---
title: "5 - Integrative analysis workshop with TCGAbiolinks and ELMER - Analysis GUI"
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    css: style.css
bibliography: bibliography.bib    
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---

```{r, echo = FALSE,hide=TRUE, message=FALSE,warning = FALSE}
library(ELMER.data)
library(ELMER)
```

<br>

# Introduction

In this section, we will perform the same analysis performed using ELMER,
but instead of doing it programmatically we will use TCGAbiolinksGUI [@Silva147496].

First we will launch the TCGAbiolinksGUI.
```{r gui, eval=FALSE, message=FALSE,warning=F}
library(TCGAbiolinksGUI)
TCGAbiolinksGUI()
```

# Downloading data

Please download this two objects:

* [Gene expression Summarized Experiment](https://github.com/BioinformaticsFMRP/Bioc2017.TCGAbiolinks.ELMER/raw/master/data/lusc.exp.rda)
* [DNA methylation Summarized Experiment](https://github.com/BioinformaticsFMRP/Bioc2017.TCGAbiolinks.ELMER/raw/master/data/lusc.met.rda)

# Analysis


## Create MultiAssayExperiment object

<div class="panel panel-info">
<div class="panel-body">
To create the MultiAssayExperiment object go to `Integrative analysis/ELMER/Create input data`.
</div>
</div>

![](figures/elmer_data_menu.png)

<br />
<div class="panel panel-info">
<div class="panel-body">
Select the DNA methylation object previously downloaded.
</div>
</div>

![](figures/elmer_data_dnamet_select.png)
![](figures/elmer_data_dnamet.png)


<br />
<div class="panel panel-info">
<div class="panel-body">
Select the gene expression object previously downloaded.
</div>
</div>


![](figures/elmer_data_exp_select.png)
![](figures/elmer_data_exp.png)


<br />
<div class="panel panel-info">
<div class="panel-body">
Fill the field `Save as:` and click on Create MAE object.
</div>
</div>

![](figures/elmer_data_final.jpg)


<br />
<div class="panel panel-info">
<div class="panel-body">
The object will be created.
</div>
</div>

![](figures/elmer_data_saved.png)

<br />

## Perform analysis


<br />
<div class="panel panel-info">
<div class="panel-body">
To perform ELMER analysis go to `Integrative analysis/ELMER/Analysis`.
</div>
</div>

![](figures/elmer_analysis_menu.png)

<br />
<div class="panel panel-info">
<div class="panel-body">
Select the MAE data created in the previous section.
</div>
</div>

![](figures/elmer_analysis_data.png)


<br />
<div class="panel panel-info">
<div class="panel-body">
Select the groups that will be analysed:  Primary solid Tumor and Solid Tissue Normal.
</div>
</div>

![](figures/elmer_analysis_groups.png)

<br />
<div class="panel panel-info">
<div class="panel-body">
We will identify probes that are hypomethylated in Primary solid Tumor compared to Solid Tissue Normal.
</div>
</div>

![](figures/elmer_analysis_diffmeth.png)

<br />
<div class="panel panel-info">
<div class="panel-body">
For the significant differently methylated probes identified before we will correlated
with the 20 nearest genes. Change the value of the field `Number of permutations` to `100`,
`Raw P-value cut-off` to `0.05` and `Empirical P value cut-off` to `0.01`.
</div>
</div>


![](figures/elmer_analysis_pair.png)


<br />
<div class="panel panel-info">
<div class="panel-body">
There will be no changes in the step 3.
</div>
</div>

![](figures/elmer_analysis_motif.png)

<br />
<div class="panel panel-info">
<div class="panel-body">
There will be no changes in the step 4.
</div>
</div>

![](figures/elmer_analysis_TF.png)

<br />
<div class="panel panel-info">
<div class="panel-body">
Click on `Run the analysis`.
</div>
</div>


![](figures/elmer_analysis_final.png)


<br />
<div class="panel panel-info">
<div class="panel-body">
If the analysis identified significant regulatory TF the results will be saved into an R object.
</div>
</div>

![](figures/elmer_analysis_message.png)

<br />

## Visualize results


<br />
<div class="panel panel-info">
<div class="panel-body">
To visualize the results go to `Integrative analysis/ELMER/Visualize results`.
</div>
</div>

![](figures/elmer_visualize_results.png)



<br />
<div class="panel panel-info">
<div class="panel-body">
Click on `Select results` and select the object created on the previous section.
</div>
</div>

![](figures/elmer_visualize_select1.png)
![](figures/elmer_visualize_select2.png)


<br />
<div class="panel panel-info">
<div class="panel-body">
Or the avarage DNA methylation levels of probes of a Motif vs the expression of a 
TF.
</div>
</div>

![](figures/elmer_visualize_plot_scatter_byTF.jpg)


<br />
<div class="panel panel-info">
<div class="panel-body">
For each enriched motif you can verify the ranking of sigificances between the correlation
of DNA methylation level on the significant paired probes with that motif vs the TF expression (for all human TF).  
</div>
</div>

![](figures/elmer_visualize_tfraningplot.jpg)


<br />
<div class="panel panel-info">
<div class="panel-body">
The enrichement of each motif can be visualized.
</div>
</div>

![](figures/elemer_visualize_motif_enrichment.jpg)

<br />
<div class="panel panel-info">
<div class="panel-body">
You can take a look for a gene which was the probe linked.
</div>
</div>

![](figures/elemer_visualize_schematic_gene.jpg)

<br />
<div class="panel panel-info">
<div class="panel-body">
You can see the plot and its neraby genes.
</div>
</div>

![](figures/elemer_visualize_schematic_probe.jpg)

<br />
<div class="panel panel-info">
<div class="panel-body">
It is possible to visualize the table with the significant differently methylated probes. 
</div>
</div>

![](figures/elmer_visualize_table_sigprobes.jpg)


<br />
<div class="panel panel-info">
<div class="panel-body">
It is possible to visualize the table with the enriched motifs.
</div>
</div>

![](figures/elmer_visualize_table_enriched_motif.jpg)


<br />
<div class="panel panel-info">
<div class="panel-body">
It is possible to visualize the table with the candidates regulatory TF.
</div>
</div>

![](figures/elmer_visualize_table_tf.jpg)

# Session Info

```{r sessioninfo, eval=TRUE}
sessionInfo()
```

# Bibliography
