---
title: |
  | Epistatic Nested Effects Models:
  | Inferring mixed epistasis from indirect measurements of knock-out screens
author: "Martin Pirkl, Madeline Diekmann, Marlies van der Wees, Niko Beerenwinkel, Holger Fröhlich, Florian Markowetz"
date: "`r Sys.Date()`"
graphics: yes
header-includes:
    - \usepackage{xcolor}
output: BiocStyle::html_document
vignette: >
    %\VignetteIndexEntry{epiNEM}
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding{UTF-8}
---

# Introduction

The method in this package is an extension of the classic Nested Effects
Models provided in the package \emph{nem}. Nested Effects Models is a pathway
reconstruction method, which takes into account effects of downstream genes.
Those effects are observed for every knock-out of an upstream pathway gene, and
the nested structure of observed effects can then be used to reconstruct the
pathway structure.
However, classic Nested Effects Models do not account for double knock-outs.
In this package \emph{epiNEM}, one additional layer of complexity is added.
For every two genes, acting on one gene together, the relationship is evaluated
and added to the model as a logical gate. Genetic relationships are represented
by the logics OR (no relationship), AND (functional overlap),
NOT (masking or inhibiting) and XOR (mutual prevention from acting on gene C).
Please see the references for a more detailed description of NEMs and epiNEMs.

Simulation and application results are imported from pre-calculated data sets
to shorten the runtime of this vignette.

# Loading epiNEM

```{r, global_options, include=FALSE}
knitr::opts_chunk$set(message=FALSE)
fig.cap1 <- "Toy example result for a double mutant (A,B) and one modulator
(C)."
fig.cap2 <- "Distribution of inferred logics for each double knock-out."
fig.cap3 <- "Ranked modulators for one double knock-out."
fig.cap4 <- "Perfect binary effects matrix for each logic."
fig.cap5 <- ""
fig.cap6 <- "Global results for the van Wageningen data set. See the help pages
of plot.epiScreen() and HeatmapOP() for additional parameters."
fig.cap7 <- "Results for one double knock-out of the van Wageningen data set.
See ?plot.epiScreen for further parameters."
fig.cap8 <- "Global result for the Sameith data set."
fig.cap9 <- "Example for one knock-out of the Sameith data set."
fig.cap10 <- "Density of the string-db interaction scores (van Wageningen).
Background (turqoise) and inferred by epiNEM (pink)."
fig.cap11 <- "Density of the string-dbinteraction scores (Sameith). Baackground
(turqoise) and inferred by epiNEM (pink)."
fig.cap12 <- "Density plot for graph-based GO similarity score
(van Wageningen)."
fig.cap13 <- "Density plot for graph-based GO similarity score (Sameith)."
fig.cap14 <- "Enrichment of van Wageningen modulators by KEGG pathways.
Colors refer to false discovery rates. NAs are colored in grey."
fig.cap15 <- "Enrichment of Sameith modulators by KEGG pathways. Colors
refer to false discovery rates. NAs are colored in grey."
fig.cap16 <- "Effect reporter KEGG pathway enrichment (van Wageningen).
Colors refer to false discovery rates. NAs are colored in grey."
fig.cap17 <- "Effect reporter KEGG pathway enrichment (Sameith). Colors
refer to false discovery rates. NAs are colored in grey."
```
```{r}
library(epiNEM)
```

# Quick start

The data should be in the form of binary effects stemming from
knock-out data including single and double perturbations with
effect reporters, e.g. genes, as rows and perturbations as columns.
A one in row i and column j denotes an effect of reporter i for
perturbation j. These effects are usually derived from comparing
control/wild-type experiments with respective perturbation experiments
(e.g. differential expression with edgeR, Robinson *et al.*, 2010).
The binary data is the main input for the epiNEM function, which
contains the inference algorithm to estimate the underlying network
structure of the data. For up to four signalling genes (=different
perturbation targets) exhaustive search is available. For five or
more a greedy search is implemented as an alternative.

```{r}
data <- matrix(sample(c(0,1), 100*4, replace = TRUE), 100, 4)
colnames(data) <- c("A", "A.B", "B", "C")
rownames(data) <- paste("E", 1:100, sep = "_")
print(head(data))
res <- epiNEM(data, method = "exhaustive")
```
```{r, fig.width = 7, fig.height = 7, fig.cap=fig.cap1}
plot(res)
```

The plot shows the inferred network with the signaling genes (bright red),
the inferred logic (green) and attachment of the number of effect
reporters (grey).

Alternatively the input can be a larger matrix with single and double
knock-outs. EpiNEM can perform a systematic analysis to identify most
likely modulators for a signaling gene pair of a double knock-out.

```{r}
data <- matrix(sample(c(0,1), 100*9, replace = TRUE), 100, 9)
colnames(data) <- c("A.B", "A.C", "B.C", "A", "B", "C", "D", "E", "G")
rownames(data) <- paste("E", 1:100, sep = "_")
res <- epiScreen(data)
```
```{r, fig.width = 4, fig.height = 4, fig.cap = fig.cap2}
plot(res)
```
```{r, fig.width = 5, fig.height = 4, fig.cap = fig.cap3}
plot(res, global = FALSE, ind = 1)
```

If the "global" parameter is set to FALSE, detailed results are plotted
for each or a specific ("ind" parameter) pair of a double knock-out.

The results (logics) of the knock-out screens have been annotated
according to the following legend of effects (black), where the
x-axis denotes the gene and the y-axis denotes the type of knock-out. E.g.
if C is regulated via the OR gate, it is effected by by both single and
the double knock-out.

```{r, warning=FALSE, fig.width = 9, fig.height=5, fig.cap = fig.cap4}
epiAnno()
```

# Simulations

We compare epiNEM to original NEMs (Markowetz *et al.*, 2005), Boolean Nested
Effects Models (B-NEM, [link](https://github.com/MartinFXP/B-NEM),
Pirkl *et al.*, 2016), Aracne (Margolin *et al.*, 2006) and the PC algorithm
(Kalisch *et al.*, 2007) for 100 four node networks and respective data
experiencing different levels of noise. The evaluation is done by accounting
for the accuracy of discovered edges and in the case of B-NEM and epiNEM also
the truth tables and logical gates.

We simulate code based on a ground truth and infer an optimal network
with all aforementioned methods solely based on the data.
The B-NEM package is necessary to run the full simulation:
[link](https://github.com/MartinFXP/B-NEM).
If the comparison to B-NEM is omitted, the B-NEM package is not
necessary for epiNEM to function.

B-NEM is a method for general Boolean network inference based on multivariate
read-out from combinatorial perturbation experiments. EpiNEM is specifically
designed for knock-out screens including double and single knock-outs. Thus
epiNEM is faster especially for large scale screens and more
accurate, because it is designed for triples.

We boxplot running time, accuracy for the inferred edges,
logical gates and expected data (truth table) for all five respectively
two methods.

```{r}
data(sim)
```
```{r, fig.width = 7, fig.height = 7, fig.cap = "Simulation results."}
plot(sim)
```

The plot shows that as expected B-NEM is the slowest followed by epiNEM,
while NEM, ARACNE and the PC algorithm are much faster. However, the last
three can not derive any logics in the network structure. epiNEM and
B-NEM show almost equally high accuracy of inferred edges and truth tables
(expected data). However, epiNEM reaches a much higher accuracy for inferred
logic gates, which is due to the much higher degree of equivalent network
structures in the case of B-NEM.

# Yeast knock-out screens

In this section we analyse previously published yeast knock-out
screens. The screens consist of gene expression data derived from
double and single knock-out mutants. We use epiNEM on each double
mutant combined with each single mutant.

## Identification of significant modulators for pairs of double knock-outs

The first knock-out screen is from van Wageningen et al., 2010 and
was created to investigate relationships between phosphorylation based pathways.

We applied epiNEM to all triples and infer logics and calculated the
log-likelihood.

The following plot shows the global distribution of the inferred logical gates
for all pairs. The single modulators are on the y-axis and the double
knock-out pairs are on the x-axis.

```{r, fig.width = 8, fig.height = 4, fig.cap = fig.cap6}
data(wagscreen)

doubles <- wagscreen$doubles

dataWag <- wagscreen$dataWag

## clean up the results:

if (length(grep("fus3|ptp2.ptc2", wagscreen$doubles)) > 0) {
    wagscreen$doubles <- wagscreen$doubles[-grep("fus3|ptp2.ptc2",
                                                 wagscreen$doubles)]
    
    wagscreen$dataWag <- wagscreen$dataWag[, -grep("fus3|ptp2.ptc2",
                                                   colnames(wagscreen$dataWag))]
    
    wagscreen$ll <- wagscreen$ll[, -grep("fus3|ptp2.ptc2",
                                         colnames(wagscreen$ll))]
    
    wagscreen$logic <- wagscreen$logic[, -grep("fus3|ptp2.ptc2",
                                               colnames(wagscreen$logic))]
}

plot(wagscreen, xrot = 45, borderwidth = 0)
```

We can also plot the significant results for every explicit triple with
log-likelihoods on the y-axis and ranked triples on the x-axis. The
"ind" parameter, if set, selects a subset, i.e. double knock-out.

```{r, fig.width = 8, fig.height = 4, fig.cap = fig.cap7}
plot(wagscreen, global = FALSE, ind = 3, cexGene = 0.7, cexLegend = 0.9,
     off = 0.2)
```

The second knock-out screen by Sameith et al., 2015 was created to
investigate growth based genetic interactions.

Again, we plot the global distribution of logics followed by
the explicit results for every identified modulator of one double
knock-out.

```{r, fig.width = 10, fig.height = 6, fig.cap = fig.cap8}
data(samscreen)

doubles <- samscreen$doubles

dataSam <- samscreen$dataSam

plot(samscreen, xrot = 45, cexCol = 0.6, borderwidth = 0)
```
```{r, fig.width = 8, fig.height = 4, fig.cap = fig.cap9}
plot(samscreen, global = FALSE, ind = 23, cexGene = 0.7, cexLegend = 0.9,
     off = 0.2)
```

## Validation of identified interactions

### String-db interactions

We plot the densities of the string-db interaction scores of our
identified modulators and the background density to visualize the significance
of the interaction identified by epiNEM.

We plot the densities of the distributions of the background distribution (blue)
and the scores of a pair from the triplets by epiNEM (pink).
Additionally we calculate, if the difference between the two score
distributions is significant (Mann-Whitney test).

We do this for both the van Wageningen et al., and the Sameith et al.,
data sets.

```{r, fig.width = 8, fig.height = 5, fig.cap = fig.cap10}
library(STRINGdb)

## get_STRING_species(version="10", species_name=NULL)[26, ] # 4932

string_db <- STRINGdb$new( version="11", species=4932, score_threshold=0,
                          input_directory="")
data(wageningen_string)

string.scores <- wageningen_string$string.scores
string.names <- wageningen_string$string.names

tmp <- string_db$get_interactions(
    string_db$mp(unique(unlist(strsplit(colnames(dataWag), "\\.")))))

stsc <- unlist(string.scores)

denspval <- wilcox.test(stsc, unlist(tmp$combined_score),
                        alternative = "greater")$p.value

for (i in 100:1) {

    if (denspval < 10^(-i)) {

        denspval <- paste("< ", 10^(-i), sep = "")

    } 

}

plot(density(stsc), col = "#00000000",
     ylim = c(0, max(c(max(density(stsc)$y),
                       max(density(unlist(tmp$combined_score))$y)))),
     main = paste("van Wageningen String-db interaction scores", sep = ""),
     xlab = "",
     cex.main = 1.5)
polygon(density(stsc), col = "#ff000066")

legend("topright", legend=paste("p-value", denspval, " "), cex = 1.5)

mtext = mtext("A", side = 3, line = 1, outer = FALSE, cex = 3, adj = 0,
              at = par("usr")[1] - (par("usr")[2]-par("usr")[1])*0.1)

lines(density(unlist(tmp$combined_score)), col = "#00000000")
polygon(density(unlist(tmp$combined_score)), col = "#00ffff66")

data(sameith_string)

string.scores2 <- sameith_string$string.scores2
string.names2 <- sameith_string$string.names2

tmp <- string_db$get_interactions(
    string_db$mp(unique(unlist(strsplit(colnames(dataSam), "\\.")))))

stsc <- unlist(string.scores2)

denspval <- wilcox.test(stsc, unlist(tmp$combined_score),
                        alternative = "greater")$p.value

for (i in 100:1) {

    if (denspval < 10^(-i)) {

        denspval <- paste("< ", 10^(-i), sep = "")

    } 

}

plot(density(stsc), col = "#00000000",
     ylim = c(0, max(c(max(density(stsc)$y),
                       max(density(unlist(tmp$combined_score))$y)))),
     main = paste("Sameith String-db interaction scores", sp = ""),
     xlab = "",
     cex.main = 1.5)
polygon(density(stsc), col = "#ff000066")

legend("topright", legend=paste("p-value", denspval, " "), cex = 1.5)

mtext = mtext("B", side = 3, line = 1, outer = FALSE, cex = 3, adj = 0,
              at = par("usr")[1] - (par("usr")[2]-par("usr")[1])*0.1)

lines(density(unlist(tmp$combined_score)), col = "#00000000")
polygon(density(unlist(tmp$combined_score)), col = "#00ffff66")
```

### Graph based GO similarity scores

We perform a similar analysis to the STRING interactions with a graph-based
GO term similarity score (Wang et al., 2007) implemented in the R-package
GOSemSim (Yu et al., 2010). This score is more flexible and allows us to
calculate the similarity of GO terms between the parent double knock-out
pair and the identified modulator instead of just pairwise interactions. For
the average GO term similarity epiNEMs did not identify more significant
interactions. However, the maximum similar GO term between parents and modulator
is significantly higher on average than by random guessing (Fig. 14, 15).

```{r, fig.width = 8, fig.height = 5, fig.cap = fig.cap12}
data(wageningen_GO)

GOepi <- wageningen_GO$epi
GOall <- wageningen_GO$all

denspval <- wilcox.test(GOepi, GOall, alternative = "greater")$p.value

for (i in 100:1) {
    if (i <= 2) {
        for (j in 1:9) {
            if (denspval < j*10^(-i)) {
                denspval <- paste("< ", j*10^(-i), sep = "")
            }
        }
    } else {
        if (denspval < 10^(-i)) {
            denspval <- paste("< ", 10^(-i), sep = "")
        }
    }
}

plot(density(GOepi), col = "#00000000",
     ylim = c(0, max(c(max(density(GOepi)$y),
                       max(density(unlist(GOall))$y)))),
     main = "van Wageningen Go similarity scores",
     xlab = "",
     cex.main = 1.5)
polygon(density(GOepi), col = "#ff000066")

legend("topleft", legend=paste("p-value", denspval, "   "), cex = 1.5)

mtext = mtext("C", side = 3, line = 1, outer = FALSE, cex = 3, adj = 0,
              at = par("usr")[1] - (par("usr")[2]-par("usr")[1])*0.1)

lines(density(unlist(GOall)), col = "#00000000")
polygon(density(unlist(GOall)), col = "#00ffff66")
```
```{r, fig.width = 8, fig.height = 5, fig.cap = fig.cap13}
data(sameith_GO)

GOepi2 <- sameith_GO$epi
GOall2 <- sameith_GO$all

denspval <- wilcox.test(GOepi2, GOall2, alternative = "greater")$p.value

for (i in 100:1) {
    if (i <= 2) {
        for (j in 1:9) {
            if (denspval < j*10^(-i)) {
                denspval <- paste("< ", j*10^(-i), sep = "")
            }
        }
    } else {
        if (denspval < 10^(-i)) {
            denspval <- paste("< ", 10^(-i), sep = "")
        }
    }
}

plot(density(GOepi2), col = "#00000000",
     ylim = c(0, max(c(max(density(GOepi2)$y),
                       max(density(unlist(GOall2))$y)))),
     main = "Sameith Go similarity scores",
     xlab = "",
     cex.main = 1.5)
polygon(density(GOepi2), col = "#ff000066")

legend("topleft", legend=paste("p-value", denspval, "   "), cex = 1.5)

mtext = mtext("D", side = 3, line = 1, outer = FALSE, cex = 3, adj = 0,
              at = par("usr")[1] - (par("usr")[2]-par("usr")[1])*0.1)

lines(density(unlist(GOall2)), col = "#00000000")
polygon(density(unlist(GOall2)), col = "#00ffff66")
```

## Enrichment analysis

### Enrichment of identified sets of modulators for each double knock-out

We further analyze the set of identified modulators for each pair by KEGG
pathway enrichment. Fig. 14 and 15 show heatmaps of false discovery rates.
Knock-out pairs are in the columns and enriched pathways in the rows. Red
denotes no enrichment. The most significant pathways are meiosis, cell cycle
and MAPK signaling, which are enriched for almost all pairs in both data sets.
Interestingly, these are the only pathways we find for the
Sameith \emph{et al.}, data set. For the van Wageningen \emph{et al.}, data set
we find several more pathways, which are only enriched in a subset of knock-out
pairs, e.g. glycerophospholipid metabolism is highly enriched for the
*ptp2,ptp3* and *hal5,sat4* double mutants only.

```{r, fig.width = 8, fig.height = 5, fig.cap = fig.cap14}
data(wageningen_GO)

golist <- wageningen_GO$golist

goterms <- character()

for (i in 1:length(golist)) {

    if (i %in% c(5,8)) { next() }

    goterms <- c(goterms,
                 golist[[i]]$term_description[which(golist[[i]]$pvalue_fdr
                                                    < 1)])

}

gomat <- matrix(NA, length(unique(goterms)), ncol(wagscreen$ll))

rownames(gomat) <- sort(unique(goterms))
colnames(gomat) <- colnames(wagscreen$ll)

for (i in 1:ncol(wagscreen$ll)) {

    gotmp <- golist[[i]]
    gotmp <- gotmp[order(gotmp$term_description), ]

    gomat[which(rownames(gomat) %in% golist[[i]]$term_description), i] <-
        golist[[i]][which(golist[[i]]$term_description %in% rownames(gomat)), 4]

}

if (nrow(gomat) > 20) {
    rownames(gomat) <- NULL
}

HeatmapOP(gomat,
          bordercol = "transparent",
          main = "", sub = "",
          xrot = 45, col = "RdYlBu", breaks = 100)
```
```{r, fig.width = 12, fig.height = 3, fig.cap = fig.cap15}
data(sameith_GO)

golist2 <- sameith_GO$golist

goterms <- character()

for (i in 1:length(golist2)) {

    goterms <-
        c(goterms,
          golist2[[i]]$term_description[which(golist2[[i]]$pvalue_fdr < 0.1)])

}

gomat <- matrix(NA, length(unique(goterms)), ncol(samscreen$ll))

rownames(gomat) <- sort(unique(goterms))
colnames(gomat) <- colnames(samscreen$ll)

for (i in 1:ncol(samscreen$ll)) {

    gotmp <- golist2[[i]]
    gotmp <- gotmp[order(gotmp$term_description), ]

    gomat[which(rownames(gomat) %in% golist2[[i]]$term_description), i] <-
        golist2[[i]][which(golist2[[i]]$term_description %in%
                           rownames(gomat)), 4]

}

if (nrow(gomat) > 20) {
    rownames(gomat) <- NULL
}

colnames(gomat) <- tolower(colnames(gomat))

HeatmapOP(gomat,
          bordercol = "transparent",
          main = "", sub = "",
          xrot = 45, cexCol = 0.5, col = "RdYlBu", breaks = 100)
```

### Enrichment of effect reporters for each identified modulator

EpiNEM calculates the a posteriori attachment for each effect reporter. Thus,
for each significant modulator and corresponding double mutant pair we can
identify genes directly regulated by that modulator. As a summary of enriched
GO terms, Fig. 16 and 17 show enriched pathways (rows) for effect reporters of
the respective modulators (columns). The false discovery rates are averaged
over all double mutant pairs which identified the modulator as significant.
Interestingly, we find the previously significant pathways like Meiosis and MAPK
signaling, but at a lower rate than before (Fig. 16). However, overall all
modulators in both data sets show strong enrichment in a similar set of
pathways.

```{r, fig.height = 7, fig.width = 14, fig.cap = fig.cap16}
gos <- unique(wageningen_GO$gos)

egenego <- wageningen_GO$egenego

gomat <- array(NA, c(length(gos), nrow(wagscreen$ll), ncol(wagscreen$ll)))

rownames(gomat) <- sort(gos)

colnames(gomat) <- rownames(wagscreen$ll)

dimnames(gomat)[[3]] <- colnames(wagscreen$ll)

for (i in 1:length(wagscreen$targets)) {
    if (length(wagscreen$targets[[i]]) == 0) { next() }
    for (j in 1:length(wagscreen$targets[[i]])) {
        if (dim(egenego[[i]][[j]])[1] > 0) {
            gomat[which(rownames(gomat) %in%
                        egenego[[i]][[j]]$term_description),
                  which(dimnames(gomat)[[2]] %in%
                        names(wagscreen$targets[[i]])[j]), i] <-
                egenego[[i]][[j]]$pvalue_fdr[
                                     order(egenego[[i]][[j]]$term_description)]
        }
    }
}

gomat <- apply(gomat, c(1,2), mean, na.rm = TRUE)

gomat <- gomat[order(apply(gomat, 1, function(x)
    return(sum(is.na(x) == FALSE)))), ]

gomat <- gomat[, rev(order(apply(gomat, 2, function(x)
    return(sum(is.na(x) == FALSE)))))]

gomat <- gomat[, which(apply(gomat, 2,
                             function(x) return(any(is.na(x) == FALSE))))]

HeatmapOP(gomat, xrot = 45, Colv = FALSE, Rowv = FALSE,
          col = "RdYlBu", main = "", sub = "", breaks = 100)
```
```{r, fig.height = 9, fig.width = 16, fig.cap = fig.cap17}
gos2 <- unique(sameith_GO$gos)

egenego2 <- sameith_GO$egenego

gomat <- array(NA, c(length(gos2), nrow(samscreen$ll), ncol(samscreen$ll)))

rownames(gomat) <- sort(gos2)

colnames(gomat) <- rownames(samscreen$ll)

dimnames(gomat)[[3]] <- colnames(samscreen$ll)

for (i in 1:length(samscreen$targets)) {
    if (length(samscreen$targets[[i]]) == 0) { next() }
    for (j in 1:length(samscreen$targets[[i]])) {
        if (dim(egenego2[[i]][[j]])[1] > 0) {
            gomat[which(rownames(gomat) %in%
                        egenego2[[i]][[j]]$term_description),
                  which(dimnames(gomat)[[2]] %in%
                        names(samscreen$targets[[i]])[j]), i] <-
                egenego2[[i]][[j]]$pvalue_fdr[
                                      order(
                                          egenego2[[i]][[j]]$term_description)]
        }
    }
}

gomat <- apply(gomat, c(1,2), mean, na.rm = TRUE)

gomat <- gomat[order(apply(gomat, 1, function(x)
    return(sum(is.na(x) == FALSE)))), ]

gomat <- gomat[, rev(order(apply(gomat, 2, function(x)
    return(sum(is.na(x) == FALSE)))))]

gomat <- gomat[, which(apply(gomat, 2,
                             function(x) return(any(is.na(x) == FALSE))))]

colnames(gomat) <- tolower(colnames(gomat))

HeatmapOP(gomat, xrot = 45, Colv = FALSE, Rowv = FALSE,
          col = "RdYlBu", main = "", sub = "", breaks = 100)
```

# Creation of data objects

In this section we show the R code, which was used to create the simulation and
application results.

```{r, eval=FALSE}
###### simulation:

## install_github("MartinFXP/B-NEM"); library(bnem)

library(nem)

library(minet)

library(pcalg)

runs <- 100

noiselvls <- c(0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5)

random <- list(FPrate = 0.1, FNrate = noiselvls,
               single = 4, double = 1, reporters = 100, replicates = 3)

do <- c("n", "p", "a", "e", "b")

maxTime <- FALSE

forcelogic <- TRUE

epinemsearch <- "greedy"

nIterations <- 3

bnemsearch <- "genetic"

simresults <- SimEpiNEM(runs, do, random, maxTime, forcelogic,
epinemsearch, bnemsearch, nIterations)

sim <- simresults

###### yeast van Wageningen et al.:

file <- paste("http://www.holstegelab.nl/",
              "publications/sv/signaling_redundancy/downloads/DataS1.txt",
              sep = "")

data <- read.delim(file)

dataM <- data[-(1), (1+(1:(324/2))*2)]

dataP <- data[-(1), (2+(1:(324/2))*2)]

data[, 2] <- as.character(data[, 2])

rndup <- which(duplicated(data[, 2]) == TRUE)

data[rndup, 2] <- paste(data[rndup, 2], "_dup", sep = "")

rownames(dataM) <- rownames(dataP) <- data[2:nrow(data), 2]

dataM <- dataM[-1, ]

dataP <- dataP[-1, ]

dataM <- apply(dataM, c(1,2), as.numeric)

dataP <- apply(dataP, c(1,2), as.numeric)

dataBin <- dataM

sig <- 0.05

cutoff <- log2(1.7)

dataBin[which(dataP < sig & dataP > 0 & abs(dataM) >= cutoff)] <- 1

dataBin[which(dataP >= sig | dataP == 0 | abs(dataM) < cutoff)] <- 0

dataBin <- dataBin[-which(apply(dataBin, 1, max) == 0), ]

dataBinWag <- dataBin

colnames(dataBin) <- gsub(".del.vs..wt", "", colnames(dataBin))

colnames(dataBin) <- gsub(".del", "", colnames(dataBin))

doubles <- colnames(dataBin)[grep("\\.", colnames(dataBin))]

if (length(grep("vs", doubles)) > 0) {
    doubles <- sort(doubles[-grep("vs", doubles)])
} else { doubles <- sort(doubles) }

doubles.genes <- unique(unlist(strsplit(doubles, "\\.")))

if (length(grep("\\.", colnames(dataBin))) > 0) {
    singles <- colnames(dataBin)[-grep("\\.", colnames(dataBin))]
} else { singles <- sort(singles) }

singles <- unique(sort(singles))

wagscreen <- epiScreen(dataBin[, -grep("fus3\\.|ptp2.ptc2", colnames(dataBin))])

wagscreen$dataWag <- dataBin[, -grep("fus3.|ptp2.ptc2", colnames(dataBin))]

###### yeast Sameith et al.:

file <- paste("http://www.holstegelab.nl/",
              "publications/GSTF_geneticinteractions/",
              "downloads/del_mutants_limma.txt", sep = "")

data <- read.delim(file)

data <- apply(data, c(1,2), as.character)

dataM <- data[-1, which(data[1, ] %in% "M")]

dataM <- apply(dataM, c(1,2), as.numeric)

dataP <- data[-1, which(data[1, ] %in% "p.value")]

dataP <- apply(dataP, c(1,2), as.numeric)

rownames(dataM) <- rownames(dataP) <- data[2:nrow(data), 1]

dataBin <- dataM

sig <- 0.01

cutoff <- log2(1.5)

dataBin[which(dataP < sig & dataP > 0 & abs(dataM) >= cutoff)] <- 1

dataBin[which(dataP >= sig | dataP == 0 | abs(dataM) < cutoff)] <- 0

dataBin <- dataBin[-which(apply(dataBin, 1, max) == 0), ]

colnames(dataBin) <- gsub("\\.\\.\\.", "\\.", colnames(dataBin))

doubles <- colnames(dataBin)[grep("\\.", colnames(dataBin))]

if (length(grep("vs", doubles)) > 0) {
    doubles <- sort(doubles[-grep("vs", doubles)])
} else { doubles <- sort(doubles) }

doubles.genes <- unique(unlist(strsplit(doubles, "\\.")))

if (length(grep("\\.", colnames(dataBin))) > 0) {
    singles <- colnames(dataBin)[-grep("\\.", colnames(dataBin))]
} else { singles <- sort(singles) }

singles <- unique(sort(singles))

samscreen <- epiScreen(dataBin)

samscreen$dataSam <- dataBin

## String-db interaction scores:

library(STRINGdb)

get_STRING_species(version="10", species_name=NULL)[26, ] # 4932

string_db <- STRINGdb$new( version="10", species=4932, score_threshold=0,
                          input_directory="")

llmat <- wagscreen$ll

logicmat <- wagscreen$logic

string.scores <- list()

string.names <- character()

for (i in 1:ncol(llmat)) {
    if (sum(!(llmat[, i] %in% c(0,-Inf))) > 0) { 
        top30 <- llmat[, i]
        top30[which(top30 == 0)] <- -Inf
        top30 <- top30[which(!(llmat[, i] %in% c(0,-Inf)))]
        top30 <- top30[order(top30,decreasing = TRUE)[1:min(30, sum(!(llmat[, i]
            %in% c(0,-Inf))))]]

        doubles <- unlist(strsplit(colnames(llmat)[i], "\\."))

        for (j in names(top30)) {
            tmp <- string_db$get_interactions(string_db$mp(c(doubles[1], j)))
            string.scores <- c(string.scores, tmp$combined_score)
            string.names <- c(string.names, paste(sort(c(doubles[1], j)),
                                                  collapse = "_"))
            tmp <- string_db$get_interactions(string_db$mp(c(doubles[2], j)))
            string.scores <- c(string.scores, tmp$combined_score)
            string.names <- c(string.names, paste(sort(c(doubles[2], j)),
                                                  collapse = "_"))
        }

    } else {
        next()
    }
}


llmat <- samscreen$ll

logicmat <- samscreen$logic

string.scores2 <- list()

string.names2 <- character()

for (i in 1:ncol(llmat)) {

    if (sum(!(llmat[, i] %in% c(0,-Inf))) > 0) { 
        top30 <- llmat[, i]
        top30[which(top30 == 0)] <- -Inf
        top30 <- top30[which(!(llmat[, i] %in% c(0,-Inf)))]
        top30 <- top30[order(top30, decreasing = TRUE)
                       [1:min(30, sum(!(llmat[, i] %in% c(0,-Inf))))]]

        doubles <- unlist(strsplit(colnames(llmat)[i], "\\."))

        for (j in names(top30)) {
            tmp <- string_db$get_interactions(string_db$mp(c(doubles[1], j)))
            string.scores2 <- c(string.scores2, tmp$combined_score)
            string.names2 <- c(string.names2, paste(sort(c(doubles[1], j)),
                                                    collapse = "_"))
            tmp <- string_db$get_interactions(string_db$mp(c(doubles[2], j)))
            string.scores2 <- c(string.scores2, tmp$combined_score)
            string.names2 <- c(string.names2, paste(sort(c(doubles[2], j)),
                                                    collapse = "_"))
        }

    } else {
        next()
    }

}

## graph based GO similarity scores:

library(GOSemSim)
library(AnnotationHub)
library(org.Sc.sgd.db)

ystGO <- godata("org.Sc.sgd.db", ont = "BP",
                keytype = keytypes(org.Sc.sgd.db)[11], computeIC = FALSE)

## van Wageningen et al.:

GOepi <- numeric()

for (i in 1:ncol(wagscreen$ll)) {
    if (i %in% grep("fus3|ptp2.ptc2", colnames(wagscreen$ll))) { next() }
    pair <- toupper(unlist(strsplit(colnames(wagscreen$ll)[i], "\\.")))
    for (j in which(!is.infinite(wagscreen$ll[, i]) == TRUE &
                    wagscreen$ll[, i] != 0)) {
        tmp <- clusterSim(pair, toupper(rownames(wagscreen$ll)[j]),
                       semData = ystGO, combine = "max")
        if (!is.na(tmp[1])) {
            GOepi <- c(GOepi, tmp)
        }
    }
}

GOall <- numeric()

for (i in colnames(wagscreen$ll)) {
    pair <- toupper(unlist(strsplit(i, "\\.")))
    for (j in rownames(wagscreen$ll)) {
        tmp <- clusterSim(pair, toupper(j), semData = ystGO, combine = "max")
        if (!is.na(tmp[1])) {
            GOall <- c(GOall, tmp)
        }
    }
}

## Sameith et al.:

GOepi2 <- numeric()

for (i in 1:ncol(samscreen$ll)) {
    if (i %in% grep("fus3|ptp2.ptc2", colnames(samscreen$ll))) { next() }
    pair <- toupper(unlist(strsplit(colnames(samscreen$ll)[i], "\\.")))
    for (j in which(!is.infinite(samscreen$ll[, i]) == TRUE &
                    samscreen$ll[, i] != 0)) {
        tmp <- clusterSim(pair, toupper(rownames(samscreen$ll)[j]),
                       semData = ystGO, combine = "max")
        if (!is.na(tmp[1])) {
            GOepi2 <- c(GOepi2, tmp)
        }
    }
}

GOall2 <- numeric()

for (i in colnames(samscreen$ll)) {
    pair <- toupper(unlist(strsplit(i, "\\.")))
    for (j in rownames(samscreen$ll)) {
        tmp <- clusterSim(pair, toupper(j), semData = ystGO, combine = "max")
        if (!is.na(tmp[1])) {
            GOall2 <- c(GOall2, tmp)
        }
    }
}

###### Go enrichment analysis:

## van Wageningen et al.:

string_db$set_background(
    string_db$mp(unique(c(unlist(strsplit(colnames(wagscreen$ll), "\\.")),
                          rownames(wagscreen$ll)))))

golist <- list()

for (i in 1:ncol(wagscreen$ll)) {
    golist[[i]] <- string_db$get_enrichment(string_db$mp(unique(
        c(unlist(strsplit(colnames(wagscreen$ll)[i], "\\.")),
          rownames(wagscreen$ll)[which(!(wagscreen$logic[, i] %in%
                                         c("NOINFO", "NOEPI")))]))),
        category = "KEGG", methodMT = "fdr", iea = TRUE)
}

string_db$set_background(string_db$mp(rownames(wagscreen$dataWag)))

egenego <- list()

gos <- character()

for (i in 1:length(wagscreen$targets)) {
    egenego[[i]] <- list()
    if (length(wagscreen$targets[[i]]) == 0) { next() }
    for (j in 1:length(wagscreen$targets[[i]])) {
        egenego[[i]][[j]] <- string_db$get_enrichment(
            string_db$mp(wagscreen$targets[[i]][[j]]),
            category = "KEGG", methodMT = "fdr", iea = TRUE)
        if (dim(egenego[[i]][[j]])[1] > 0) {
            gos <- c(gos, egenego[[i]][[j]]$term_description)
        }
    }
}
## Sameith et al.:

string_db$set_background(string_db$mp(unique(c(unlist(
    strsplit(colnames(samscreen$ll), "\\.")), rownames(samscreen$ll)))))

golist2 <- list()

for (i in 1:ncol(samscreen$ll)) {
    golist2[[i]] <- string_db$get_enrichment(string_db$mp(
        unique(c(unlist(strsplit(colnames(samscreen$ll)[i], "\\.")),
                 rownames(samscreen$ll)
                 [which(!(samscreen$logic[, i] %in% c("NOINFO", "NOEPI")))]))),
        category = "KEGG", methodMT = "fdr", iea = TRUE)
}

string_db$set_background(string_db$mp(rownames(samscreen$dataWag)))

egenego2 <- list()

gos2 <- character()

for (i in 1:length(samscreen$targets)) {
    egenego2[[i]] <- list()
    if (length(samscreen$targets[[i]]) == 0) { next() }
    for (j in 1:length(samscreen$targets[[i]])) {
        egenego2[[i]][[j]] <- string_db$get_enrichment(
            string_db$mp(samscreen$targets[[i]][[j]]),
            category = "KEGG", methodMT = "fdr", iea = TRUE)
        if (dim(egenego2[[i]][[j]])[1] > 0) {
            gos2 <- c(gos2, egenego2[[i]][[j]]$term_description)
        }
    }
}
``` 

# Session information

```{r}
sessionInfo()
```

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Margolin AA, Nemenman I, Basso K, Wiggins C, Stolovitzky G, {Dalla Favera} R,
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