---
title: "MethReg: estimating regulatory potential of DNA methylation in gene transcription"
author:
  - name: Tiago Chedraoui Silva
    affiliation: University of Miami Miller School of Medicine
    email: txs902 at miami.edu
  - name: Lily Wang
    affiliation: University of Miami Miller School of Medicine
    email: lily.wangg at gmail.com
package: MethReg
output: 
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bibliography: bibliography.bib    
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# Introduction

Transcription factors (TFs) are proteins that facilitate the transcription of 
DNA into RNA. A number of recent studies have observed that the binding of TFs 
onto DNA can be affected by DNA methylation, and in turn, DNA methylation can 
also be added or removed by proteins associated with transcription 
factors [@bonder2017disease; @banovich2014methylation; @zhu2016transcription].

To provide functional annotations for differentially methylated regions (DMRs) 
and differentially methylated CpG sites (DMS), `MethReg` performs integrative 
analyses using matched DNA methylation and gene expression along with 
Transcription Factor Binding Sites (TFBS) data. MethReg evaluates, prioritizes 
and annotates DNA methylation regions (or sites) with high regulatory potential 
that works synergistically with TFs to regulate target gene expressions, 
without any additional ChIP-seq data. 

The results from `MethReg` can be used to generate testable hypothesis on the 
synergistic collaboration of DNA methylation changes and TFs in gene regulation.
`MethReg` can be used either to evaluate regulatory potentials of candidate 
regions or to search for methylation coupled TF regulatory processes in the entire genome.

# Installation 

`MethReg` is a Bioconductor package and can be installed through `BiocManager::install()`.

```{r, eval = FALSE}
if (!"BiocManager" %in% rownames(installed.packages()))
     install.packages("BiocManager")
BiocManager::install("MethReg", dependencies = TRUE)
```

After the package is installed, it can be loaded into R workspace by

```{r setup, eval = TRUE}
library(MethReg)
```

# MethReg workflow

The figure below illustrates the workflow for MethReg. 
Given matched array DNA methylation data and RNA-seq gene expression data, 
MethReg additionally incorporates TF binding information from ReMap2020  [@remap2020] 
or the JASPAR2020 [@JASPAR2020; @fornes2020jaspar] database,
and optionally additional TF-target gene interaction databases, 
to perform both promoter and distal (enhancer) analysis. 

In the unsupervised mode, MethReg analyzes all CpGs on the Illumina arrays. 
In the supervised mode, MethReg analyzes and prioritizes differentially methylated CpGs identified in EWAS. 

There are three main steps: (1) create a dataset with triplets of CpGs, TFs that bind near the CpGs, 
and putative target genes, (2) for each triplet (CpG, TF, target gene), apply integrative 
statistical models to DNA methylation, target gene expression, and TF expression values, 
and (3) visualize and interpret results from statistical models to estimate individual 
and joint impacts of DNA methylation and TF on target gene expression, as well as 
annotate the roles of TF and CpG methylation in each triplet. 

The results from 
the statistical models will also allow us to identify a list of CpGs that work 
synergistically with TFs to influence target gene expression.

```{r workflow, fig.cap = "Figure 2 Workflow of MethReg. Data - MethReg required inputs are: (1) array-based DNA methylation data (HM450/EPIC) with beta-values, (2) RNA-seq data with normalized counts, (3) estimated TF activities from the RNA-seq data using GSVA or VIPER  software. Creating triplets – there are multiple ways to create a CpG-TF-target gene triplet: (1) CpGs can be mapped to human TFs by using TF motifs from databases such as JASPAR or ReMap and scanning the CpGs region to identify if there is a binding site (2) CpG can be mapped to target genes using a distance-based approach, CpGs will be linked to a gene if it is on promoter region (+-1K bp away from the TSS), for a distal CpG it can be linked to either all genes within a fixed width (i.e. 500k bp), or a fixed number of genes upstream and downstream of the CpG location (3) TF-target genes can be retrieved from external databases (i.e. Cistrome Cancer; Dorothea). Using two different pairs (i.e. CpG-TF, TF-target gene), triplets can then be created. Analysis – each triplet will be evaluated using a robust linear model in which TF activity is estimated from GSVA/Viper software and DNAm.group is a binary variable used to model if the sample CpG belongs to the top 25% methylation levels or the lowest 25% methylation levels. Results – MethReg will output the prioritized triplets and classify both the TF role on the gene expression (repressor or activator) and the DNAm effect on the TF activity (Enhancing or attenuating).", echo = FALSE, fig.width = 13}
png::readPNG("workflow_methReg.png") %>% grid::grid.raster()
```

# Analysis illustration

## Input data

For illustration, we will use chromosome 21 data from 38 TCGA-COAD (colon cancer) samples.

### Input DNA methylation dataset

The DNA methylation dataset is a matrix or SummarizedExperiment object with 
methylation beta or M-values 
(The samples are in the columns and methylation regions or probes are in the rows). 
If there are potential confounding factors (e.g. batch effect, age, sex) in the dataset, 
this matrix would contain residuals from fitting linear regression 
instead (see details **Section 5** "Controlling effects from confounding variables" below). 

#### Analysis for individual CpGs data

We will analyze all CpGs on chromosome 21 in this vignette. 

However, oftentimes, the methylation data can also be, for example, 
**differentially methylated sites** (DMS) or **differentially methylated regions** (DMRs)
obtained in an epigenome-wide association study (EWAS) study.

```{R warning=FALSE}
data("dna.met.chr21")
```

```{R}
dna.met.chr21[1:5,1:5]
```

We will first create a SummarizedExperiment object with the function
`make_dnam_se`. This function will use the Sesame R/Bioconductor package 
to map the array probes into genomic regions. You cen set human genome version
(hg38 or hg19) and the array type ("450k" or "EPIC")

```{R}
dna.met.chr21.se <- make_dnam_se(
  dnam = dna.met.chr21,
  genome = "hg38",
  arrayType = "450k",
  betaToM = FALSE, # transform beta to m-values 
  verbose = FALSE # hide informative messages
)
```


```{R}
dna.met.chr21.se
SummarizedExperiment::rowRanges(dna.met.chr21.se)[1:4,1:4]
```


#### Analysis of DMRs

Differentially Methylated Regions (DMRs) associated with phenotypes such 
as tumor stage can be obtained from R packages such as 
`coMethDMR`, `comb-p`, `DMRcate` and many others. 
The methylation levels in multiple CpGs within the DMRs need to be 
summarized (e.g. using medians), then the analysis for 
DMR will proceed in the same way 
as those for CpGs. 

### Input gene expression dataset

The gene expression dataset is a matrix with log2 transformed and 
normalized gene expression values. 
If there are potential confounding factors (e.g. batch effect, age, sex) in the dataset, 
this matrix can also contain residuals from linear regression instead 
(see **Section 6** "Controlling effects from confounding variables" below).

The samples are in the columns and the genes are in the rows. 

```{R}
data("gene.exp.chr21.log2")
gene.exp.chr21.log2[1:5,1:5]
```

We will also create a SummarizedExperiment object for the gene expression data.
This object will contain the genomic information for each gene.

```{R}
gene.exp.chr21.se <- make_exp_se(
  exp = gene.exp.chr21.log2,
  genome = "hg38",
  verbose = FALSE
)
gene.exp.chr21.se
SummarizedExperiment::rowRanges(gene.exp.chr21.se)[1:5,]
```

###  Creating triplet dataset
#### Creating triplet dataset using distance based approaches and JASPAR2020

In this section, **regions** refer to the regions where CpGs are located. 

The function `create_triplet_distance_based` provides three different methods to 
link a region to a target gene: 

1. Mapping the region to the closest gene (`target.method = "genes.promoter.overlap"`)
2. Mapping the region to a specific number of genes upstream down/upstream 
of the region (`target.method = "nearby.genes"`) [@silva2019elmer].
3. Mapping the region to all the genes within a window 
(default size = 500 kbp around the region, i.e. +- 250 kbp from start or end 
of the region) (`target.method = "window"`) [@reese2019epigenome].


```{r plot, fig.cap = "Different target linking strategies", echo = FALSE}
png::readPNG("mapping_target_strategies.png") %>% grid::grid.raster()
```


For the analysis of probes in gene promoter region, we recommend setting 
`method = "genes.promoter.overlap"`, or 
`method = "closest.gene"`. 
For the analysis of probes in distal regions, we recommend setting either 
`method = "window"` or `method = "nearby.genes"`. 
Note that the distal analysis will be more time and resource consuming. 


To link regions to TF using JASPAR2020, MethReg uses `motifmatchr` [@motifmatchr] to scan 
these regions for occurrences of motifs in the database. JASPAR2020 is an 
open-access database of curated, non-redundant transcription 
factor (TF)-binding profiles [@JASPAR2020; @fornes2020jaspar], which contains 
more the 500 human TF motifs.

The argument `motif.search.window.size` will be used to extend the region when scanning
for the motifs, for example, a `motif.search.window.size` of `50` will add `25` bp 
upstream and `25` bp downstream of the original region.

As an example, the following scripts link CpGs with the probes in gene 
promoter region (method 1. above)

```{R, message = FALSE, results = "hide"}
triplet.promoter <- create_triplet_distance_based(
  region = dna.met.chr21.se,
  target.method = "genes.promoter.overlap",
  genome = "hg38",
  target.promoter.upstream.dist.tss = 2000,
  target.promoter.downstream.dist.tss = 2000,
  motif.search.window.size = 500,
  motif.search.p.cutoff  = 1e-08,
  cores = 1  
)
```

Alternatively, we can also link each probe with genes within 
$500 kb$ window (method 2). 

```{R, message = FALSE, results = "hide"}
# Map probes to genes within 500kb window
triplet.distal.window <- create_triplet_distance_based(
  region = dna.met.chr21.se,
    genome = "hg38", 
    target.method = "window",
    target.window.size = 500 * 10^3,
    target.rm.promoter.regions.from.distal.linking = TRUE,
    motif.search.window.size = 500,
    motif.search.p.cutoff  = 1e-08,
    cores = 1
)
```

For method 3, to map probes to 5 nearest upstream and downstream genes:  

```{R, message = FALSE, results = "hide"}
# Map probes to 5 genes upstream and 5 downstream
triplet.distal.nearby.genes <- create_triplet_distance_based(
  region = dna.met.chr21.se,
    genome = "hg38", 
    target.method = "nearby.genes",
    target.num.flanking.genes = 5,
    target.window.size = 500 * 10^3,
    target.rm.promoter.regions.from.distal.linking = TRUE,
    motif.search.window.size = 500,
    motif.search.p.cutoff  = 1e-08,
    cores = 1  
)
```

#### Creating triplet dataset using distance based approaches and REMAP2020


Instead of using JASPAR2020 motifs, we will be using REMAP2020 catalogue of 
TF peaks which can be access using the package `ReMapEnrich`.

```{r, eval = FALSE}
if (!"BiocManager" %in% rownames(installed.packages()))
     install.packages("BiocManager")
BiocManager::install("remap-cisreg/ReMapEnrich", dependencies = TRUE)
```

To download  REMAP2020 catalogue (~1Gb) the following functions are used:

```{R, eval = FALSE}
library(ReMapEnrich)
remapCatalog2018hg38 <- downloadRemapCatalog("/tmp/", assembly = "hg38")
remapCatalog <- bedToGranges(remapCatalog2018hg38)
```

The function `create_triplet_distance_based` will accept any Granges with TF 
information in the same format as the `remapCatalog` one.

```{R, eval = FALSE}
#-------------------------------------------------------------------------------
# Triplets promoter using remap
#-------------------------------------------------------------------------------
triplet.promoter.remap <- create_triplet_distance_based(
  region = dna.met.chr21.se,
  genome = "hg19",
  target.method =  "genes.promoter.overlap",
  TF.peaks.gr = remapCatalog,
  motif.search.window.size = 500,
  max.distance.region.target = 10^6,
) 
```

#### Creating triplet dataset using regulon-based approaches

The human regulons from the dorothea database will be used as an example: 

```{r, eval = FALSE}
if (!"BiocManager" %in% rownames(installed.packages()))
     install.packages("BiocManager")
BiocManager::install("dorothea", dependencies = TRUE)
```


```{R}
regulons.dorothea <- dorothea::dorothea_hs
regulons.dorothea %>% head
```

Using the regulons, you can calculate enrichment scores for each TF across 
all samples using dorothea and viper.

```{R}
rnaseq.tf.es <- get_tf_ES(
  exp = gene.exp.chr21.se %>% SummarizedExperiment::assay(),
  regulons = regulons.dorothea
)
```

Finally, triplets can be identified using TF-target from regulon databases with the function `create_triplet_regulon_based`.

```{R, message = FALSE, results = "hide"}
  triplet.regulon <- create_triplet_regulon_based(
    region = dna.met.chr21.se,
    genome = "hg38",  
    motif.search.window.size = 500,
    tf.target = regulons.dorothea,
    max.distance.region.target = 10^6 # 1Mbp
  ) 
```

```{R}
triplet.regulon %>% head
```

#### Example of triplet data frame

The triplet is a data frame with the following columns:

* `target`: gene identifier (obtained from row names of the gene expression matrix),
* `regionID`: region/CpG identifier (obtained from row names of the DNA methylation matrix)
* `TF`: gene identifier (obtained from the row names of the gene expression matrix)


```{R}
str(triplet.promoter)
triplet.promoter$distance_region_target_tss %>% range
triplet.promoter %>% head
```

Note that there may be multiple rows for a CpG region, when multiple 
target gene and/or TFs are found close to it. 

## Evaluating the regulatory potential of CpGs (or DMRs) 

Because TF binding to DNA can be influenced by (or influences) 
DNA methylation levels nearby [@yin2017impact], 
target gene expression levels are often resulted from the synergistic effects 
of both TF and DNA methylation. In other words, TF activities in gene 
regulation is often affected by DNA methylation. 

Our goal then is to highlight DNA methylation regions (or CpGs) where 
these synergistic DNAm and TF collaborations occur. 
We will perform analyses using the 3 datasets described above in Section 3:

* An input DNA methylation matrix
* An input Gene expression matrix
* The created triplet data frame

### Analysis using model with methylation by TF interaction

The function `interaction_model` assess the regulatory impact of 
DNA methylation on TF regulation of target genes via the following approach: 

**considering DNAm values as a binary variable** - we define a binary variable 
`DNAm Group` for DNA methylation values (high = 1, low = 0). 
That is, samples with the  highest DNAm levels (top 25 percent) has high = 1, 
samples with lowest DNAm levels (bottom 25 pecent) has high = 0.  

Note that in this implementation, only samples with DNAm values in 
the first and last quartiles are considered.

$$log_2(RNA target) \sim log_2(TF) + \text{DNAm Group} + log_2(TF) * \text{DNAm Group}$$
 
```{R interaction_model, message = FALSE, results = "hide", eval = TRUE}
results.interaction.model <- interaction_model(
    triplet = triplet.promoter, 
    dnam = dna.met.chr21.se,
    exp = gene.exp.chr21.se,
    sig.threshold = 0.05,
    fdr = TRUE,
    stage.wise.analysis = FALSE,
    filter.correlated.tf.exp.dnam = TRUE,
    filter.triplet.by.sig.term = FALSE
)
```

The output of `interaction_model` function will be a data frame with the following variables:

* `<variable>_pvalue`: p-value for a tested variable (methylation or TF), given the other variables included in the model.
* `<variable>_estimate`: estimated effect for a variable. If estimate > 0, increasing values 
of the variable corresponds to increased outcome values (target gene expression). 
If estimate < 0, increasing values of the variable correspond to decreased target gene expression levels.


The following columns are provided for the results of fitting **quartile model** to triplet data:

* direct effect of methylation:  
  + `RLM_DNAmGroup_pvalue`: p-value for binary DNA methylation variable
  + `RLM_DNAmGroup_estimate`: estimated DNA methylation effect

* direct effect of TF: 
  + `RLM_TF_pvalue` : p-value for TF expression
  + `RLM_TF_estimate`: estimated TF effect
  
* synergistic effects of methylation and TF: 
  + `RLM_DNAmGroup:TF_pvalue`: : p-value for DNA methylation by TF interaction
  + `RLM_DNAmGroup:TF_estimate`: estimated DNA methylation by TF interaction effect

```{R}
# Results for quartile model
results.interaction.model %>% dplyr::select(
  c(1,4,5,grep("RLM",colnames(results.interaction.model)))
  ) %>% head
```

### Stratified analysis by high and low DNA methylation levels

For triplets with significant $log_2(TF) × DNAm$ interaction effect identified 
above, we can further assess how gene regulation by TF changes when DNAm 
is high or low. To this end, the function 
`stratified_model` fits two separate models (see below) to only 
samples with the highest DNAm levels (top 25 percent), and then to
only samples with lowest DNAm levels (bottom 25 percent), separately.

$$\text{Stratified Model: } log_2(RNA target) \sim log_2(TF)$$


```{R stratified_model, message = FALSE, warning = FALSE, results = "hide", eval = TRUE}
results.stratified.model <- stratified_model(
    triplet = results.interaction.model,
    dnam = dna.met.chr21.se,
    exp = gene.exp.chr21.se
)
```

```{R}
results.stratified.model %>% head
```

### Visualization of data  

The functions `plot_interaction_model` 
will create figures to visualize the data, 
in a way that corresponds to the linear model we considered above. 
It requires the output from the function `interaction_model` (a dataframe),
the DNA methylation matrix and the gene expression matrix as input. 


```{R plot_interaction_model, eval = TRUE, message = FALSE, results = "hide", warning = FALSE}
plots <- plot_interaction_model(
    triplet.results = results.interaction.model[1,], 
    dnam = dna.met.chr21.se, 
    exp = gene.exp.chr21.se
)
```

```{R, fig.height = 8, fig.width = 13, eval = TRUE, fig.cap = "An example output from MethReg."}
plots
```


The first row of the figures shows pairwise associations between DNA methylation, 
TF and target gene expression levels. 

The second row of the figures show how much TF activity on target 
gene expression levels vary by DNA methylation levels. When TF by methylation 
interaction is significant (Section 4.1), we expect the association between TF 
and target gene expression vary depending on whether DNA methylation is low or high. 

In this example, when DNA methylation is low, target gene expression is relatively 
independent of the amount of TF available. On the other hand, when DNA methylation 
level is high, more abundant TF corresponds to increased gene expression (an activator TF). 
One possibility is that DNA methylation might enhance TF binding in this case. 
_**This is an example where DNA methylation and TF work synergistically to affect target gene expression**_. 

While the main goal of MethReg is to prioritize methylation CpGs, also 
note that without stratifying by DNA methylation, the overall TF-target 
effects (p = 0.142) is not as significant as the association in stratified 
analysis in high methylation samples (p = 0.00508). 
_**This demonstrates that by additionally modeling DNA methylation,**_
_**we can also nominate TF – target associations that might have been missed otherwise**_. 

## Results interpretation

Shown below are some expected results from fitting Models 1 & 2 described in 
**Section 4.1** above, depending on TF binding preferences. Please note that 
there can be more possible scenarios than those listed here, therefore, 
careful evaluation of the statistical models and visualization of data as 
described in **Section 4** are needed to gain a good understanding of the 
multi-omics data. 

```{r scenarios, fig.cap =  "Scenarios modeled by MethReg. A and B: DNA methylation decreases TF activity. In A TF is a repressor of the target gene, while in B TF is an activator. C and D: DNA methylation increases TF activity. In C TF is a repressor of the target gene, while in D TF is an activator. E and F: DNA methylation inverts the TF role. In E when DNA methylation levels are low the TF works as a repressor, when DNA methylation levels are high the TF acts as an activator. In F when the DNA methylation levels are low the TF works as an activator, when methylation levels are high the TF acts a repressor.", echo = FALSE, fig.width=13}
png::readPNG("scenarios.png")  %>% grid::grid.raster()
```


# Controlling effects from confounding variables

Both gene expressions and DNA methylation levels can be affected by age, sex, 
shifting in cell types, batch effects and other confounding (or covariate) variables. 
In this section, we illustrate analysis workflow that reduces confounding effects,
by first extracting the residual data with the function `get_residuals`, 
before fitting the models discussed above in Section 4. 

The `get_residuals` function will use gene expression (or DNA methylation data) 
and phenotype data as input. To remove confounding effects in gene expression data, 
we use the `get_residuals` function which extract residuals after fitting the
following model for gene expression data: 
$$log_2(RNA target) \sim covariate_{1} + covariate_{2} + ... + covariate_{N}$$
or the following model for methylation data: 

$$methylation.Mvalues \sim covariate_{1} + covariate_{2} + ... + covariate_{N}$$


```{R residuals, results = "hide", eval = FALSE}
data("gene.exp.chr21.log2")
data("clinical")
metadata <- clinical[,c("sample_type","gender")]

gene.exp.chr21.residuals <- get_residuals(gene.exp.chr21, metadata) %>% as.matrix()
```

```{R, eval = FALSE}
gene.exp.chr21.residuals[1:5,1:5]
```

```{R, results = "hide", eval = FALSE}
data("dna.met.chr21")
dna.met.chr21 <- make_se_from_dnam_probes(
  dnam = dna.met.chr21,
  genome = "hg38",
  arrayType = "450k", 
  betaToM = TRUE
)
dna.met.chr21.residuals <- get_residuals(dna.met.chr21, metadata) %>% as.matrix()
```

```{R, eval = FALSE}
dna.met.chr21.residuals[1:5,1:5]
```

The models described in **Section 4.1** can then be applied to these residuals 
data using the `interaction_model` function:  

```{R, message = FALSE, results = "hide", eval = FALSE}
results <- interaction_model(
    triplet = triplet, 
    dnam = dna.met.chr21.residuals, 
    exp = gene.exp.chr21.residuals
)
```

# Calculating enrichment scores

## Using dorothea and viper 

This example shows how to use dorothea regulons and viper to calculate 
enrichment scores for each TF across all samples.

```{R}
regulons.dorothea <- dorothea::dorothea_hs
regulons.dorothea %>% head
```

```{R, message = FALSE, results = "hide"}
rnaseq.tf.es <- get_tf_ES(
  exp = gene.exp.chr21.se %>% SummarizedExperiment::assay(),
  regulons = regulons.dorothea
)
```

```{R}
rnaseq.tf.es[1:4,1:4]
```

## Using dorothea and GSVA

```{R, message = FALSE, results = "hide"}
regulons.dorothea <- dorothea::dorothea_hs
regulons.dorothea$tf <- MethReg:::map_symbol_to_ensg(
  gene.symbol = regulons.dorothea$tf,
  genome = "hg38"
)
regulons.dorothea$target <- MethReg:::map_symbol_to_ensg(
  gene.symbol = regulons.dorothea$target,
  genome = "hg38"
)
split_tibble <- function(tibble, col = 'col') tibble %>% split(., .[, col])
regulons.dorothea.list <- regulons.dorothea %>% na.omit() %>% 
  split_tibble('tf') %>% 
  lapply(function(x){x[[3]]})
```



```{R, message = FALSE, results = "hide", eval = FALSE}
library(GSVA)
rnaseq.tf.es.gsva <- gsva(
  expr = gene.exp.chr21.se %>% SummarizedExperiment::assay(), 
  gset.idx.list = regulons.dorothea.list, 
  method = "gsva",
  kcdf = "Gaussian",
  abs.ranking = TRUE,
  min.sz = 5,
  max.sz = Inf,
  parallel.sz = 1L,
  mx.diff = TRUE,
  ssgsea.norm = TRUE,
  verbose = TRUE
)
```

# Session information
```{R,size = 'tiny'}
sessionInfo()
```

# Bibliography