1) of a node and the total reduced degree of all nodes at a distance d from the node. This centrality measure is for the first time provided in an R package.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating Collective Influence
<- collective.influence(graph = My_graph,
ci vertices = GraphVertices,
mode = "all", d=3)
head(ci)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 9918 70560 39078 675 10716 7350
```

Collective Influence could be also calculated for *directed* graphs via specifying the `mode`

parameter.

ClusterRank is a local centrality measure that makes a connection between local and semi-local characteristics of a node and at the same time removes the negative effects of local clustering.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculating ClusterRank
<- clusterRank(graph = My_graph,
cr vids = GraphVertices,
directed = FALSE, loops = TRUE)
head(cr)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 63.459812 5.185675 21.111776 1.280000 135.098278 81.255195
```

ClusterRank could be also calculated for *directed* graphs via specifying the `directed`

parameter.

The function `cond.prob.analysis`

assesses the conditional probability of deviation of two centrality measures (or any other two continuous variables) from their corresponding means in opposite directions.

```
# Preparing the data
<- centrality.measures
MyData
# Assessing the conditional probability
<- cond.prob.analysis(data = MyData,
My.conditional.prob nodes.colname = rownames(MyData),
Desired.colname = "BC",
Condition.colname = "NC")
print(My.conditional.prob)
#> $ConditionalProbability
#> [1] 51.61871
#>
#> $ConditionalProbability_split.half.sample
#> [1] 51.73611
```

- As you can see in the results, the whole data is also randomly splitted into half in order to further test the validity of conditional probability assessments.
*The higher the conditional probability the more two centrality measures behave in contrary manners*.

The function `double.cent.assess`

could be used to automatically assess both the distribution mode of centrality measures (two continuous variables) and the nature of their association. The analyses done through this formula are as follows:

**Normality assessment**:- Variables with
**lower than**5000 observations:*Shapiro-Wilk test* - Variables with
**over**5000 observations:*Anderson-Darling test*

- Variables with
**Assessment of non-linear/non-monotonic correlation**:*Non-linearity assessment*: Fitting a generalized additive model (GAM) with integrated smoothness approximations using the`mgcv`

package*Non-monotonicity assessment*: Comparing the squared coefficients of the correlation based on Spearman’s rank correlation analysis and ranked regression test with non-linear splines.- Squared coefficient of Spearman’s rank correlation
**>**R-squared ranked regression with non-linear splines:*Monotonic* - Squared coefficient of Spearman’s rank correlation
**<**R-squared ranked regression with non-linear splines:*Non-monotonic*

- Squared coefficient of Spearman’s rank correlation

**Dependence assessment**:*Hoeffding’s independence test*: Hoeffding’s test of independence is a test based on the population measure of deviation from independence which computes a D Statistics ranging from -0.5 to 1: Greater D values indicate a higher dependence between variables.*Descriptive non-linear non-parametric dependence test*: This assessment is based on non-linear non-parametric statistics (NNS) which outputs a dependence value ranging from 0 to 1. For further details please refer to the NNS R package^{4}: Greater values indicate a higher dependence between variables.

**Correlation assessment**: As the correlation between most of the centrality measures follows a non-monotonic form, this part of the assessment is done based on the NNS statistics which itself calculates the correlation based on partial moments and outputs a correlation value ranging from -1 to 1. For further details please refer to the NNS R package.**Assessment of conditional probability of deviation from means**This step assesses the conditional probability of deviation of two centrality measures (or any other two continuous variables) from their corresponding means in opposite directions.- The independent centrality measure (variable) is considered as the condition variable and the other as the desired one.
- As you will see in the results, the whole data is also randomly splitted into half in order to further test the validity of conditional probability assessments.
*The higher the conditional probability the more two centrality measures behave in contrary manners*.

```
# Preparing the data
<- centrality.measures
MyData
# Association assessment
<- double.cent.assess(data = MyData,
My.metrics.assessment nodes.colname = rownames(MyData),
dependent.colname = "BC",
independent.colname = "NC")
print(My.metrics.assessment)
#> $Summary_statistics
#> BC NC
#> Min. 0.000000000 1.2000
#> 1st Qu. 0.000000000 66.0000
#> Median 0.000000000 156.0000
#> Mean 0.005813357 132.3443
#> 3rd Qu. 0.000340000 179.3214
#> Max. 0.529464720 192.0000
#>
#> $Normality_results
#> p.value
#> BC 1.415450e-50
#> NC 9.411737e-30
#>
#> $Dependent_Normality
#> [1] "Non-normally distributed"
#>
#> $Independent_Normality
#> [1] "Non-normally distributed"
#>
#> $GAM_nonlinear.nonmonotonic.results
#> edf p-value
#> 8.992406 0.000000
#>
#> $Association_type
#> [1] "nonlinear-nonmonotonic"
#>
#> $HoeffdingD_Statistic
#> D_statistic P_value
#> Results 0.01770279 1e-08
#>
#> $Dependence_Significance
#> Hoeffding
#> Results Significantly dependent
#>
#> $NNS_dep_results
#> Correlation Dependence
#> Results -0.7948106 0.8647164
#>
#> $ConditionalProbability
#> [1] 55.35386
#>
#> $ConditionalProbability_split.half.sample
#> [1] 55.90331
```

**Note**: It should also be noted that as a single regression line does not fit all models with a certain degree of freedom, based on the size and correlation mode of the variables provided, this function might return an error due to incapability of running step 2. In this case, you may follow each step manually or as an alternative run the other function named `double.cent.assess.noRegression`

which does not perform any regression test and consequently it is not required to determine the dependent and independent variables.

The function `double.cent.assess.noRegression`

could be used to automatically assess both the distribution mode of centrality measures (two continuous variables) and the nature of their association. The analyses done through this formula are as follows:

**Normality assessment**:- Variables with
**lower than**5000 observations:*Shapiro-Wilk test* - Variables with
**over**5000 observations:*Anderson–Darling test*

- Variables with
**Dependence assessment**:*Hoeffding’s independence test*: Hoeffding’s test of independence is a test based on the population measure of deviation from independence which computes a D Statistics ranging from -0.5 to 1: Greater D values indicate a higher dependence between variables.*Descriptive non-linear non-parametric dependence test*: This assessment is based on non-linear non-parametric statistics (NNS) which outputs a dependence value ranging from 0 to 1. For further details please refer to the NNS R package: Greater values indicate a higher dependence between variables.

**Correlation assessment**: As the correlation between most of the centrality measures follows a non-monotonic form, this part of the assessment is done based on the NNS statistics which itself calculates the correlation based on partial moments and outputs a correlation value ranging from -1 to 1. For further details please refer to the NNS R package.**Assessment of conditional probability of deviation from means**This step assesses the conditional probability of deviation of two centrality measures (or any other two continuous variables) from their corresponding means in opposite directions.- The
`centrality2`

variable is considered as the condition variable and the other (`centrality1`

) as the desired one. - As you will see in the results, the whole data is also randomly splitted into half in order to further test the validity of conditional probability assessments.
*The higher the conditional probability the more two centrality measures behave in contrary manners*.

- The

```
# Preparing the data
<- centrality.measures
MyData
# Association assessment
<- double.cent.assess.noRegression(data = MyData,
My.metrics.assessment nodes.colname = rownames(MyData),
centrality1.colname = "BC",
centrality2.colname = "NC")
print(My.metrics.assessment)
#> $Summary_statistics
#> BC NC
#> Min. 0.000000000 1.2000
#> 1st Qu. 0.000000000 66.0000
#> Median 0.000000000 156.0000
#> Mean 0.005813357 132.3443
#> 3rd Qu. 0.000340000 179.3214
#> Max. 0.529464720 192.0000
#>
#> $Normality_results
#> p.value
#> BC 1.415450e-50
#> NC 9.411737e-30
#>
#> $Centrality1_Normality
#> [1] "Non-normally distributed"
#>
#> $Centrality2_Normality
#> [1] "Non-normally distributed"
#>
#> $HoeffdingD_Statistic
#> D_statistic P_value
#> Results 0.01770279 1e-08
#>
#> $Dependence_Significance
#> Hoeffding
#> Results Significantly dependent
#>
#> $NNS_dep_results
#> Correlation Dependence
#> Results -0.7948106 0.8647164
#>
#> $ConditionalProbability
#> [1] 55.35386
#>
#> $ConditionalProbability_split.half.sample
#> [1] 55.68163
```

`influential`

network nodes**IVI** : `IVI`

is the first integrative method for the identification of network most influential nodes in a way that captures all network topological dimensions. The `IVI`

formula integrates the most important local (i.e. degree centrality and ClusterRank), semi-local (i.e. neighborhood connectivity and local H-index) and global (i.e. betweenness centrality and collective influence) centrality measures in such a way that both synergizes their effects and removes their biases.

```
# Preparing the data
<- centrality.measures
MyData
# Calculation of IVI
<- ivi.from.indices(DC = MyData$DC,
My.vertices.IVI CR = MyData$CR,
NC = MyData$NC,
LH_index = MyData$LH_index,
BC = MyData$BC,
CI = MyData$CI)
head(My.vertices.IVI)
#> [1] 24.670056 8.344337 18.621049 1.017768 29.437028 33.512598
```

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculation of IVI
<- ivi(graph = My_graph, vertices = GraphVertices,
My.vertices.IVI weights = NULL, directed = FALSE, mode = "all",
loops = TRUE, d = 3, scaled = TRUE)
head(My.vertices.IVI)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 39.53878 19.94999 38.20524 1.12371 100.00000 47.49356
```

IVI could be also calculated for *directed* and/or *weighted* graphs via specifying the `directed`

, `mode`

, and `weights`

parameters.

Check out our paper^{5} for a more complete description of the IVI formula and all of its underpinning methods and analyses.

The following tutorial video demonstrates how to simply calculate the IVI value of all of the nodes within a network.

The `cent_network.vis`

is a function for the visualization of a network based on applying a centrality measure to the size and color of network nodes. The centrality of network nodes could be calculated by any means and based on any centrality index. Here, we demonstrate the visualization of a network according to IVI values.

```
# Reconstructing the graph
set.seed(70)
<- igraph::sample_gnm(n = 50, m = 120, directed = TRUE)
My_graph
# Calculating the IVI values
<- ivi(My_graph, directed = TRUE)
My_graph_IVI
# Visualizing the graph based on IVI values
<- cent_network.vis(graph = My_graph,
My_graph_IVI_Vis cent.metric = My_graph_IVI,
directed = TRUE,
plot.title = "IVI-based Network",
legend.title = "IVI value")
My_graph_IVI_Vis
```

The above figure illustrates a simple use case of the function `cent_network.vis`

. You can apply this function to directed/undirected and/or weighted/unweighted networks. Also, a complete flexibility (list of arguments) have been provided for the adjustment of colors, transparencies, sizes, titles, etc. Additionally, several different layouts have been provided that could be conveniently applied to a network.

In the case of highly crowded networks, the “

grid” layout would be most appropriate.

The following tutorial video demonstrates how to visualize a network based on the centrality of nodes (e.g. their `IVI`

values).

A shiny app has also been developed for the calculation of IVI as well as IVI-based network visualization, which is accessible using the following command.

`influential::runShinyApp("IVI")`

You can also access the shiny app online at the Influential Software Package server.

Sometimes we seek to identify not necessarily the most influential nodes but the nodes with most potential in spreading of information throughout the network.

**Spreading score** : `spreading.score`

is an integrative score made up of four different centrality measures including ClusterRank, neighborhood connectivity, betweenness centrality, and collective influence. Also, Spreading score reflects the spreading potential of each node within a network and is one of the major components of the `IVI`

.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculation of Spreading score
<- spreading.score(graph = My_graph,
Spreading.score vertices = GraphVertices,
weights = NULL, directed = FALSE, mode = "all",
loops = TRUE, d = 3, scaled = TRUE)
head(Spreading.score)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 42.932497 38.094111 45.114648 1.587262 100.000000 49.193292
```

Spreading score could be also calculated for *directed* and/or *weighted* graphs via specifying the `directed`

, `mode`

, and `weights`

parameters. The results could be conveniently illustrated using the centrality-based network visualization function.

In some cases we want to identify not the nodes with the most sovereignty in their surrounding local environments.

**Hubness score** : `hubness.score`

is an integrative score made up of two different centrality measures including degree centrality and local H-index. Also, Hubness score reflects the power of each node in its surrounding environment and is one of the major components of the `IVI`

.

```
# Preparing the data
<- coexpression.data
MyData
# Reconstructing the graph
<- graph_from_data_frame(MyData)
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculation of Hubness score
<- hubness.score(graph = My_graph,
Hubness.score vertices = GraphVertices,
directed = FALSE, mode = "all",
loops = TRUE, scaled = TRUE)
head(Hubness.score)
#> ADAMTS9-AS2 C8orf34-AS1 CADM3-AS1 FAM83A-AS1 FENDRR LANCL1-AS1
#> 84.299719 46.741660 77.441514 8.437142 92.870451 88.734131
```

Spreading score could be also calculated for *directed* graphs via specifying the `directed`

and `mode`

parameters. The results could be conveniently illustrated using the centrality-based network visualization function.

`SIRIR`

model**SIRIR** : `SIRIR`

is achieved by the integration of susceptible-infected-recovered (SIR) model with the leave-one-out cross validation technique and ranks network nodes based on their true universal influence on the network topology and spread of information. One of the applications of this function is the assessment of performance of a novel algorithm in identification of network influential nodes.

```
# Reconstructing the graph
<- sif2igraph(Path = "Sample_SIF.sif")
My_graph
# Extracting the vertices
<- V(My_graph)
GraphVertices
# Calculation of influence rank
<- sirir(graph = My_graph,
Influence.Ranks vertices = GraphVertices,
beta = 0.5, gamma = 1, no.sim = 10, seed = 1234)
```

difference.value | rank | |
---|---|---|

MRAP | 49.7 | 1 |

FOXM1 | 49.5 | 2 |

ATAD2 | 49.5 | 2 |

POSTN | 49.4 | 4 |

CDC7 | 49.3 | 5 |

ZWINT | 42.1 | 6 |

MKI67 | 41.9 | 7 |

FN1 | 41.9 | 7 |

ASPM | 41.8 | 9 |

ANLN | 41.8 | 9 |

**ExIR** : `ExIR`

is a model for the classification and ranking of top candidate features. The input data could come from any type of experiment such as transcriptomics and proteomics. This model is based on multi-level filtration and scoring based on several supervised and unsupervised analyses followed by the classification and integrative ranking of top candidate features. Using this function and depending on the input data and specified arguments, the user can get a graph object and one to four tables including:

**Drivers**: Prioritized drivers are supposed to have the highest impact on the progression of a biological process or disease under investigation.**Biomarkers**: Prioritized biomarkers are supposed to have the highest sensitivity to different conditions under investigation and the severity of each condition.**DE-mediators**: Prioritized DE-mediators are those features that are differentially expressed/abundant but in a fluctuating manner and play mediatory roles between drivers.**nonDE-mediators**: Prioritized nonDE-mediators are those features that are not differentially expressed/abundant but have associations with and play mediatory roles between drivers.

First, prepare your data. Suppose we have the data for time-course transcriptomics and we have previously performed differential expression analysis for each step-wise pair of time-points. Also, we have performed trajectory analysis to identify the genes that have significant alterations across all time-points.

```
# Prepare sample data
<- paste("gene", c(1:20000), sep = "_")
gene.names
set.seed(60)
<- data.frame(logFC = rnorm(n = 700, mean = 2, sd = 4),
tp2.vs.tp1.DEGs FDR = runif(n = 700, min = 0.0001, max = 0.049))
set.seed(60)
rownames(tp2.vs.tp1.DEGs) <- sample(gene.names, size = 700)
set.seed(70)
<- data.frame(logFC = rnorm(n = 1300, mean = -1, sd = 5),
tp3.vs.tp2.DEGs FDR = runif(n = 1300, min = 0.0011, max = 0.039))
set.seed(70)
rownames(tp3.vs.tp2.DEGs) <- sample(gene.names, size = 1300)
set.seed(80)
<- data.frame(R_squared = runif(n = 800, min = 0.1, max = 0.85))
regression.data
set.seed(80)
rownames(regression.data) <- sample(gene.names, size = 800)
```

Use the function `diff_data.assembly`

to automatically generate the Diff_data table for the `ExIR`

model.

```
<- diff_data.assembly(tp2.vs.tp1.DEGs,
my_Diff_data
tp3.vs.tp2.DEGs,
regression.data)
c(1:10),] my_Diff_data[
```

Have a look at the top 10 rows of the `Diff_data`

data frame:

Diff_value1 | Sig_value1 | Diff_value2 | Sig_value2 | Diff_value3 | |
---|---|---|---|---|---|

gene_17331 | 4.9 | 0 | 0 | 1 | 0 |

gene_12546 | 4.0 | 0 | 0 | 1 | 0 |

gene_12837 | -0.3 | 0 | 0 | 1 | 0 |

gene_18522 | 1.4 | 0 | 0 | 1 | 0 |

gene_6260 | -4.9 | 0 | 0 | 1 | 0 |

gene_2722 | -4.9 | 0 | 0 | 1 | 0 |

gene_19882 | 6.3 | 0 | 0 | 1 | 0 |

gene_2790 | 3.3 | 0 | 0 | 1 | 0 |

gene_17011 | -1.6 | 0 | 0 | 1 | 0 |

gene_8321 | 3.8 | 0 | 0 | 1 | 0 |

Now, prepare a sample normalized experimental data matrix

```
set.seed(60)
<- matrix(data = runif(n = 1000000, min = 2, max = 300),
MyExptl_data nrow = 50, ncol = 20000,
dimnames = list(c(paste("cancer_sample", c(1:25), sep = "_"),
paste("normal_sample", c(1:25), sep = "_")),
gene.names))
# Log transform the data to bring them closer to normal distribution
<- log2(MyExptl_data)
MyExptl_data
c(1:5, 45:50),c(1:5)] MyExptl_data[
```

Have a look at top 5 cancer and normal samples (rows) of the `Exptl_data`

:

gene_1 | gene_2 | gene_3 | gene_4 | gene_5 | |
---|---|---|---|---|---|

cancer_sample_1 | 8 | 8 | 8 | 8 | 8 |

cancer_sample_2 | 7 | 8 | 6 | 8 | 8 |

cancer_sample_3 | 8 | 7 | 8 | 7 | 8 |

cancer_sample_4 | 8 | 7 | 7 | 7 | 8 |

cancer_sample_5 | 6 | 4 | 8 | 5 | 4 |

normal_sample_20 | 8 | 7 | 7 | 8 | 8 |

normal_sample_21 | 8 | 7 | 8 | 6 | 8 |

normal_sample_22 | 8 | 8 | 8 | 7 | 6 |

normal_sample_23 | 7 | 6 | 8 | 7 | 8 |

normal_sample_24 | 8 | 8 | 7 | 5 | 7 |

normal_sample_25 | 5 | 7 | 8 | 8 | 6 |

Now add the “condition” column to the Exptl_data table.

```
<- as.data.frame(MyExptl_data)
MyExptl_data $condition <- c(rep("C", 25), rep("N", 25)) MyExptl_data
```

`ExIR`

modelFinally, prepare the other required input data for the `ExIR`

model.

```
#The table of differential/regression previously prepared
my_Diff_data
#The column indices of differential values in the Diff_data table
<- c(1,3)
Diff_value
#The column indices of regression values in the Diff_data table
<- 5
Regr_value
#The column indices of significance (P-value/FDR) values in
# the Diff_data table
<- c(2,4)
Sig_value
#The matrix/data frame of normalized experimental
# data previously prepared
MyExptl_data
#The name of the column delineating the conditions of
# samples in the Exptl_data matrix
<- "condition"
Condition_colname
#The desired list of features
set.seed(60)
<- sample(gene.names, size = 1000) #Optional
MyDesired_list
#Running the ExIR model
<- exir(Desired_list = MyDesired_list,
My.exir Diff_data = my_Diff_data, Diff_value = Diff_value,
Regr_value = Regr_value, Sig_value = Sig_value,
Exptl_data = MyExptl_data, Condition_colname = Condition_colname,
seed = 60, verbose = FALSE)
names(My.exir)
#> [1] "Driver table" "DE-mediator table" "nonDE-mediator table" "Biomarker table" "Graph"
class(My.exir)
#> [1] "ExIR_Result"
```

Have a look at the heads of the output tables of ExIR:

**Drivers**

Score | Z.score | Rank | P.value | P.adj | Type | |
---|---|---|---|---|---|---|

gene_17331 | 4.837927 | -0.03142096 | 135 | 0.5125331 | 0.6623473 | Accelerator |

gene_12546 | 1.769802 | -0.38897583 | 659 | 0.6513530 | 0.6623473 | Accelerator |

gene_12837 | 6.631165 | 0.17756047 | 93 | 0.4295341 | 0.6623473 | Decelerator |

gene_18522 | 2.126212 | -0.34744030 | 467 | 0.6358697 | 0.6623473 | Accelerator |

gene_6260 | 1.927154 | -0.37063825 | 551 | 0.6445465 | 0.6623473 | Decelerator |

gene_2722 | 3.066216 | -0.23789354 | 326 | 0.5940182 | 0.6623473 | Decelerator |

**Biomarkers**

Score | Z.score | Rank | P.value | P.adj | Type | |
---|---|---|---|---|---|---|

gene_17331 | 2.612152 | 0.34642339 | 11 | 0.3645123 | 0.5264108 | Up-regulated |

gene_12546 | 1.000005 | -0.06624912 | 539 | 0.5264103 | 0.5264108 | Up-regulated |

gene_12837 | 1.000735 | -0.06606216 | 352 | 0.5263358 | 0.5264108 | Down-regulated |

gene_18522 | 1.000003 | -0.06624973 | 615 | 0.5264105 | 0.5264108 | Up-regulated |

gene_6260 | 1.000007 | -0.06624851 | 477 | 0.5264100 | 0.5264108 | Down-regulated |

gene_2722 | 1.001196 | -0.06594418 | 339 | 0.5262889 | 0.5264108 | Down-regulated |

**nonDE-mediators**

Score | Z.score | Rank | P.value | P.adj | |
---|---|---|---|---|---|

gene_12988 | 1.000000 | -0.1219667 | 46 | 0.5485373 | 0.5485373 |

gene_9173 | 1.000670 | -0.1218875 | 44 | 0.5485059 | 0.5485373 |

gene_5014 | 1.022135 | -0.1193471 | 33 | 0.5474998 | 0.5485373 |

gene_7165 | 1.000000 | -0.1219667 | 46 | 0.5485373 | 0.5485373 |

gene_2687 | 1.000000 | -0.1219667 | 46 | 0.5485373 | 0.5485373 |

gene_1873 | 1.000000 | -0.1219667 | 46 | 0.5485373 | 0.5485373 |

**DE-mediators**

Score | Z.score | Rank | P.value | P.adj | |
---|---|---|---|---|---|

gene_10754 | 62.570886 | 0.93490127 | 2 | 0.1749196 | 0.7871382 |

gene_9479 | 30.044859 | -0.02057836 | 4 | 0.5082090 | 0.8088852 |

gene_18046 | 49.518892 | 0.55148787 | 3 | 0.2906496 | 0.8088852 |

gene_18954 | 1.802499 | -0.85022165 | 8 | 0.8023991 | 0.8088852 |

gene_13827 | 7.488785 | -0.68318215 | 6 | 0.7527541 | 0.8088852 |

gene_1992 | 1.000000 | -0.87379573 | 9 | 0.8088852 | 0.8088852 |

The following tutorial video demonstrates how to run the `ExIR`

model on a sample experimental data.

You can also computationally simulate knockout and/or up-regulation of the top candidate features outputted by ExIR to evaluate the impact of their manipulations on the flow of information/signaling and integrity of the network prior to taking them to your lab bench.

The `exir.vis`

is a function for the visualization of the output of the ExIR model. The function simply gets the output of the ExIR model as a single argument and returns a plot of the top 10 prioritized features of all classes. Here, we visualize the top five candidates of the results of the `ExIR`

model obtained in the previous step .

```
<- exir.vis(exir.results = My.exir,
My.exir.Vis n = 5,
y.axis.title = "Gene")
My.exir.Vis
```

However, a complete flexibility (list of arguments) has been provided for the adjustment of all of the visual features of the plot and selection of the desired classes, feature types, and the number of top candidates.

The following tutorial video demonstrates how to visualize the results of `ExIR`

model.

A shiny app has also been developed for Running the ExIR model, visualization of its results as well as computational simulation of knockout and/or up-regulation of its top candidate outputs, which is accessible using the following command.

`influential::runShinyApp("ExIR")`

You can also access the shiny app online at the Influential Software Package server.

The `comp_manipulate`

is a function for the simulation of feature (gene, protein, etc.) knockout and/or up-regulation in cells. This function works based on the SIRIR (SIR-based Influence Ranking) model and could be applied on the output of the ExIR model or any other independent association network. For feature (gene/protein/etc.) knockout the SIRIR model is used to remove the feature from the network and assess its impact on the flow of information (signaling) within the network. On the other hand, in case of up-regulation a node similar to the desired node is added to the network with exactly the same connections (edges) as of the original node. Next, the SIRIR model is used to evaluate the difference in the flow of information/signaling after adding (up-regulating) the desired feature/node compared with the original network. In case you are applying this function on the output of ExIR model, you may note that as the gene/protein knockout would impact on the integrity of the under-investigation network as well as the networks of other overlapping biological processes/pathways, it is recommended to select those features that simultaneously have the highest (most significant) ExIR-based rank and lowest knockout rank. In contrast, as the up-regulation would not affect the integrity of the network, you may select the features with highest (most significant) ExIR-based and up-regulation-based ranks. Altogether, it is recommended to select the features with the highest (most significant) ExIR-based (major drivers or mediators of the under-investigation biological process/disease) and *Up-regulation*-based (having higher impact on the signaling within the under-investigation network when up-regulated) ranks, but with the lowest *Knockout*-based rank (having the lowest disturbance to the under-investigation as well as other overlapping networks). Below is an example of running this function on the same ExIR output generated above.

```
# Select which genes to knockout
set.seed(60)
<- sample(igraph::as_ids(V(My.exir$Graph)), size = 5)
ko_vertices
# Select which genes to up-regulate
set.seed(1234)
<- sample(igraph::as_ids(V(My.exir$Graph)), size = 5)
upregulate_vertices
<- comp_manipulate(exir_output = My.exir,
Computational_manipulation ko_vertices = ko_vertices,
upregulate_vertices = upregulate_vertices,
beta = 0.5, gamma = 1, no.sim = 100, seed = 1234)
```

Have a look at the heads of the output tables:

**Knockout**

Feature_name | Rank | Manipulation_type | |
---|---|---|---|

2 | gene_280 | 1 | Knockout |

1 | gene_4798 | 2 | Knockout |

4 | gene_276 | 3 | Knockout |

3 | gene_16459 | 4 | Knockout |

5 | gene_7535 | 5 | Knockout |

**Up-regulation**

Feature_name | Rank | Manipulation_type |
---|---|---|

gene_6433 | 1 | Up-regulation |

gene_8426 | 1 | Up-regulation |

gene_6687 | 1 | Up-regulation |

gene_1274 | 1 | Up-regulation |

gene_11555 | 1 | Up-regulation |

**Combined**

Feature_name | Rank | Manipulation_type | |
---|---|---|---|

2 | gene_280 | 1 | Knockout |

1 | gene_4798 | 2 | Knockout |

4 | gene_276 | 3 | Knockout |

3 | gene_16459 | 4 | Knockout |

11 | gene_6433 | 5 | Up-regulation |

21 | gene_8426 | 5 | Up-regulation |

31 | gene_6687 | 5 | Up-regulation |

41 | gene_1274 | 5 | Up-regulation |

51 | gene_11555 | 5 | Up-regulation |

5 | gene_7535 | 10 | Knockout |

Csardi G., Nepusz T.

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*Survival analysis and functional annotation of long non-coding RNAs in lung adenocarcinoma*. J Cell Mol Med. 2019;23:5600–5617. (PMID: 31211495)↩︎Maslov S., Sneppen K.

*Specificity and stability in topology of protein networks*. Science. 2002; 296: 910-913 (PMID:11988575)↩︎Salavaty A, Ramialison M, Currie PD.

*Integrated Value of Influence: An Integrative Method for the Identification of the Most Influential Nodes within Networks*. Patterns. 2020.08.14. (Read online)↩︎