---
title: "Vignette of the esetVis package"
author: "Laure Cougnaud"
date: "`r Sys.Date()`"
output: 
  rmarkdown::html_document:
    css: custom.css
    toc: true
    toc_float:
      collapsed: true
    number_sections: true
references:
- id: spm
  title: Spectral mapping, a technique for classifying biological activity profiles of chemical compounds
  author:
  - family: P.J.
    given: Lewi
  type: article-journal
  volume: 26
  page: 1295-1300
  publisher: Arzneimittel Forschung (Drug Research)
  issued:
    year: 1976
- id: tsne
  title: Visualizing High-Dimensional Data Using t-SNE
  author:
  - family: van der Maaten
    given: L.J.P.
  type: article-journal
  volume: 26
  page: 2579-2605
  publisher: Journal of Machine Learning Research
  issued:
    year: 2008
- id: ldaFisher
  title: The Use of Multiple Measurements in Taxonomic Problems
  author:
  - family: Fisher
    given: R. A.
  type: article-journal
  volume: 7 (2)
  page: 179-188
  publisher: Annals of Eugenics
  issued:
    year: 1936
vignette: >
  %\VignetteIndexEntry{esetVis package}
  %\VignetteEngine{knitr::rmarkdown}
  \usepackage[utf8]{inputenc}
---

# Introduction

This document explains the functionalities available in the
**esetVis** package.


This package contains wrapper functions for three types of visualization:
**spectral map**[@spm], **tsne**[@tsne] and **linear discriminant
analysis**[@ldaFisher] for data contained in a _expressionSet
Bioconductor_  (or an _SummarizedExperiment_) object. The visualizations are
available in two types: **static**, via the
[ggplot2](https://cran.r-project.org/package=ggplot2)
package or **interactive**, via the
[ggvis](https://cran.r-project.org/package=ggvis)
or [plotly](https://cran.r-project.org/package=plotly)
packages.

```{r options, echo = FALSE}
	
	library(knitr)
	opts_chunk$set(echo = TRUE, results = 'asis', warning = FALSE, 
		error = FALSE, message = FALSE, cache = FALSE,
		fig.width = 8, fig.height = 7,
		#out.width = '0.7\\textwidth', 
		fig.align = 'center')#, out.height = 0.5\\textwidth', fig.path = 'graphs/') 
	options(width = 170)#, stringsAsFactors = FALSE
	options(warn = 1)#instead of warn = 0 by default -> to have place where warnings occur in the call to Sweave function
	
```

```{r loadLibraries, results = 'hide', echo = FALSE}

	library(esetVis)
#	library(xtable)
	library(pander)

```

# Example dataset

## ExpressionSet object

The _ALL_ dataset contains microarray results from 128 patients with
acute lymphoblastic leukemia (ALL). The data is contained in a
_Bioconductor_ `ExpressionSet` object.  Extra gene annotation is
added to the object, via the annotation package `hgu95av2.db`.

```{r ALL-loadAndFormatAllDataset}

	library(ALL)
	data(ALL)
	
	# to get gene annotation from probe IDs (from the paper HGU95aV2 gene chip was used)
	library("hgu95av2.db")
	library("AnnotationDbi")
	probeIDs <- featureNames(ALL)
	geneInfo <- AnnotationDbi::select(hgu95av2.db, probeIDs, 
		c("ENTREZID", "SYMBOL", "GENENAME"), "PROBEID")
	# 482 on the 12625 probe IDs don't have ENTREZ ID/SYMBOL/GENENAME

	# remove genes with duplicated annotation: 1214
	geneInfoWthtDuplicates <- geneInfo[!duplicated(geneInfo$PROBEID), ]

	# remove genes without annotation: 482
	genesWthtAnnotation <- rowSums(is.na(geneInfoWthtDuplicates)) > 0
	geneInfoWthtDuplicatesAndWithAnnotation <- geneInfoWthtDuplicates[!genesWthtAnnotation, ]
	
	probeIDsWithAnnotation <- featureNames(ALL)[featureNames(ALL) %in% 
		geneInfoWthtDuplicatesAndWithAnnotation$PROBEID]
	ALL <- ALL[probeIDsWithAnnotation, ]
	
	fData <- geneInfoWthtDuplicatesAndWithAnnotation[
		match(probeIDsWithAnnotation, geneInfoWthtDuplicatesAndWithAnnotation$PROBEID), ]
	rownames(fData) <- probeIDsWithAnnotation
	fData(ALL) <- fData

	# grouping variable: B = B-cell, T = T-cell
	groupingVariable <- pData(ALL)$BT
	
	# create custom palette
	colorPalette <- c("dodgerblue", 
		colorRampPalette(c("white","dodgerblue2", "darkblue"))(5)[-1], 
		"red", colorRampPalette(c("white", "red3", "darkred"))(5)[-1])
	names(colorPalette) <- levels(groupingVariable)
	color <- groupingVariable; levels(color) <- colorPalette
	
	# reformat type of the remission
	remissionType <- ifelse(is.na(ALL$remission), "unknown", as.character(ALL$remission))
	ALL$remissionType <- factor(remissionType,
		levels = c("unknown", "CR", "REF"), 
		labels = c("unknown", "achieved", "refractory"))

```

```{r ALL-loadAndFormatAllDataset-extraNotes, echo = FALSE}

	fvarMetadata(ALL)$labelDescription <- paste(rownames(fvarMetadata(ALL)),
		"obtained from the Bioconductor hgu95av2.db package")	

```

```{r rmAnnotObjectsFromMemory, echo = FALSE}

	rm(list = c("fData", ls(pattern = "^(geneInfo|probeIDs)")));tmp <- gc(verbose = FALSE)
	#for testing: sort(sapply(ls(), function(x) object.size(get(x))))

```

Following tables detail some sample and gene annotation contained in the _ALL_
`ExpressionSet` used in the vignette.

```{r ALL-someDataCharacteristics, echo = FALSE}

	varLabelsUsed <- c("cod", "sex", "age", "BT", "remissionType")
	pander(head(pData(ALL)[, varLabelsUsed]),
		caption = "subset of the **phenoData** of the ALL dataset for the first genes")
	
	pander(head(fData(ALL)), 
        caption = "**featureData** of the ALL dataset for the first genes")

```

## SummarizedExperiment object

The functions of the package also supports object
of class:
[`SummarizedExperiment`](http://bioconductor.org/packages/release/bioc/html/SummarizedExperiment.html).
Note: In this case, the functions `fData`, `pData`, `exprs` should be replaced
by their corresponding functions `rowData`, `colData` and `assay`.

# Spectral map: `esetSpectralMap`

## Simple spectral map

The function `esetSpectralMap` creates a **spectral
map**[@spm] for the dataset. Some default parameters are set, e.g.
to print the top 10 samples and top 10 genes, to display the first two
dimensions of the analysis...

The resulting biplot contains two components:

* in the background, a cloud of the genes coordinates (plotted with the
  [hexbin](https://cran.r-project.org/web/packages/hexbin/index.html) package)
* in the foreground, each sample of the data is represented as a single
  point/symbol

Here is an example for the _ALL_ dataset, with the default parameters.

```{r ALL-esetSimple}

	print(esetSpectralMap(eset = ALL))

```

## Additional sample information

Several annotation variables are available in the `eSet`.

### General

Four different aesthetics `[aes]` can be used to display these variables:

* **color**, with the tag `color`
* **transparency**, with the tag `alpha`
* **size**, with the tag `size`
* **shape**, with the tag `shape`

For each of this aesthetic `[aes]`, several parameters are available:

* `[aes]Var`: name of the column of the `phenoData` of the
  `eSet` used for the aesthetic, i.e. `colorVar`
* `[aes]`: palette/specified shape/size used for the aesthetic, i.e.
  `color`

### Custom size and transparency

Custom size and the transparency (variables `sizeVar` and `alphaVar`) can be
specified:

* if the size/transparency is a numerical
variable (numeric or integer), the range of the size/transparency can be
specified respectively with the arguments `sizeRange` and
`alphaRange`
* in the other cases (factor or character), custom
size/transparency can be specified directly respectively via the `size`
and `alpha`   arguments

In the example, the type and stage of the disease (variable
_BT_) is used for coloring, the remission type for the transparency, the
_sex_ for the shape and the _age_ for the size of the points. A custom color
palette is specified via the `color` argument.

```{r ALL-esetSampleAnnotation1}

	print(esetSpectralMap(eset = ALL, 
		title = "Acute lymphoblastic leukemia dataset \n Spectral map \n Sample annotation (1)",
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex", 
		sizeVar = "age", sizeRange = c(0.1, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topSamples = 0, topGenes = 0, cloudGenes = TRUE))

```

Just for the demonstration, another visualization of the same dataset,
using this time a continuous variable: _age_ for coloring and transparency, a
factor for the size and _BT_ for the shape.
Because the output is a `ggplot` object, any additional customization
not implemented via specific parameters, the plot can be modified
with additional `ggplot2` functions, e.g. with the color of the gradient.

```{r ALL-esetSampleAnnotation2, fig.height = 8}

	gg <- esetSpectralMap(eset = ALL, 
		title = "Acute lymphoblastic leukemia dataset \n Spectral map \n Sample annotation (2)",
		colorVar = "age",
		shapeVar = "BT", shape = 15+1:nlevels(ALL$BT),
		sizeVar = "remissionType", size = c(2, 4, 6),
		alphaVar = "age", alphaRange = c(0.2, 0.8),
		topSamples = 0, topGenes = 0, cloudGenes = TRUE)
	# change color of gradient
	library(ggplot2)
	gg <- gg + scale_color_gradientn(colours = 
		colorRampPalette(c("darkgreen", "gold"))(2)
	)
	print(gg)

```

## Custom gene representation

Several parameters related to the gene visualization are available:

* gene subset: only a subset of the genes (at least two) can be displayed, via
  the argument `psids`
* gene cloud:
  + inclusion/removal of the gene cloud via `cloudGenes`
  + number of bins specification via `cloudGenesNBins`
  + cloud color via `cloudGenesColor`
  + legend:
    - inclusion/removal of the gene legend via `cloudGenesIncludeLegend`
    - title legend specified via `cloudGenesTitleLegend`
    
The spectral map is done only on the subset of the genes, with the number of
bins, color, and legend title modified.
    
```{r ALL-customGeneRepresentation}

	print(esetSpectralMap(eset = ALL,
		psids = 1:1000,
		title = "Acute lymphoblastic leukemia dataset \n Spectral map \n Custom cloud genes",
		topSamples = 0, topGenes = 0, 
		cloudGenes = TRUE, cloudGenesColor = "red", cloudGenesNBins = 50,
		cloudGenesIncludeLegend = TRUE, cloudGenesTitleLegend = "number of features"))

```

## Label outlying elements

###  Parameters

Three different kind of elements can be labelled in the
plot: **genes, samples and gene sets**.

For each `[element]`, several parameters are available:

* `top[Elements]`: number of elements to label
* `top[Elements]Var` (not available for gene sets): column of the
corresponding element in the `eSet` used for labelling, in
`phenoData` for sample and `featureData` for gene. If not
specified, the feature/sample names of the `eSet` are used
* `top[Elements]Just`: label justification
* `top[Elements]Cex`: label size
* `top[Elements]Color`: label color

### Method to select top elements

The **distance (sum of squared coordinates) of the gene/sample/gene set to the
origin of the plot** is used to rank the elements, and to extract the top
'outlying' ones.

### Package used for static plot

By default (and if installed), the package
[ggrepel](https://cran.r-project.org/web/packages/ggrepel/index.htm)
is used for text labelling (as in this vignette), to avoid overlapping labels.
The text labels can also be displayed with the
[ggplot2](https://cran.r-project.org/web/packages/ggplot2/index.html)
by setting the parameter `packageTextLabel` to `ggplot2` (default
`ggrepel`).

### Example

In the example, the top genes are labelled with gene symbols (_SYMBOL_
column of the phenoData of the eSet), and the top samples with patient
identifiers (_cod_ column of the phenoData of the eSet).

```{r ALL-genesSamplesAnnotation}

	print(esetSpectralMap(eset = ALL, 
		title = paste("Acute lymphoblastic leukemia dataset \n",
			"Spectral map \n Label outlying samples and genes"),
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topGenes = 10, topGenesVar = "SYMBOL",
		topGenesCex = 2, topGenesColor = "darkgrey",
		topSamples = 15, topSamplesVar = "cod", topSamplesColor = "chocolate4",
		topSamplesCex = 3
	))

```

## Gene sets annotation

Genes can be grouped into biologically meaningful gene sets, which can be
labelled in the biplot.

Compared to previous section, two additional parameters are
available: 

* `geneSets` (**required**): list of
gene sets. Each element in the list should contain genes identifiers, and the
  list should be named
* `geneSetsVar`: column of the featureData of the eSet used to map the gene
  identifiers contained in the `geneSets` object
* `geneSetsMaxNChar`: number of characters used in gene sets labels

The `geneSets` can be created with the `getGeneSetsForPlot`
function (wrapper around the `getGeneSets` function of the
[MLP](https://www.bioconductor.org/packages/release/bioc/html/MLP.html)
package), which can extract gene sets available in the Gene Ontology (Biological
Process, Molecular Function and Cellular Component) and KEGG databases. Custom
gene set lists can also be provided.

In the following example, only the pathways from the _GOBP_ database are
used.

```{r ALL-extractGeneSets}

	geneSets <- getGeneSetsForPlot(
		entrezIdentifiers = fData(ALL)$ENTREZID, 
		species = "Human", 
		geneSetSource = 'GOBP',
		useDescription = TRUE)

```

Then this list of gene sets is provided to the `esetSpectralMap`
function.

```{r ALL-geneSetAnnotation}

	print(esetSpectralMap(eset = ALL, 
		title = "Acute lymphoblastic leukemia dataset \n Spectral map \n Gene set annotation",
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topGenes = 0,
		topGeneSets = 4, geneSets = geneSets, geneSetsVar = "ENTREZID", geneSetsMaxNChar = 30,
		topGeneSetsCex = 2, topGeneSetsColor = "purple"))

```

Note: because of the inherent hierarchical structure of the Gene Ontology
database, sets of genes can be very similar, which can result in close (even
overlapping) labels in the visualization.

```{r rmGeneSetsFromMemory, echo = FALSE}

	rm(geneSets);tmp <- gc(verbose = FALSE)

```

## Dimensions of the biplot

In all previous plots, only the first dimensions of the principal component
analysis were visualized, this can be specified via the `dim` parameter.
The third and fourth dimensions are visualized in the next Figure. This
parameter is only available for the spectral map visualization.

```{r ALL-dimensionsBiPlot}

	print(esetSpectralMap(eset = ALL, 
		title = "Acute lymphoblastic leukemia dataset \n Spectral map \n Dimensions of the PCA",
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(0.5, 4),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		dim = c(3, 4)))

```

## Implementation

The function uses the `mpm` and `plot.mpm` function from the
`mpm` package. Some default parameters are set for these two functions,
they can be changed via the `mpm.args` and `plot.mpm.args`
arguments. For further details, refer to the documentation of these two
functions.

Note: One important argument is `logtrans` in `mpm`
function, which indicates if the **data should be first log-transformed**
before the computation. It is set by default to FALSE, **assuming that the
data are already in the log scale** (it is the case for the _ALL_
dataset).

## Interactive spectral map

All plots available in the `esetVis` package can be **static or
interactive**.

The argument `typePlot` can be set respectively to
`static` (by default), in this case `ggplot2` is used, or
`interactive`, in this case either the `ggvis` or
`plotly` package is used.

Two functionalities of the interactive plot can be of
interest for such high-dimensional data:

* **hover** to check sample annotation.
  By default, only the sample variables used for the aesthetics are displayed
  when hovering on a specific sample dot. Additional sample variables
  (contained in `phenoData`) displayed in the hover can be specified via the
  `interactiveTooltipExtraVars` parameter.
* **zoom** to focus on specific sample in high-dimensional
  dataset

### plotly

```{r ALL-interactivePlot-plotly}
esetSpectralMap(
	eset = ALL, 
	title = paste("Acute lymphoblastic leukemia dataset - spectral map"),
	colorVar = "BT", color = unname(colorPalette),
	shapeVar = "sex",
	alphaVar = "remissionType",
	typePlot = "interactive", packageInteractivity = "plotly",
	figInteractiveSize = c(700, 600),
	topGenes = 10, topGenesVar = "SYMBOL",
	topSamples = 10,
	# use all sample variables for hover
	interactiveTooltipExtraVars = varLabels(ALL)
)
```

### ggvis

Note: as `ggvis` plot requires to have a R session running, only a static
version of the plot is included.

```{r ALL-interactivePlot-ggvis, cache = FALSE}
library(ggvis)

# embed a static version of the plot
esetSpectralMap(
	eset = ALL,
	title = paste("Acute lymphoblastic leukemia dataset - spectral map"),
	colorVar = "BT", color = unname(colorPalette),
	shapeVar = "sex",
	alphaVar = "remissionType",
	typePlot = "interactive", packageInteractivity = "ggvis",
	figInteractiveSize = c(700, 600),
	sizeVar = "age", sizeRange = c(2, 6),
	# use all phenoData variables for hover
	interactiveTooltipExtraVars = varLabels(ALL)
)
```

# Tsne: `esetTsne`

## General

Another unsupervised visualization is available in the package:
**t-Distributed Stochastic Neighbor Embedding** (_tsne_). The
function `esetTnse` uses the `Rtsne` function of the same
package.

Most of the previous parameters discussed for the spectral map are
available for this visualization, at the exception of:

* parameter linked to gene annotation/labelling. The gene annotation is  not
  (yet) mapped to the sample coordinated obtained as output from the
  `Rtsne` function
* parameter specific to the spectral map, i.e. `dim`

Arguments to the `Rtsne` function can be specified via the
`Rtsne.args` argument.

Here is an example of tsne, for the same dataset and same annotation/labelling.

<!-- takes 8s  -->
```{r ALL-tsne}

	print(esetTsne(eset = ALL, 
		title = "Acute lymphoblastic leukemia dataset \n Tsne",
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topSamplesVar = "cod"
	))

```

## Additional pre-processing step

The tsne can be quite time-consuming, especially for big data. As the
`Rtsne` function used in the background can also uses an object of class
`dist`, the data can be pre-transformed before running the `tsne`
analysis. The argument `fctTransformDataForInputTsne` enables to specify
a custom function to pre-transform the data.

```{r ALL-tsne-preProcessing}

	print(esetTsne(eset = ALL, 
		title = "Acute lymphoblastic leukemia dataset \n Tsne",
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topSamplesVar = "cod",
		fctTransformDataForInputTsne = 
			function(mat)	as.dist((1 - cor(mat))/2)
	))

```

# Linear discriminant analysis: `esetLda`

Another visualization, this time semi-supervised (as a variable is used for the
computation), is included: **Linear Discriminant Analysis**. This uses the
`lda` function from the `MASS` package.

This function maximizes the variance between levels of a factor, here describing
some sample annotation, specified via the `ldaVar` argument.

As this analysis can be quite time consuming,
for the demonstration, the analysis is run only a random feature subset
of the data. 

## All samples

The `returnAnalysis` parameter can be used, to extract the
analysis which will be used as input for the `esetPlotWrapper` function,
used in the background. It is available also for the `esetSpectralMap`
and `esetTnse` functions.

```{r ALL-Lda, fig.keep = 'first'}

	# extract random features, because analysis is quite time consuming
	retainedFeatures <- sample(featureNames(ALL), size = floor(nrow(ALL)/5))
	
	# run the analysis
	outputEsetLda <- esetLda(eset = ALL[retainedFeatures, ], ldaVar = "BT",
		title = paste("Acute lymphoblastic leukemia dataset \n",
			"Linear discriminant analysis on BT variable \n",
			"(Subset of the feature data)"),
		colorVar = "BT", color = colorPalette,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topSamplesVar = "cod", topGenesVar = "SYMBOL",
		returnAnalysis = TRUE
	)

	# extract and print the ggplot object
	print(outputEsetLda$plot)
```

The top elements (here genes and samples) labelled in the plot can be accessed
via the `topElements` slot of the object. These are labelled with the
identifiers used in the plot and named with sample/gene identifiers of the
`eset`.

```{r ALL-Lda-topElements}	

	# extract top elements labelled in the plot
	pander(t(data.frame(topGenes = outputEsetLda$topElements[["topGenes"]])))
	pander(t(data.frame(topSamples = outputEsetLda$topElements[["topSamples"]])))

```

When `returnAnalysis` is set to TRUE, the output of the function can be
used as input to the `esetPlotWrapper` function, and extra parameters can
then be modified.

Here the variable used for the shape of the points for the samples is changed to
type of remission (_remissionType_ column).

```{r ALL-Lda-changeSomeParameters}

	# to change some plot parameters
	esetPlotWrapper(
		dataPlotSamples = outputEsetLda$analysis$dataPlotSamples,
		dataPlotGenes = outputEsetLda$analysis$dataPlotGenes,
		esetUsed = outputEsetLda$analysis$esetUsed,
		title = paste("Acute lymphoblastic leukemia dataset \n",
			"Linear discriminant analysis on BT variable (2) \n",
			"(Subset of the feature data)"),
		colorVar = "BT", color = colorPalette,
		shapeVar = "remissionType", 
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topSamplesVar = "cod", topGenesVar = "SYMBOL"
	)

```

```{r rmOutputEsetLdaFromMemory, echo = FALSE}

	rm(outputEsetLda);tmp <- gc(verbose = FALSE)

```

## Data sample subset

From the previous visualization (obtained on a subset of the genes), the biggest
difference between all levels of the type/stage of the disease seems to reside
between all B-cells (tagged _B_) and T-cells (tagged _T_). It might be
interesting to focus on a subset of the data, e.g. only one cell type.

```{r ALL-Lda-BcellOnly}

	# keep only 'B-cell' samples
	ALLBCell <- ALL[, grep("^B", ALL$BT)]
	ALLBCell$BT <- factor(ALLBCell$BT)
	colorPaletteBCell <- colorPalette[1:nlevels(ALLBCell$BT )]
	
	print(esetLda(eset = ALLBCell[retainedFeatures, ], ldaVar = "BT",
		title = paste("Acute lymphoblastic leukemia dataset \n",
			"Linear discriminant analysis on BT variable \n B-cell only",
			"(Subset of the feature data)"
		),
		colorVar = "BT", color = colorPaletteBCell,
		shapeVar = "sex",
		sizeVar = "age", sizeRange = c(2, 6),
		alphaVar = "remissionType", alpha = c(0.3, 0.6, 0.9),
		topSamplesVar = "cod", topGenesVar = "SYMBOL",
	))

```

```{r rmALLBCellFromMemory, echo = FALSE}

	rm(ALLBCell);tmp <- gc(verbose = FALSE)

```
The subcell type which seems to differ the most within all B-cell type is
the
first one: _B1_ (most of these samples at the right side of the plot).

# References
