GOplot/0000755000175100001440000000000012677016126011504 5ustar hornikusersGOplot/inst/0000755000175100001440000000000012676706463012472 5ustar hornikusersGOplot/inst/CITATION0000644000175100001440000000116312676706421013622 0ustar hornikuserscitHeader("To cite GOplot in publications use:") citEntry(entry = "Article", title = "GOplot: an R package for visually combining expression data with functional analysis", author = personList(as.person("Wencke Walter"), as.person("Fatima Sanchez-Cabo"), as.person("Mercedes Ricote")), journal = "Bioinformatics", year = "2015", textVersion = paste("Walter, Wencke, Fatima Sanchez-Cabo, and Mercedes Ricote.", "GOplot: an R package for visually combining expression data with functional analysis.", "Bioinformatics (2015): btv300.") ) GOplot/inst/doc/0000755000175100001440000000000012676706463013237 5ustar hornikusersGOplot/inst/doc/GOplot_vignette.Rmd0000644000175100001440000006135412676706463017025 0ustar hornikusers--- title: "GOplot 1.0.2" author: "Wencke Walter" date: "`r Sys.Date()`" output: rmarkdown::html_vignette: css: GOplot.css vignette: > %\VignetteIndexEntry{GOplot_0.2} %\VignetteEngine{knitr::rmarkdown} \usepackage[utf8]{inputenc} --- A manual to exploit the possibilities and limitations of the R package GOplot. ##Introduction The GOplot package concentrates on the visualization of biological data. More precisely, the package will help combine and integrate expression data with the results of a functional analysis. The package cannot be used to perform any of these analyses. It is for visualization purpose only. In all the scientific fields we visualize information to meet a basic need- to tell a story. Attributable to space restrictions and a general need to present everything neat and tidy most of the times it is simply not possible to actually tell a story. Therefore, we use vision to communicate information. A well designed and elaborated figure provides the beholder with high-dimensional information in a much smaller space than for example a table. The idea of the package is to provide the user with functions that allow a quick examination of large amounts of data, expose trends and find patterns & correlations within the data. Effective data visualization is an important tool in the decision making process and helps to find further pieces of the puzzle picturing the answer of your biological question. Based on that you will be able to confirm or falsify your hypotheses. You might even start to look in a different direction to investigate your topic relying on the insight a new visualization provides. The plotting functions of the package were developed with a hierarchical structure in mind; starting with a general overview and closing with definite subsets of selected genes and terms. To explain the idea let us use an example. ##The toy example GOplot comes with a manually compiled data set. Selected samples were downloaded from gene expression omnibus (accession number: *[GSE47067](http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47067)*). As a brief summary, the data set contains the transcriptomic information of endothelial cells from two steady state tissues (brain and heart). More detailed information can be found in the paper by *[Nolan et al. 2013](http://www.ncbi.nlm.nih.gov/pubmed/23871589)*. The data was normalized and a statistical analysis was performed to determine differentially expressed genes. *DAVID* functional annotation tool was used to perform a gene- annotation enrichment analysis of the set of differentially expressed genes (adjusted p-value < 0.05). The data set contains the five following items: ```{r table1, echo = FALSE, results = 'asis'} toy<-data.frame(Name=c('EC$eset','EC$genelist','EC$david','EC$genes','EC$process'),Description=c('Data frame of normalized expression values of brain and heart endothelial cells (3 replicates)','Data frame of differentially expressed genes (adjusted p-value < 0.05)','Data frame of results from a functional analysis of the differentially expressed genes performed with DAVID','Data frame of selected genes with logFC','Character vector of selected enriched biological processes'),Dimension=c('20644 x 7','2039 x 7','174 x 5','37 x 2','7')) knitr::kable(toy, colnames=c('Name','Description','Dimension (row, col)')) ``` ##Getting started As a first step we want to get an overview of the enriched GO terms of our differentially expressed genes. But before we start plotting we need to bring the data in the right format for the plotting functions. In general, the data object of the plotting functions can be created manually, but the package includes a function that does the job for you. The *circle_dat* function combines the result of the functional analysis with a list of selected genes and their logFC. Most likely a list of differentially expressed genes. *circle_dat* takes two data frames as an input. The first one contains the results of the functional analysis and should have at least four columns (category, term, genes, adjusted p-value). Additionally, a data frame of the selected genes and their logFC is needed. This data frame can be, for example, the result from a statistical analysis performed with *limma*. Let us have a look at the mentioned data frames. ```{r glimpse, warning = FALSE, message = FALSE} library(GOplot) # Load the dataset data(EC) # Get a glimpse of the data format of the results of the functional analysis... head(EC$david) # ...and of the data frame of selected genes head(EC$genelist) ``` Now, that we know what the input data looks like it's time to use the *cirlce_dat* function to create the plotting object. ```{r circ_object, warning = FALSE, message = FALSE} # Generate the plotting object circ <- circle_dat(EC$david, EC$genelist) ``` The **circ** object has eight columns with the following names: * category * ID * term * count * gene * logFC * adj_pval * zscore Since most of the gene- annotation enrichment analysis are based on the gene ontology database the package was build with this structure in mind, but is not restricted to it. As explained by *[Ashburner et al.](http://www.ncbi.nlm.nih.gov/pubmed/10802651)* in their paper from the year 2000, gene ontology is structured as an acyclic graph and it provides terms covering different areas. These terms are grouped into three independent categories: BP (biological process), CC (cellular component) or MF (molecular function). The first column of the **circ** object contains this information, which was already given in the input. For more information on the structure of gene ontology, have a look at the documentation section of the gene ontology consortium [website](http://geneontology.org/page/ontology-documentation). All the terms from inside the gene ontology database come with a GO **ID** and a GO **term** description. The **ID** column of the circ object is optional. So in case you want to use a functional analysis tool that is not based on gene ontology you won't have an **ID** column. The term description column does contain just that: a description of the term and the performance of the implemented functions does not depend on possible resemblance with gene ontology terms. **Count** is the number of genes assigned to a term. **Gene** names and their **logFC** are taken from the input list of selected genes. The significance of a term is indicated by the adjusted p-value (**adj_pval**). Terms with an adjusted p-value < 0.05 are considered as significantly enriched and are more likely to provide reliable information. The last column contains the **zscore**, an easy to calculate value to give you a hint if the biological process (/molecular function/cellular components) is more likely to be decreased (negative value) or increased (positive value). $$zscore=\frac{(up-down)}{\sqrt{count}}$$ Whereas *up* and *down* are the number of assigned genes up-regulated (logFC>0) in the data or down- regulated (logFC<0), respectively. ##The plots ###The modified barplot (GOBar) Since we do not really know what to expect from our data the aim of the first figure should be to display as many terms as possible without going too much into details. Nevertheless, the figure shall help us to pick the interesting and valuable terms. Therefore, we need to have some parameters to quantify the importance. Since the majority of scientific sampled data is plotted using bar charts a modified version of the normal barplot function, named *GOBar*, is included. The *GOBar* function allows the user to quickly create an appealing barplot. ```{r GOBar, warning = FALSE, message = FALSE, fig.width = 8.3, fig.height = 6} # Generate a simple barplot GOBar(subset(circ, category == 'BP')) ``` On the y-axis the significance of the terms is shown and the bars are ordered according to their z-score. If you want, you can change the order by setting the argument *order.by.zscore* to FALSE. In this case the bars are ordered based on their significance. Additionally, the barplot can be easily faceted according to the categories of the terms using the argument *display* of the plotting function (output not shown). ```{r GOBar2, eval = FALSE, warning = FALSE, message = FALSE} # Facet the barplot according to the categories of the terms GOBar(circ, display = 'multiple') ``` To add a title use *title* and to change the colour scale of the z-score use the argument *zsc.col* (output not shown). ```{r GOBar3, eval = FALSE, warning = FALSE, message = FALSE} # Facet the barplot, add a title and change the colour scale for the z-score GOBar(circ, display = 'multiple', title = 'Z-score coloured barplot', zsc.col = c('yellow', 'black', 'cyan')) ``` Barplots are common and very easy to read, but they might not be the absolute solution. Another possibility to display an overview for high- dimensional data is the bubble plot. ###The bubble plot (GOBubble) The bubble plot is another possibility to get an overview of the enriched terms. The z-score is assigned to the x-axis and the negative logarithm of the adjusted p-value to the y-axis, as in the barplot (the higher the more significant). The area of the displayed circles is proportional to the number of genes (circ$count) assigned to the term and the colour corresponds to the category. The help page of the plotting function (?GOBubble) lists all the arguments to change the layout of the plot. As a default the circles are labeled with the term ID. Therefore, a table connecting the IDs and terms is displayed on the right side by default. You can hide it by setting the argument *table.legend* to FALSE. If you want to display the term description instead set the argument *ID* to FALSE. Not all the circles are labeld due to the limited space and the overlap of the circles. A threshold for the labeling is set (default=5) based on the negative logarithm of the adjusted p-value. ```{r GOBubble1, warning = FALSE, message = FALSE, fig.keep = 'none'} # Generate the bubble plot with a label threshold of 3 GOBubble(circ, labels = 3) ``` ![GOBubble1.](GOBubble1.png) To add a title, change the colour of the circles, facet the plot and to change the label threshold use the following arguments: ```{r GOBubble2, warning = FALSE, message = FALSE, fig.keep = 'none'} # Add a title, change the colour of the circles, facet the plot according to the categories and change the label threshold GOBubble(circ, title = 'Bubble plot', colour = c('orange', 'darkred', 'gold'), display = 'multiple', labels = 3) ``` ![GOBubble2.](GOBubble2.png) For the facet plot it is also possible to colour the background of the panels according to the displayed category by setting *bg.col* to TRUE. ```{r GOBubble3, warning = FALSE, message = FALSE, fig.keep = 'none'} # Colour the background according to the category GOBubble(circ, title = 'Bubble plot with background colour', display = 'multiple', bg.col = T, labels = 3) ``` ![GOBubble3.](GOBubble3.png) A new function, *reduce_overlap*, was included in the updated version of the package to reduce the number of redundant terms. So far, the implemented method is very simple + slow and needs further refinement. Nevertheless, by reducing the number of redundant terms the readability of plots, like the bubble plot, improves significantly. The function deletes all terms that have a gene overlap greater than or equal to a set threshold. The function keeps one term per group as a representative without taking into consideration the GO hierarchy. ```{r GOBubble4, warning = FALSE, message = FALSE, fig.keep = 'none', eval = FALSE} # Reduce redundant terms with a gene overlap >= 0.75... reduced_circ <- reduce_overlap(circ, overlap = 0.75) # ...and plot it GOBubble(reduced_circ, labels = 2.8) ``` ![GOBubble4.](GOBubble4.png) ### Circular visualization of the results of gene- annotation enrichment analysis (GOCircle) The overview plots shall help to decide which of the terms are the most interesting to us. Of course, this decision depends although on the hypothesis and ideas you want to confirm with your data. Not always are the most significant terms the ones you are interested in. So, after manually selecting a set of valuable terms (EC$process) the next figure should provide us with more details on this specific terms. One of the major issues we figured out by presenting the plots was: it was sometimes difficult to interpret the information the z-score provides. Since the measure is not that common. As shown above it is simply the number of up- regulated genes minus the number of down- regulated genes divided by the square root of the count. The *GOCircle* plot emphasizes this fact. ```{r GOCircle1, warning = FALSE, message = FALSE, fig.keep = 'none'} # Generate a circular visualization of the results of gene- annotation enrichment analysis GOCircle(circ) ``` ![Circle plot.](GOCirc.png) The outer circle shows a scatter plot for each term of the logFC of the assigned genes. Red circles display up- regulation and blue ones down- regulation by default. The colours can be changed with the argument *lfc.col*. Therefore, it is easier to understand, why in some cases highly significant terms have a z-score close to zero. A z-score of zero does not mean that the term is not important. At least not as long as it is significantly enriched. It just shows that the z-score is a crude measure, because obviously the score does not take into account the functional level and activation dependencies of the single genes within a process. You can change the layout of the plot with various arguments, see ?GOCirlce.The *nsub* argument needs a little bit more explanation to be used wisely. First of all, it can be a numeric or a character vector. If it is a character vector then it contains the IDs or term descriptions of the processes you want to display (output not shown). ```{r GOCircle2, eval = FALSE} # Generate a circular visualization of selected terms IDs <- c('GO:0007507', 'GO:0001568', 'GO:0001944', 'GO:0048729', 'GO:0048514', 'GO:0005886', 'GO:0008092', 'GO:0008047') GOCircle(circ, nsub = IDs) ``` If *nsub* is a numeric vector then the number defines how many terms are displayed. It starts with the first row of the input data frame (output not shown). ```{r GOCircle3, eval = FALSE} # Generate a circular visualization for 10 terms GOCircle(circ, nsub = 10) ``` This kind of visualization is only useful for a smaller set of terms. The maximum number of terms lies around 12. While the number of terms decreases the amount of displayed information increases. ### Display of the relationship between genes and terms (GOChord) Based on the **[Circos](http://circos.ca/)** plots designed by *[Martin Krzywinski](http://mkweb.bcgsc.ca/)* the *GOChord* plotting function was implemented. It displays the relationship between a list of selected genes and terms, as well as the logFC of the genes. As an input a binary membership matrix is necessary. You can build the matrix on your own or you use the implemented function *chord_dat* which does the job for you. The function takes three arguments: *data*, *genes* and *process*, of which from the last two only one is mandatory. So, the *circle_dat* combined your expression data with the results from the functional analysis. The bar and bubble plot allowed you to get a first impression of your data and now, you selected a list of genes and processes you think are valuable. *GOCircle* adds a layer to display the expression values of the genes assigned to the terms, but it lacks the information of the relationship between the genes and the terms. It is not easy to figure out if some of the genes are linked to multiple processes. The chord plot fills the void left by *GOCircle*. ```{r GOChord1, warning = FALSE, message = FALSE} # Define a list of genes which you think are interesting to look at. The item EC$genes of the toy # sample contains the data frame of selected genes and their logFC. Have a look... head(EC$genes) # Since we have a lot of significantly enriched processes we selected some specific ones (EC$process) EC$process # Now it is time to generate the binary matrix chord <- chord_dat(circ, EC$genes, EC$process) head(chord) ``` Rows are genes and columns are terms. A '0' indicates that the gene is not assigned to the term; a '1' the opposite. As mentioned before it is possible to leave either the *genes* or the *process* argument out. If you pass on the *process* argument the binary matrix is build for the list of selected genes and all the processes with at least one assigned gene. On the other hand, if you just provide a set of processes without limiting the list of genes, the binary matrix is generated for all the genes which are assigned to at least one of the processes from your list (output not shown). ```{r GOChord2, eval=FALSE, warning = FALSE, message = FALSE} # Generate the matrix with a list of selected genes chord <- chord_dat(data = circ, genes = EC$genes) # Generate the matrix with selected processes chord <- chord_dat(data = circ, process = EC$process) ``` Be aware that a pass on either *genes* or *process* might lead to a large binary matrix which results in a confusing visualization. The chart was designed for smaller subsets of high-dimensional data. Like the other plotting functions *GOChord* provides the user with a lot of arguments to change the layout of the plot, see ?GOChord. Most of the arguments address the adjustment of the font size of the labels, the space between them, the colour scale for the logFC and the colour of the ribbons. Despite the asthetics there are two other arguments: *gene.order* and *nlfc*. The first argument defines the order of the genes with the three possible options: 'logFC', 'alphabetical', 'none'. Actually the options are quite self- explanatory. Sometimes you are performing the differential expression analysis for multiple conditions and/or batches. Therefore, you want to include more than one logFC value per gene. To adjust to this situation you should use the *nlfc* argument. It is a numeric value and it defines the number of logFC columns within your binary membership matrix. The default is '1' assuming that most of the time you just have one contrast and one logFC value per gene. ```{r GOChord3, warning = FALSE, message = FALSE, fig.keep = 'none'} # Create the plot GOChord(chord, space = 0.02, gene.order = 'logFC', gene.space = 0.25, gene.size = 5) ``` ![Chord1.](GOChord1.png) The *space* argument defines the space between the coloured rectangles representing the logFC. Also the font size of the gene labels (*gene.size*) and the space (*gene.space*) between them was changed. The genes were ordered according to their logFC values setting *gene.order* to 'logFC'. Sometimes the plot gets a bit crowded and you would like to reduce the number of displayed genes or processes. You can do this automatically by making use of the *limit* argument. Limit is a vector with two cutoff values (default = c(0, 0)). The first value defines the minimum (>=) number of terms a gene has to be assigned to. The second value determines the number of genes assigned to a selected term. For example, to display only genes which are assigned to at least three processes you would use the following line of code (output not shown): ```{r GOChord4, warning = FALSE, message = FALSE, fig.keep = 'none'} # Display only genes which are assigned to at least three processes GOChord(chord, limit = c(3, 0), gene.order = 'logFC') ``` ### Heatmap of genes and terms (GOHeat) Thanks to a very nice suggestion from *[Maureen Sartor, Ph.D.](http://sartorlab.ccmb.med.umich.edu/)* I implemented *GOHeat*. The *GOHeat* function generates a heatmap of the relationship between genes and terms similar to *GOChord*. Biological processes are displayed in rows and genes in columns. Each column is divided into smaller rectangles and the colouring of the tiles depends on the presence or abscence of logFC values. In addition genes are clustered to highlight groups of genes with similar annotated functions. Basically the function has two modes depending on the *nlfc* argument. If *nlfc = 0*, so no logFC values are available, the colouring encodes for the overall number of processes the respective gene is assigned to. Let's have a look at an example... ```{r GOHeat1, warning = FALSE, message = FALSE, fig.keep = 'none'} # First, we use the chord object without logFC column to create the heatmap GOHeat(chord[,-8], nlfc = 0) ``` ![Heat1.](GOHeat_nolfc.png) In case of *nlfc = 1* the colour corresponds to the logFC of the gene... ```{r GOHeat2, warning = FALSE, message = FALSE, fig.keep = 'none'} # First, we use the chord object without logFC column to create the heatmap GOHeat(chord, nlfc = 1, fill.col = c('red', 'yellow', 'green')) ``` ![Heat2.](GOHeat_lfc.png) ### Golden eye (GOCluster) The idea behind the *GOCluster* function is to visualize as much information as possible. Here is an example: ```{r GOCluster, warning=FALSE, eval=FALSE, message=FALSE, fig.keep='none'} GOCluster(circ, EC$process, clust.by = 'logFC', term.width = 2) ``` ![GOCluster.](GOCluster.png) Hierarchical clustering is a popular method for gene expression analysis due to its unsupervised nature assuring an unbiased result. Genes are grouped together based on their expression patterns, thus clusters are likely to contain sets of co-regulated or functionally related genes. *GOCluster* performs the hierarchical clustering of the gene expression profiles using the *hclust* method in core R. If you want to change the distance metric or the clustering algorithm use the arguments *metric* and *clust*, respectively. The resulting dendrogram is transformed with the help of *ggdendro* to be suitable for a visualization with *ggplot2*. As before a circular layout was chosen, because it is not only effective but also visually appealing. The first ring next to the dendrogram represents the logFC of the genes, which are actually the leaves of the clustering tree. In case you are interested in more than one contrast the *nlfc* argument is also available for this function. By default it is set to '1', so only one ring is drawn. Like always the logFC values are colour- coded with an user- definable colour scale (*lfc.col*). The next ring represents the terms assigned to the genes. For aesthetic reasons the terms should be reduced to a reasonable number with the argument *process*. The terms are colour- coded as well and you can change the default colours by using the argument *term.col*. Once again, the plotting function provides you with a bunch of arguments to change the layout of the plot and you can check them out on the help page, ?GOCluster. Probably the most important argument of the function is *clust.by*. It expects a character vector specifying if the clustering should be done for gene expression pattern ('logFC', as in the figure above) or functional categories ('terms'). ```{r GOCluster2, warning=FALSE, eval=FALSE, message=FALSE, fig.keep='none'} GOCluster(circ, EC$process, clust.by = 'term', lfc.col = c('darkgoldenrod1', 'black', 'cyan1')) ``` ![GOCluster2.](GOCluster2.png) ### Venn diagram (GOVenn) In this biological context we implemented a Venn diagram that can be used to detect relations between various lists of differentially expressed genes or to explore the intersection of genes of multiple terms from the functional analysis. The Venn diagram does not only display the number of overlap genes, but it also displays the information about the gene expression patterns (commonly up- regulated, commonly down- regulated or contra- regulated). At the moment, maximal three datasets are aloud as an input. The input data frame contains at least two columns: one for the gene names and one for the logFC value. ```{r GOVenn, warning=FALSE, message=FALSE, fig.keep='none'} l1 <- subset(circ, term == 'heart development', c(genes,logFC)) l2 <- subset(circ, term == 'plasma membrane', c(genes,logFC)) l3 <- subset(circ, term == 'tissue morphogenesis', c(genes,logFC)) GOVenn(l1,l2,l3, label = c('heart development', 'plasma membrane', 'tissue morphogenesis')) ``` ![Venn diagram.](GOVenn.png) For example, heart development and tissue morphogenesis share a set of 22 genes, whereas 5 are commonly up-regulated and 17 are commonly down-regulated. The important thing to notice is, that the pie charts don't display redundant information. Thus, if you compare three datasets the genes which are shared by all datasets (pie chart in the middle) are not included in the other pie charts. The following [link](https://wwalter.shinyapps.io/Venn/) refers to the shinyapp of this tool. The web tool is slightly more interactive since the circles are area-proportional to the number of genes of the dataset and the small pie charts can be moved with sliders. It has also all the other options of the *GOVenn* function to change the layout of the plot. You can easily download the picture and gene lists. GOplot/inst/doc/GOplot_vignette.R0000644000175100001440000001305612676706463016500 0ustar hornikusers## ----table1, echo = FALSE, results = 'asis'------------------------------ toy<-data.frame(Name=c('EC$eset','EC$genelist','EC$david','EC$genes','EC$process'),Description=c('Data frame of normalized expression values of brain and heart endothelial cells (3 replicates)','Data frame of differentially expressed genes (adjusted p-value < 0.05)','Data frame of results from a functional analysis of the differentially expressed genes performed with DAVID','Data frame of selected genes with logFC','Character vector of selected enriched biological processes'),Dimension=c('20644 x 7','2039 x 7','174 x 5','37 x 2','7')) knitr::kable(toy, colnames=c('Name','Description','Dimension (row, col)')) ## ----glimpse, warning = FALSE, message = FALSE--------------------------- library(GOplot) # Load the dataset data(EC) # Get a glimpse of the data format of the results of the functional analysis... head(EC$david) # ...and of the data frame of selected genes head(EC$genelist) ## ----circ_object, warning = FALSE, message = FALSE----------------------- # Generate the plotting object circ <- circle_dat(EC$david, EC$genelist) ## ----GOBar, warning = FALSE, message = FALSE, fig.width = 8.3, fig.height = 6---- # Generate a simple barplot GOBar(subset(circ, category == 'BP')) ## ----GOBar2, eval = FALSE, warning = FALSE, message = FALSE-------------- # # Facet the barplot according to the categories of the terms # GOBar(circ, display = 'multiple') ## ----GOBar3, eval = FALSE, warning = FALSE, message = FALSE-------------- # # Facet the barplot, add a title and change the colour scale for the z-score # GOBar(circ, display = 'multiple', title = 'Z-score coloured barplot', zsc.col = c('yellow', 'black', 'cyan')) ## ----GOBubble1, warning = FALSE, message = FALSE, fig.keep = 'none'------ # Generate the bubble plot with a label threshold of 3 GOBubble(circ, labels = 3) ## ----GOBubble2, warning = FALSE, message = FALSE, fig.keep = 'none'------ # Add a title, change the colour of the circles, facet the plot according to the categories and change the label threshold GOBubble(circ, title = 'Bubble plot', colour = c('orange', 'darkred', 'gold'), display = 'multiple', labels = 3) ## ----GOBubble3, warning = FALSE, message = FALSE, fig.keep = 'none'------ # Colour the background according to the category GOBubble(circ, title = 'Bubble plot with background colour', display = 'multiple', bg.col = T, labels = 3) ## ----GOBubble4, warning = FALSE, message = FALSE, fig.keep = 'none', eval = FALSE---- # # Reduce redundant terms with a gene overlap >= 0.75... # reduced_circ <- reduce_overlap(circ, overlap = 0.75) # # ...and plot it # GOBubble(reduced_circ, labels = 2.8) ## ----GOCircle1, warning = FALSE, message = FALSE, fig.keep = 'none'------ # Generate a circular visualization of the results of gene- annotation enrichment analysis GOCircle(circ) ## ----GOCircle2, eval = FALSE--------------------------------------------- # # Generate a circular visualization of selected terms # IDs <- c('GO:0007507', 'GO:0001568', 'GO:0001944', 'GO:0048729', 'GO:0048514', 'GO:0005886', 'GO:0008092', 'GO:0008047') # GOCircle(circ, nsub = IDs) ## ----GOCircle3, eval = FALSE--------------------------------------------- # # Generate a circular visualization for 10 terms # GOCircle(circ, nsub = 10) ## ----GOChord1, warning = FALSE, message = FALSE-------------------------- # Define a list of genes which you think are interesting to look at. The item EC$genes of the toy # sample contains the data frame of selected genes and their logFC. Have a look... head(EC$genes) # Since we have a lot of significantly enriched processes we selected some specific ones (EC$process) EC$process # Now it is time to generate the binary matrix chord <- chord_dat(circ, EC$genes, EC$process) head(chord) ## ----GOChord2, eval=FALSE, warning = FALSE, message = FALSE-------------- # # Generate the matrix with a list of selected genes # chord <- chord_dat(data = circ, genes = EC$genes) # # Generate the matrix with selected processes # chord <- chord_dat(data = circ, process = EC$process) ## ----GOChord3, warning = FALSE, message = FALSE, fig.keep = 'none'------- # Create the plot GOChord(chord, space = 0.02, gene.order = 'logFC', gene.space = 0.25, gene.size = 5) ## ----GOChord4, warning = FALSE, message = FALSE, fig.keep = 'none'------- # Display only genes which are assigned to at least three processes GOChord(chord, limit = c(3, 0), gene.order = 'logFC') ## ----GOHeat1, warning = FALSE, message = FALSE, fig.keep = 'none'-------- # First, we use the chord object without logFC column to create the heatmap GOHeat(chord[,-8], nlfc = 0) ## ----GOHeat2, warning = FALSE, message = FALSE, fig.keep = 'none'-------- # First, we use the chord object without logFC column to create the heatmap GOHeat(chord, nlfc = 1, fill.col = c('red', 'yellow', 'green')) ## ----GOCluster, warning=FALSE, eval=FALSE, message=FALSE, fig.keep='none'---- # GOCluster(circ, EC$process, clust.by = 'logFC', term.width = 2) ## ----GOCluster2, warning=FALSE, eval=FALSE, message=FALSE, fig.keep='none'---- # GOCluster(circ, EC$process, clust.by = 'term', lfc.col = c('darkgoldenrod1', 'black', 'cyan1')) ## ----GOVenn, warning=FALSE, message=FALSE, fig.keep='none'--------------- l1 <- subset(circ, term == 'heart development', c(genes,logFC)) l2 <- subset(circ, term == 'plasma membrane', c(genes,logFC)) l3 <- subset(circ, term == 'tissue morphogenesis', c(genes,logFC)) GOVenn(l1,l2,l3, label = c('heart development', 'plasma membrane', 'tissue morphogenesis')) GOplot/inst/doc/GOplot_vignette.html0000644000175100001440000651054512676706463017256 0ustar hornikusers GOplot 1.0.2

A manual to exploit the possibilities and limitations of the R package GOplot.

Introduction

The GOplot package concentrates on the visualization of biological data. More precisely, the package will help combine and integrate expression data with the results of a functional analysis. The package cannot be used to perform any of these analyses. It is for visualization purpose only. In all the scientific fields we visualize information to meet a basic need- to tell a story. Attributable to space restrictions and a general need to present everything neat and tidy most of the times it is simply not possible to actually tell a story. Therefore, we use vision to communicate information. A well designed and elaborated figure provides the beholder with high-dimensional information in a much smaller space than for example a table. The idea of the package is to provide the user with functions that allow a quick examination of large amounts of data, expose trends and find patterns & correlations within the data. Effective data visualization is an important tool in the decision making process and helps to find further pieces of the puzzle picturing the answer of your biological question. Based on that you will be able to confirm or falsify your hypotheses. You might even start to look in a different direction to investigate your topic relying on the insight a new visualization provides. The plotting functions of the package were developed with a hierarchical structure in mind; starting with a general overview and closing with definite subsets of selected genes and terms. To explain the idea let us use an example.

The toy example

GOplot comes with a manually compiled data set. Selected samples were downloaded from gene expression omnibus (accession number: GSE47067). As a brief summary, the data set contains the transcriptomic information of endothelial cells from two steady state tissues (brain and heart). More detailed information can be found in the paper by Nolan et al. 2013. The data was normalized and a statistical analysis was performed to determine differentially expressed genes. DAVID functional annotation tool was used to perform a gene- annotation enrichment analysis of the set of differentially expressed genes (adjusted p-value < 0.05). The data set contains the five following items:

Name Description Dimension
EC$eset Data frame of normalized expression values of brain and heart endothelial cells (3 replicates) 20644 x 7
EC$genelist Data frame of differentially expressed genes (adjusted p-value < 0.05) 2039 x 7
EC$david Data frame of results from a functional analysis of the differentially expressed genes performed with DAVID 174 x 5
EC$genes Data frame of selected genes with logFC 37 x 2
EC$process Character vector of selected enriched biological processes 7

Getting started

As a first step we want to get an overview of the enriched GO terms of our differentially expressed genes. But before we start plotting we need to bring the data in the right format for the plotting functions. In general, the data object of the plotting functions can be created manually, but the package includes a function that does the job for you. The circle_dat function combines the result of the functional analysis with a list of selected genes and their logFC. Most likely a list of differentially expressed genes. circle_dat takes two data frames as an input. The first one contains the results of the functional analysis and should have at least four columns (category, term, genes, adjusted p-value). Additionally, a data frame of the selected genes and their logFC is needed. This data frame can be, for example, the result from a statistical analysis performed with limma. Let us have a look at the mentioned data frames.

library(GOplot)
# Load the dataset
data(EC)
# Get a glimpse of the data format of the results of the functional analysis... 
head(EC$david)
##   Category         ID                             Term
## 1       BP GO:0007507                heart development
## 2       BP GO:0001944          vasculature development
## 3       BP GO:0001568         blood vessel development
## 4       BP GO:0048729             tissue morphogenesis
## 5       BP GO:0048514       blood vessel morphogenesis
## 6       BP GO:0051336 regulation of hydrolase activity
##                                                                                                                                                                                                                                                                                                                                                                              Genes
## 1       DLC1, NRP2, NRP1, EDN1, PDLIM3, GJA1, TTN, GJA5, ZIC3, TGFB2, CERKL, GATA6, COL4A3BP, GAB1, SEMA3C, MKL2, SLC22A5, MB, PTPRJ, RXRA, VANGL2, MYH6, TNNT2, HHEX, MURC, MIB1, FOXC2, FOXC1, ADAM19, MYL2, TCAP, EGLN1, SOX9, ITGB1, CHD7, HEXIM1, PKD2, NFATC4, PCSK5, ACTC1, TGFBR2, NF1, HSPG2, SMAD3, TBX1, TNNI3, CSRP3, FOXP1, KCNJ8, PLN, TSC2, ATP6V0A1, TGFBR3, HDAC9
## 2 GNA13, ACVRL1, NRP1, PGF, IL18, LEPR, EDN1, GJA1, FOXO1, GJA5, TGFB2, WARS, CERKL, APOE, CXCR4, ANG, SEMA3C, NOS2, MKL2, FGF2, RAPGEF1, PTPRJ, RECK, EFNB2, VASH1, PNPLA6, THY1, MIB1, NUS1, FOXC2, FOXC1, CAV1, CDH2, MEIS1, WT1, CDH5, PTK2, FBXW8, CHD7, PLCD1, PLXND1, FIGF, PPAP2B, MAP2K1, TBX4, TGFBR2, NF1, TBX1, TNNI3, LAMA4, MEOX2, ECSCR, HBEGF, AMOT, TGFBR3, HDAC7
## 3        GNA13, ACVRL1, NRP1, PGF, IL18, LEPR, EDN1, GJA1, FOXO1, GJA5, TGFB2, WARS, CERKL, APOE, CXCR4, ANG, SEMA3C, NOS2, MKL2, FGF2, RAPGEF1, PTPRJ, RECK, VASH1, PNPLA6, THY1, MIB1, NUS1, FOXC2, FOXC1, CAV1, CDH2, MEIS1, WT1, CDH5, PTK2, FBXW8, CHD7, PLCD1, PLXND1, FIGF, PPAP2B, MAP2K1, TBX4, TGFBR2, NF1, TBX1, TNNI3, LAMA4, MEOX2, ECSCR, HBEGF, AMOT, TGFBR3, HDAC7
## 4                                   DLC1, ENAH, NRP1, PGF, ZIC2, TGFB2, CD44, ILK, SEMA3C, RET, AR, RXRA, VANGL2, LEF1, TNNT2, HHEX, MIB1, NCOA3, FOXC2, FOXC1, TGFB1I1, WNT5A, COBL, BBS4, FGFR3, TNC, BMPR2, CTNND1, EGLN1, NR3C1, SOX9, TCF7L1, IGF1R, FOXQ1, MACF1, HOXA5, BCL2, PLXND1, CAR2, ACTC1, TBX4, SMAD3, FZD3, SHANK3, FZD6, HOXB4, FREM2, TSC2, ZIC5, TGFBR3, APAF1
## 5                                                                                            GNA13, CAV1, ACVRL1, NRP1, PGF, IL18, LEPR, EDN1, GJA1, CDH2, MEIS1, WT1, TGFB2, WARS, PTK2, CERKL, APOE, CXCR4, ANG, SEMA3C, PLCD1, NOS2, MKL2, PLXND1, FIGF, FGF2, PTPRJ, TGFBR2, TBX4, NF1, TBX1, TNNI3, PNPLA6, VASH1, THY1, NUS1, MEOX2, ECSCR, AMOT, HBEGF, FOXC2, FOXC1, HDAC7
## 6                                                                               CAV1, XIAP, AGFG1, ADORA2A, TNNC1, TBC1D9, LEPR, ABHD5, EDN1, ASAP2, ASAP3, SMAP1, TBC1D12, ANG, TBC1D14, MTCH1, TBC1D13, TBC1D4, TBC1D30, DHCR24, HIP1, VAV3, NOS1, NF1, MYH6, RICTOR, TBC1D22A, THY1, PLCE1, RNF7, NDEL1, CHML, IFT57, ACAP2, TSC2, ERN1, APAF1, ARAP3, ARAP2, ARAP1, HTR2A, F2R
##      adj_pval
## 1 0.000002170
## 2 0.000010400
## 3 0.000007620
## 4 0.000119000
## 5 0.000720000
## 6 0.001171166
# ...and of the data frame of selected genes
head(EC$genelist)
##        ID    logFC   AveExpr        t  P.Value adj.P.Val        B
## 1 Slco1a4 6.645388 1.2168670 88.65515 1.32e-18  2.73e-14 29.02715
## 2 Slc19a3 6.281525 1.1600468 69.95094 2.41e-17  2.49e-13 27.62917
## 3     Ddc 4.483338 0.8365231 65.57836 5.31e-17  3.65e-13 27.18476
## 4 Slco1c1 6.469384 1.3558865 59.87613 1.62e-16  8.34e-13 26.51242
## 5  Sema3c 5.515630 2.3252117 58.53141 2.14e-16  8.81e-13 26.33626
## 6 Slc38a3 4.761755 0.9218670 54.11559 5.58e-16  1.76e-12 25.70308

Now, that we know what the input data looks like it’s time to use the cirlce_dat function to create the plotting object.

# Generate the plotting object
circ <- circle_dat(EC$david, EC$genelist)

The circ object has eight columns with the following names:

Since most of the gene- annotation enrichment analysis are based on the gene ontology database the package was build with this structure in mind, but is not restricted to it. As explained by Ashburner et al. in their paper from the year 2000, gene ontology is structured as an acyclic graph and it provides terms covering different areas. These terms are grouped into three independent categories: BP (biological process), CC (cellular component) or MF (molecular function). The first column of the circ object contains this information, which was already given in the input. For more information on the structure of gene ontology, have a look at the documentation section of the gene ontology consortium website. All the terms from inside the gene ontology database come with a GO ID and a GO term description. The ID column of the circ object is optional. So in case you want to use a functional analysis tool that is not based on gene ontology you won’t have an ID column. The term description column does contain just that: a description of the term and the performance of the implemented functions does not depend on possible resemblance with gene ontology terms. Count is the number of genes assigned to a term. Gene names and their logFC are taken from the input list of selected genes. The significance of a term is indicated by the adjusted p-value (adj_pval). Terms with an adjusted p-value < 0.05 are considered as significantly enriched and are more likely to provide reliable information. The last column contains the zscore, an easy to calculate value to give you a hint if the biological process (/molecular function/cellular components) is more likely to be decreased (negative value) or increased (positive value).

\[zscore=\frac{(up-down)}{\sqrt{count}}\]

Whereas up and down are the number of assigned genes up-regulated (logFC>0) in the data or down- regulated (logFC<0), respectively.

The plots

The modified barplot (GOBar)

Since we do not really know what to expect from our data the aim of the first figure should be to display as many terms as possible without going too much into details. Nevertheless, the figure shall help us to pick the interesting and valuable terms. Therefore, we need to have some parameters to quantify the importance. Since the majority of scientific sampled data is plotted using bar charts a modified version of the normal barplot function, named GOBar, is included. The GOBar function allows the user to quickly create an appealing barplot.

# Generate a simple barplot
GOBar(subset(circ, category == 'BP'))

On the y-axis the significance of the terms is shown and the bars are ordered according to their z-score. If you want, you can change the order by setting the argument order.by.zscore to FALSE. In this case the bars are ordered based on their significance. Additionally, the barplot can be easily faceted according to the categories of the terms using the argument display of the plotting function (output not shown).

# Facet the barplot according to the categories of the terms 
GOBar(circ, display = 'multiple')

To add a title use title and to change the colour scale of the z-score use the argument zsc.col (output not shown).

# Facet the barplot, add a title and change the colour scale for the z-score
GOBar(circ, display = 'multiple', title = 'Z-score coloured barplot', zsc.col = c('yellow', 'black', 'cyan'))

Barplots are common and very easy to read, but they might not be the absolute solution. Another possibility to display an overview for high- dimensional data is the bubble plot.

The bubble plot (GOBubble)

The bubble plot is another possibility to get an overview of the enriched terms. The z-score is assigned to the x-axis and the negative logarithm of the adjusted p-value to the y-axis, as in the barplot (the higher the more significant). The area of the displayed circles is proportional to the number of genes (circ$count) assigned to the term and the colour corresponds to the category. The help page of the plotting function (?GOBubble) lists all the arguments to change the layout of the plot. As a default the circles are labeled with the term ID. Therefore, a table connecting the IDs and terms is displayed on the right side by default. You can hide it by setting the argument table.legend to FALSE. If you want to display the term description instead set the argument ID to FALSE. Not all the circles are labeld due to the limited space and the overlap of the circles. A threshold for the labeling is set (default=5) based on the negative logarithm of the adjusted p-value.

# Generate the bubble plot with a label threshold of 3
GOBubble(circ, labels = 3)

GOBubble1.

To add a title, change the colour of the circles, facet the plot and to change the label threshold use the following arguments:

# Add a title, change the colour of the circles, facet the plot according to the categories and change the label threshold
GOBubble(circ, title = 'Bubble plot', colour = c('orange', 'darkred', 'gold'), display = 'multiple', labels = 3)

GOBubble2.

For the facet plot it is also possible to colour the background of the panels according to the displayed category by setting bg.col to TRUE.

# Colour the background according to the category
GOBubble(circ, title = 'Bubble plot with background colour', display = 'multiple', bg.col = T, labels = 3)

GOBubble3.

A new function, reduce_overlap, was included in the updated version of the package to reduce the number of redundant terms. So far, the implemented method is very simple + slow and needs further refinement. Nevertheless, by reducing the number of redundant terms the readability of plots, like the bubble plot, improves significantly. The function deletes all terms that have a gene overlap greater than or equal to a set threshold. The function keeps one term per group as a representative without taking into consideration the GO hierarchy.

# Reduce redundant terms with a gene overlap >= 0.75...
reduced_circ <- reduce_overlap(circ, overlap = 0.75)
# ...and plot it
GOBubble(reduced_circ, labels = 2.8)

GOBubble4.

Circular visualization of the results of gene- annotation enrichment analysis (GOCircle)

The overview plots shall help to decide which of the terms are the most interesting to us. Of course, this decision depends although on the hypothesis and ideas you want to confirm with your data. Not always are the most significant terms the ones you are interested in. So, after manually selecting a set of valuable terms (EC$process) the next figure should provide us with more details on this specific terms. One of the major issues we figured out by presenting the plots was: it was sometimes difficult to interpret the information the z-score provides. Since the measure is not that common. As shown above it is simply the number of up- regulated genes minus the number of down- regulated genes divided by the square root of the count. The GOCircle plot emphasizes this fact.

# Generate a circular visualization of the results of gene- annotation enrichment analysis
GOCircle(circ)

Circle plot.

The outer circle shows a scatter plot for each term of the logFC of the assigned genes. Red circles display up- regulation and blue ones down- regulation by default. The colours can be changed with the argument lfc.col. Therefore, it is easier to understand, why in some cases highly significant terms have a z-score close to zero. A z-score of zero does not mean that the term is not important. At least not as long as it is significantly enriched. It just shows that the z-score is a crude measure, because obviously the score does not take into account the functional level and activation dependencies of the single genes within a process. You can change the layout of the plot with various arguments, see ?GOCirlce.The nsub argument needs a little bit more explanation to be used wisely. First of all, it can be a numeric or a character vector. If it is a character vector then it contains the IDs or term descriptions of the processes you want to display (output not shown).

# Generate a circular visualization of selected terms
IDs <- c('GO:0007507', 'GO:0001568', 'GO:0001944', 'GO:0048729', 'GO:0048514', 'GO:0005886', 'GO:0008092', 'GO:0008047')
GOCircle(circ, nsub = IDs)

If nsub is a numeric vector then the number defines how many terms are displayed. It starts with the first row of the input data frame (output not shown).

# Generate a circular visualization for 10 terms
GOCircle(circ, nsub = 10)

This kind of visualization is only useful for a smaller set of terms. The maximum number of terms lies around 12. While the number of terms decreases the amount of displayed information increases.

Display of the relationship between genes and terms (GOChord)

Based on the Circos plots designed by Martin Krzywinski the GOChord plotting function was implemented. It displays the relationship between a list of selected genes and terms, as well as the logFC of the genes. As an input a binary membership matrix is necessary. You can build the matrix on your own or you use the implemented function chord_dat which does the job for you. The function takes three arguments: data, genes and process, of which from the last two only one is mandatory. So, the circle_dat combined your expression data with the results from the functional analysis. The bar and bubble plot allowed you to get a first impression of your data and now, you selected a list of genes and processes you think are valuable. GOCircle adds a layer to display the expression values of the genes assigned to the terms, but it lacks the information of the relationship between the genes and the terms. It is not easy to figure out if some of the genes are linked to multiple processes. The chord plot fills the void left by GOCircle.

# Define a list of genes which you think are interesting to look at. The item EC$genes of the toy 
# sample contains the data frame of selected genes and their logFC. Have a look...
head(EC$genes)
##      ID      logFC
## 1  PTK2 -0.6527904
## 2 GNA13  0.3711599
## 3  LEPR  2.6539788
## 4  APOE  0.8698346
## 5 CXCR4 -2.5647537
## 6  RECK  3.6926860
# Since we have a lot of significantly enriched processes we selected some specific ones (EC$process)
EC$process
## [1] "heart development"        "phosphorylation"         
## [3] "vasculature development"  "blood vessel development"
## [5] "tissue morphogenesis"     "cell adhesion"           
## [7] "plasma membrane"
# Now it is time to generate the binary matrix
chord <- chord_dat(circ, EC$genes, EC$process)
head(chord)
##       heart development phosphorylation vasculature development
## PTK2                  0               1                       1
## GNA13                 0               0                       1
## LEPR                  0               0                       1
## APOE                  0               0                       1
## CXCR4                 0               0                       1
## RECK                  0               0                       1
##       blood vessel development tissue morphogenesis cell adhesion
## PTK2                         1                    0             0
## GNA13                        1                    0             0
## LEPR                         1                    0             0
## APOE                         1                    0             0
## CXCR4                        1                    0             0
## RECK                         1                    0             0
##       plasma membrane      logFC
## PTK2                1 -0.6527904
## GNA13               1  0.3711599
## LEPR                1  2.6539788
## APOE                1  0.8698346
## CXCR4               1 -2.5647537
## RECK                1  3.6926860

Rows are genes and columns are terms. A ‘0’ indicates that the gene is not assigned to the term; a ‘1’ the opposite. As mentioned before it is possible to leave either the genes or the process argument out. If you pass on the process argument the binary matrix is build for the list of selected genes and all the processes with at least one assigned gene. On the other hand, if you just provide a set of processes without limiting the list of genes, the binary matrix is generated for all the genes which are assigned to at least one of the processes from your list (output not shown).

# Generate the matrix with a list of selected genes
chord <- chord_dat(data = circ, genes = EC$genes)
# Generate the matrix with selected processes
chord <- chord_dat(data = circ, process = EC$process)

Be aware that a pass on either genes or process might lead to a large binary matrix which results in a confusing visualization. The chart was designed for smaller subsets of high-dimensional data. Like the other plotting functions GOChord provides the user with a lot of arguments to change the layout of the plot, see ?GOChord. Most of the arguments address the adjustment of the font size of the labels, the space between them, the colour scale for the logFC and the colour of the ribbons. Despite the asthetics there are two other arguments: gene.order and nlfc. The first argument defines the order of the genes with the three possible options: ‘logFC’, ‘alphabetical’, ‘none’. Actually the options are quite self- explanatory. Sometimes you are performing the differential expression analysis for multiple conditions and/or batches. Therefore, you want to include more than one logFC value per gene. To adjust to this situation you should use the nlfc argument. It is a numeric value and it defines the number of logFC columns within your binary membership matrix. The default is ‘1’ assuming that most of the time you just have one contrast and one logFC value per gene.

# Create the plot
GOChord(chord, space = 0.02, gene.order = 'logFC', gene.space = 0.25, gene.size = 5)

Chord1.

The space argument defines the space between the coloured rectangles representing the logFC. Also the font size of the gene labels (gene.size) and the space (gene.space) between them was changed. The genes were ordered according to their logFC values setting gene.order to ‘logFC’.

Sometimes the plot gets a bit crowded and you would like to reduce the number of displayed genes or processes. You can do this automatically by making use of the limit argument. Limit is a vector with two cutoff values (default = c(0, 0)). The first value defines the minimum (>=) number of terms a gene has to be assigned to. The second value determines the number of genes assigned to a selected term. For example, to display only genes which are assigned to at least three processes you would use the following line of code (output not shown):

# Display only genes which are assigned to at least three processes
GOChord(chord, limit = c(3, 0), gene.order = 'logFC')

Heatmap of genes and terms (GOHeat)

Thanks to a very nice suggestion from Maureen Sartor, Ph.D. I implemented GOHeat. The GOHeat function generates a heatmap of the relationship between genes and terms similar to GOChord. Biological processes are displayed in rows and genes in columns. Each column is divided into smaller rectangles and the colouring of the tiles depends on the presence or abscence of logFC values. In addition genes are clustered to highlight groups of genes with similar annotated functions. Basically the function has two modes depending on the nlfc argument. If nlfc = 0, so no logFC values are available, the colouring encodes for the overall number of processes the respective gene is assigned to. Let’s have a look at an example…

# First, we use the chord object without logFC column to create the heatmap
GOHeat(chord[,-8], nlfc = 0)

Heat1.

In case of nlfc = 1 the colour corresponds to the logFC of the gene…

# First, we use the chord object without logFC column to create the heatmap
GOHeat(chord, nlfc = 1, fill.col = c('red', 'yellow', 'green'))

Heat2.

Golden eye (GOCluster)

The idea behind the GOCluster function is to visualize as much information as possible. Here is an example:

GOCluster(circ, EC$process, clust.by = 'logFC', term.width = 2)

GOCluster.

Hierarchical clustering is a popular method for gene expression analysis due to its unsupervised nature assuring an unbiased result. Genes are grouped together based on their expression patterns, thus clusters are likely to contain sets of co-regulated or functionally related genes. GOCluster performs the hierarchical clustering of the gene expression profiles using the hclust method in core R. If you want to change the distance metric or the clustering algorithm use the arguments metric and clust, respectively. The resulting dendrogram is transformed with the help of ggdendro to be suitable for a visualization with ggplot2. As before a circular layout was chosen, because it is not only effective but also visually appealing. The first ring next to the dendrogram represents the logFC of the genes, which are actually the leaves of the clustering tree. In case you are interested in more than one contrast the nlfc argument is also available for this function. By default it is set to ‘1’, so only one ring is drawn. Like always the logFC values are colour- coded with an user- definable colour scale (lfc.col). The next ring represents the terms assigned to the genes. For aesthetic reasons the terms should be reduced to a reasonable number with the argument process. The terms are colour- coded as well and you can change the default colours by using the argument term.col. Once again, the plotting function provides you with a bunch of arguments to change the layout of the plot and you can check them out on the help page, ?GOCluster. Probably the most important argument of the function is clust.by. It expects a character vector specifying if the clustering should be done for gene expression pattern (‘logFC’, as in the figure above) or functional categories (‘terms’).

GOCluster(circ, EC$process, clust.by = 'term', lfc.col = c('darkgoldenrod1', 'black', 'cyan1'))

GOCluster2.

Venn diagram (GOVenn)

In this biological context we implemented a Venn diagram that can be used to detect relations between various lists of differentially expressed genes or to explore the intersection of genes of multiple terms from the functional analysis. The Venn diagram does not only display the number of overlap genes, but it also displays the information about the gene expression patterns (commonly up- regulated, commonly down- regulated or contra- regulated). At the moment, maximal three datasets are aloud as an input. The input data frame contains at least two columns: one for the gene names and one for the logFC value.

l1 <- subset(circ, term == 'heart development', c(genes,logFC))
l2 <- subset(circ, term == 'plasma membrane', c(genes,logFC))
l3 <- subset(circ, term == 'tissue morphogenesis', c(genes,logFC))
GOVenn(l1,l2,l3, label = c('heart development', 'plasma membrane', 'tissue morphogenesis'))

Venn diagram.

For example, heart development and tissue morphogenesis share a set of 22 genes, whereas 5 are commonly up-regulated and 17 are commonly down-regulated. The important thing to notice is, that the pie charts don’t display redundant information. Thus, if you compare three datasets the genes which are shared by all datasets (pie chart in the middle) are not included in the other pie charts. The following link refers to the shinyapp of this tool. The web tool is slightly more interactive since the circles are area-proportional to the number of genes of the dataset and the small pie charts can be moved with sliders. It has also all the other options of the GOVenn function to change the layout of the plot. You can easily download the picture and gene lists.

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hornikusersGOplot/R/Helper.R0000644000175100001440000002414512676706421013260 0ustar hornikusers############## # In general # ############## # Theme blank theme_blank <- theme(axis.line = element_blank(), axis.text.x = element_blank(), axis.text.y = element_blank(), axis.ticks = element_blank(), axis.title.x = element_blank(), axis.title.y = element_blank(), panel.background = element_blank(), panel.border = element_blank(), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), plot.background = element_blank()) # Draw adjacent table for GOBubble and GOCircle draw_table <- function(data, col){ id <- term <- NULL colnames(data) <- tolower(colnames(data)) if (missing(col)){ tt1 <- ttheme_default() }else{ text.col <- c(rep(col[1], sum(data$category == 'BP')), rep(col[2], sum(data$category == 'CC')), rep(col[3], sum(data$category == 'MF'))) tt1 <- ttheme_minimal( core = list(bg_params = list(fill = text.col, col=NA, alpha= 1/3)), colhead = list(fg_params = list(col = "black"))) } table <- tableGrob(subset(data, select = c(id, term)), cols = c('ID', 'Description'), rows = NULL, theme = tt1) return(table) } ########### # GOChord # ########### # Bezier function for drawing ribbons bezier <- function(data, process.col){ x <- c() y <- c() Id <- c() sequ <- seq(0, 1, by = 0.01) N <- dim(data)[1] sN <- seq(1, N, by = 2) if (process.col[1] == '') col_rain <- grDevices::rainbow(N) else col_rain <- process.col for (n in sN){ xval <- c(); xval2 <- c(); yval <- c(); yval2 <- c() for (t in sequ){ xva <- (1 - t) * (1 - t) * data$x.start[n] + t * t * data$x.end[n] xval <- c(xval, xva) xva2 <- (1 - t) * (1 - t) * data$x.start[n + 1] + t * t * data$x.end[n + 1] xval2 <- c(xval2, xva2) yva <- (1 - t) * (1 - t) * data$y.start[n] + t * t * data$y.end[n] yval <- c(yval, yva) yva2 <- (1 - t) * (1 - t) * data$y.start[n + 1] + t * t * data$y.end[n + 1] yval2 <- c(yval2, yva2) } x <- c(x, xval, rev(xval2)) y <- c(y, yval, rev(yval2)) Id <- c(Id, rep(n, 2 * length(sequ))) } df <- data.frame(lx = x, ly = y, ID = Id) return(df) } # Check function for GOChord argument 'limit' check_chord <- function(mat, limit){ if(all(colSums(mat) >= limit[2]) & all(rowSums(mat) >= limit[1])) return(mat) tmp <- mat[(rowSums(mat) >= limit[1]),] mat <- tmp[,(colSums(tmp) >= limit[2])] mat <- check_chord(mat, limit) return(mat) } ########## # GOVenn # ########## # Calculate points to draw a circle circleFun <- function(center = c(0,0),diameter = 1, npoints = 100){ r = diameter / 2 tt <- seq(0,2*pi,length.out = npoints) xx <- center[1] + r * cos(tt) yy <- center[2] + r * sin(tt) return(data.frame(x = xx, y = yy)) } # Calculate overlap for three lists get_overlap<-function(A,B,C){ colnames(A)<-c('ID','logFC') colnames(B)<-c('ID','logFC') colnames(C)<-c('ID','logFC') UP<-NULL;DOWN<-NULL;Change<-NULL if (class(A$logFC)!='numeric'){ A$logFC<-gsub(",", ".", gsub("\\.", "", A$logFC)) A$Trend<-sapply(as.numeric(A$logFC), function(x) ifelse(x > 0,'UP','DOWN')) }else{ A$Trend<-sapply(A$logFC, function(x) ifelse(x > 0,'UP','DOWN'))} if (class(B$logFC)!='numeric'){ B$logFC<-gsub(",", ".", gsub("\\.", "", B$logFC)) B$Trend<-sapply(as.numeric(B$logFC), function(x) ifelse(x > 0,'UP','DOWN')) }else{ B$Trend<-sapply(B$logFC, function(x) ifelse(x > 0,'UP','DOWN'))} if (class(C$logFC)!='numeric'){ C$logFC<-gsub(",", ".", gsub("\\.", "", C$logFC)) C$Trend<-sapply(as.numeric(C$logFC), function(x) ifelse(x > 0,'UP','DOWN')) }else{ C$Trend<-sapply(C$logFC, function(x) ifelse(x > 0,'UP','DOWN'))} if (sum(((A$ID%in%B$ID)==T)==T)==0){ AB<-data.frame() }else{ AB<-A[(A$ID%in%B$ID)==T,which(colnames(A)%in%c('ID','logFC','Trend'))] BA<-B[(B$ID%in%A$ID)==T,which(colnames(B)%in%c('ID','logFC','Trend'))] AB<-merge(AB,BA,by="ID") rownames(AB)<-AB$ID AB<-AB[,-1] } if (sum(((A$ID%in%C$ID)==T)==T)==0){ AC<-data.frame() }else{ AC<-A[(A$ID%in%C$ID)==T,which(colnames(A)%in%c('ID','logFC','Trend'))] CA<-C[(C$ID%in%A$ID)==T,which(colnames(C)%in%c('ID','logFC','Trend'))] AC<-merge(AC,CA,by="ID") rownames(AC)<-AC$ID AC<-AC[,-1] } if (sum(((B$ID%in%C$ID)==T)==T)==0){ BC<-data.frame() }else{ BC<-B[(B$ID%in%C$ID)==T,which(colnames(B)%in%c('ID','logFC','Trend'))] CB<-C[(C$ID%in%B$ID)==T,which(colnames(C)%in%c('ID','logFC','Trend'))] BC<-merge(BC,CB,by="ID") rownames(BC)<-BC$ID BC<-BC[,-1] } if (sum(((A$ID%in%B$ID)==T & (A$ID%in%C$ID)==T))==0){ ABC<-data.frame() }else{ ABC<-A[((A$ID%in%B$ID)==T & (A$ID%in%C$ID)==T),which(colnames(A)%in%c('ID','logFC','Trend'))] BAC<-B[((B$ID%in%A$ID)==T & (B$ID%in%C$ID)==T),which(colnames(B)%in%c('ID','logFC','Trend'))] CAB<-C[((C$ID%in%A$ID)==T & (C$ID%in%B$ID)==T),which(colnames(C)%in%c('ID','logFC','Trend'))] ABC<-merge(ABC,BAC,by='ID') ABC<-merge(ABC,CAB,by='ID') rownames(ABC)<-ABC$ID ABC<-ABC[,-1] } A_only<-A[((A$ID%in%B$ID)==F & (A$ID%in%C$ID)==F),which(colnames(A)%in%c('ID','logFC','Trend'))] rownames(A_only)<-A_only$ID A_only<-A_only[,-1] B_only<-B[((B$ID%in%A$ID)==F & (B$ID%in%C$ID)==F),which(colnames(A)%in%c('ID','logFC','Trend'))] rownames(B_only)<-B_only$ID B_only<-B_only[,-1] C_only<-C[((C$ID%in%A$ID)==F & (C$ID%in%B$ID)==F),which(colnames(A)%in%c('ID','logFC','Trend'))] rownames(C_only)<-C_only$ID C_only<-C_only[,-1] UP<-c(UP,sum(A_only$Trend=='UP'));DOWN<-c(DOWN,sum(A_only$Trend=='DOWN'));Change<-c(Change,sum(A_only$Trend=='Change')) UP<-c(UP,sum(B_only$Trend=='UP'));DOWN<-c(DOWN,sum(B_only$Trend=='DOWN'));Change<-c(Change,sum(B_only$Trend=='Change')) UP<-c(UP,sum(C_only$Trend=='UP'));DOWN<-c(DOWN,sum(C_only$Trend=='DOWN'));Change<-c(Change,sum(C_only$Trend=='Change')) if (dim(AB)[1]==0){ OvAB<-data.frame() UP<-c(UP,0);DOWN<-c(DOWN,0);Change<-c(Change,0) }else{ tmp<-NULL for (t in 1:dim(AB)[1]) tmp<-c(tmp,ifelse(AB$Trend.x[t]==AB$Trend.y[t],AB$Trend.x[t],'Change')) OvAB<-data.frame(logFC_A=AB$logFC.x,logFC_B=AB$logFC.y,Trend=tmp) rownames(OvAB)<-rownames(AB) AB<-OvAB[order(OvAB$Trend),] UP<-c(UP,sum(tmp=='UP'));DOWN<-c(DOWN,sum(tmp=='DOWN'));Change<-c(Change,sum(tmp=='Change')) } if (dim(AC)[1]==0){ OvAc<-data.frame() UP<-c(UP,0);DOWN<-c(DOWN,0);Change<-c(Change,0) }else{ tmp<-NULL for (t in 1:dim(AC)[1]) tmp<-c(tmp,ifelse(AC$Trend.x[t]==AC$Trend.y[t],AC$Trend.x[t],'Change')) OvAC<-data.frame(logFC_A=AC$logFC.x,logFC_C=AC$logFC.y,Trend=tmp) rownames(OvAC)<-rownames(AC) AC<-OvAC[order(OvAC$Trend),] UP<-c(UP,sum(tmp=='UP'));DOWN<-c(DOWN,sum(tmp=='DOWN'));Change<-c(Change,sum(tmp=='Change')) } if (dim(BC)[1]==0){ OvBC<-data.frame() UP<-c(UP,0);DOWN<-c(DOWN,0);Change<-c(Change,0) }else{ tmp<-NULL for (t in 1:dim(BC)[1]) tmp<-c(tmp,ifelse(BC$Trend.x[t]==BC$Trend.y[t],BC$Trend.x[t],'Change')) OvBC<-data.frame(logFC_B=BC$logFC.x,logFC_C=BC$logFC.y,Trend=tmp) rownames(OvBC)<-rownames(BC) BC<-OvBC[order(OvBC$Trend),] UP<-c(UP,sum(tmp=='UP'));DOWN<-c(DOWN,sum(tmp=='DOWN'));Change<-c(Change,sum(tmp=='Change')) } if (dim(ABC)[1]==0){ OvABC<-data.frame() UP<-c(UP,0);DOWN<-c(DOWN,0);Change<-c(Change,0) }else{ tmp<-NULL for (t in 1:dim(ABC)[1]) tmp<-c(tmp,ifelse(((ABC$Trend.x[t]==ABC$Trend.y[t]) & (ABC$Trend.x[t]==ABC$Trend[t])),ABC$Trend.x[t],'Change')) OvABC<-data.frame(logFC_A=ABC$logFC.x,logFC_B=ABC$logFC.y,logFC_C=ABC$logFC,Trend=tmp) rownames(OvABC)<-rownames(ABC) ABC<-OvABC[order(OvABC$Trend),] UP<-c(UP,sum(tmp=='UP'));DOWN<-c(DOWN,sum(tmp=='DOWN'));Change<-c(Change,sum(tmp=='Change')) } counts<-data.frame(Contrast=c('A_only','B_only','C_only','AB','AC','BC','ABC'),Count=c(dim(A_only)[1],dim(B_only)[1],dim(C_only)[1],dim(AB)[1],dim(AC)[1],dim(BC)[1],dim(ABC)[1]),UP=UP,DOWN=DOWN,Change=Change) venn<-list(A_only=A_only,B_only=B_only,C_only=C_only,AB=AB,BC=BC,AC=AC,ABC=ABC) return(list(venn_df=counts,table=venn)) } # Caluclate overlap for two lists get_overlap2<-function(A,B){ colnames(A)<-c('ID','logFC') colnames(B)<-c('ID','logFC') UP<-NULL;DOWN<-NULL;Change<-NULL if (class(A$logFC)!='numeric'){ A$logFC<-gsub(",", ".", gsub("\\.", "", A$logFC)) A$Trend<-sapply(as.numeric(A$logFC), function(x) ifelse(x > 0,'UP','DOWN')) }else{ A$Trend<-sapply(A$logFC, function(x) ifelse(x > 0,'UP','DOWN'))} if (class(B$logFC)!='numeric'){ B$logFC<-gsub(",", ".", gsub("\\.", "", B$logFC)) B$Trend<-sapply(as.numeric(B$logFC), function(x) ifelse(x > 0,'UP','DOWN')) }else{ B$Trend<-sapply(B$logFC, function(x) ifelse(x > 0,'UP','DOWN'))} AB<-A[(A$ID%in%B$ID)==T,which(colnames(A)%in%c('ID','logFC','Trend'))] BA<-B[(B$ID%in%A$ID)==T,which(colnames(B)%in%c('ID','logFC','Trend'))] A_only<-A[(A$ID%in%B$ID)==F,which(colnames(A)%in%c('ID','logFC','Trend'))] B_only<-B[(B$ID%in%A$ID)==F,which(colnames(B)%in%c('ID','logFC','Trend'))] AB<-merge(AB,BA,by='ID') UP<-c(UP,sum(A_only$Trend=='UP'));DOWN<-c(DOWN,sum(A_only$Trend=='DOWN'));Change<-c(Change,sum(A_only$Trend=='Change')) UP<-c(UP,sum(B_only$Trend=='UP'));DOWN<-c(DOWN,sum(B_only$Trend=='DOWN'));Change<-c(Change,sum(B_only$Trend=='Change')) rownames(A_only)<-A_only$ID A_only<-A_only[,-1] A_only<-A_only[order(A_only$Trend),] rownames(B_only)<-B_only$ID B_only<-B_only[,-1] B_only<-B_only[order(B_only$Trend),] tmp<-NULL for (t in 1:dim(AB)[1]) tmp<-c(tmp,ifelse(AB$Trend.x[t]==AB$Trend.y[t],AB$Trend.x[t],'Change')) OvAB<-data.frame(logFC_A=AB$logFC.x,logFC_B=AB$logFC.y,Trend=tmp) rownames(OvAB)<-AB$ID AB<-OvAB[order(OvAB$Trend),] UP<-c(UP,sum(tmp=='UP'));DOWN<-c(DOWN,sum(tmp=='DOWN'));Change<-c(Change,sum(tmp=='Change')) counts<-data.frame(Contrast=c('A_only','B_only','AB'),Count=c(dim(A_only)[1],dim(B_only)[1],dim(AB)[1]),UP=UP,DOWN=DOWN,Change=Change) venn<-list(A_only=A_only,B_only=B_only,AB=AB) return(list(venn_df=counts,table=venn,dim=c(dim(A)[1],dim(B)[1]))) }GOplot/R/GOVenn.R0000644000175100001440000002111612676706421013170 0ustar hornikusers#' #' @name GOVenn #' @title Venn diagram of differentially expressed genes. #' @description The function compares lists of differentially expressed genes #' and illustrates possible relations.Additionally it represents the variety #' of gene expression patterns within the intersection in small pie charts #' with three segements. Clockwise are shown the number of commonly up- #' regulated, commonly down- regulated and contra- regulated genes. #' @param data1 A data frame consisting of two columns: ID, logFC #' @param data2 A data frame consisting of two columns: ID, logFC #' @param data3 A data frame consisting of two columns: ID, logFC #' @param title The title of the plot #' @param label A character vector to define the legend keys #' @param lfc.col A character vector determining the background colors of the #' pie segments representing up- and down- regulated genes #' @param circle.col A character vector to assign clockwise colors for the #' circles #' @param plot If TRUE only the venn diagram is plotted. Otherwise the function #' returns a list with two items: the actual plot and a list containing the #' overlap entries (default= TRUE) #' @details The \code{plot} argument can be used to adjust the amount of #' information that is returned by calling the function. If you are only #' interested in the actual plot of the venn diagram, \code{plot} should be #' set to TRUE. Sometimes you also want to know the elements of the #' intersections. In this case \code{plot} should be set to FALSE and the #' function call will return a list of two items. The first item, that can be #' accessed by $plot, contains the plotting information. Additionally, a list #' ($table) will be returned containing the elements of the various overlaps. #' @import ggplot2 #' @examples #' \dontrun{ #' #Load the included dataset #' data(EC) #' #' #Generating the circ object #' circ<-circular_dat(EC$david, EC$genelist) #' #' #Selecting terms of interest #' l1<-subset(circ,term=='heart development',c(genes,logFC)) #' l2<-subset(circ,term=='plasma membrane',c(genes,logFC)) #' l3<-subset(circ,term=='tissue morphogenesis',c(genes,logFC)) #' #' GOVenn(l1,l2,l3, label=c('heart development','plasma membrane','tissue morphogenesis')) #' } #' @export GOVenn<-function(data1, data2, data3, title, label, lfc.col, circle.col, plot=T){ id <- NULL if (missing(label)) label<-c('List1','List2','List3') if (missing(lfc.col)) lfc.col<-c('firebrick1','gold','cornflowerblue') if (missing(circle.col)) circle.col<-c('brown1','chartreuse3','cornflowerblue') if (missing(title)) title<-'' if (missing(data3)==F) { three<-T overlap<-get_overlap(data1,data2,data3) venn_df<-overlap$venn_df table<-overlap$table }else{ three<-F overlap<-get_overlap2(data1,data2) venn_df<-overlap$venn_df table<-overlap$table } ### calc Venn ### if (three){ center<-data.frame(x=c(0.4311,0.4308,0.6380),y=c(0.6197,0.3801,0.5001),diameter=c(0.4483,0.4483,0.4483)) outerCircle<-data.frame(x=numeric(),y=numeric(),id=numeric()) for (var in 1:3){ dat <- circleFun(c(center$x[var],center$y[var]),center$diameter[var],npoints = 100) outerCircle<-rbind(outerCircle,dat) } outerCircle$id<-rep(c(label[1],label[2],label[3]),each=100) outerCircle$id<-factor(outerCircle$id, levels=c(label[1],label[2],label[3])) }else{ center<-data.frame(x=c(0.33,0.6699),y=c(0.5,0.5),diameter=c(0.6180,0.6180)) outerCircle<-data.frame(x=numeric(),y=numeric(),id=numeric()) for (var in 1:2){ dat <- circleFun(c(center$x[var],center$y[var]),center$diameter[var],npoints = 100) outerCircle<-rbind(outerCircle,dat) } outerCircle$id<-rep(c(label[1],label[2]),each=100) outerCircle$id<-factor(outerCircle$id, levels=c(label[1],label[2])) } ### calc single pies ### if (three){ Pie<-data.frame(x=numeric(),y=numeric(),id=numeric()) dat <- circleFun(c(center$x[1],max(subset(outerCircle,id==label[1])$y)-0.05),0.1,npoints = 100) Pie<-rbind(Pie,dat) dat <- circleFun(c(center$x[2],min(subset(outerCircle,id==label[2])$y)+0.05),0.1,npoints = 100) Pie<-rbind(Pie,dat) dat <- circleFun(c(max(subset(outerCircle,id==label[3])$x)-0.05,center$y[3]),0.1,npoints = 100) Pie<-rbind(Pie,dat) Pie$id<-rep(1:3,each=100) UP<-Pie[c(1:50,100:150,200:250),] Down<-Pie[c(50:100,150:200,250:300),] }else{ Pie<-data.frame(x=numeric(),y=numeric(),id=numeric()) dat <- circleFun(c(min(subset(outerCircle,id==label[1])$x)+0.05,center$y[1]),0.1,npoints = 100) Pie<-rbind(Pie,dat) dat <- circleFun(c(max(subset(outerCircle,id==label[2])$x)-0.05,center$y[2]),0.1,npoints = 100) Pie<-rbind(Pie,dat) Pie$id<-rep(1:2,each=100) UP<-Pie[c(1:50,100:150),] Down<-Pie[c(50:100,150:200),] } ### calc single pie text ### if (three){ x<-c();y<-c() for (i in unique(Pie$id)){ x<-c(x,rep((min(subset(Pie,id==i)$x)+max(subset(Pie,id==i)$x))/2,2)) y<-c(y,(min(subset(Pie,id==i)$y)+max(subset(Pie,id==i)$y))/2+0.02) y<-c(y,(min(subset(Pie,id==i)$y)+max(subset(Pie,id==i)$y))/2-0.02) } pieText<-data.frame(x=x,y=y,label=c(venn_df$UP[1],venn_df$DOWN[1],venn_df$UP[2],venn_df$DOWN[2],venn_df$UP[3],venn_df$DOWN[3])) }else{ x<-c();y<-c() for (i in unique(Pie$id)){ x<-c(x,rep((min(subset(Pie,id==i)$x)+max(subset(Pie,id==i)$x))/2,2)) y<-c(y,(min(subset(Pie,id==i)$y)+max(subset(Pie,id==i)$y))/2+0.02) y<-c(y,(min(subset(Pie,id==i)$y)+max(subset(Pie,id==i)$y))/2-0.02) } pieText<-data.frame(x=x,y=y,label=c(venn_df$UP[1],venn_df$DOWN[1],venn_df$UP[2],venn_df$DOWN[2])) } ### calc overlap pies ### if (three){ smc<-data.frame(x=c(0.6,0.59,0.31,0.5),y=c(0.66,0.34,0.5,0.5)) PieOv<-data.frame(x=numeric(),y=numeric()) PieOv<-rbind(PieOv,circleFun(c(smc$x[1],smc$y[1]),0.06,npoints = 100)) PieOv<-rbind(PieOv,circleFun(c(smc$x[2],smc$y[2]),0.06,npoints = 100)) PieOv<-rbind(PieOv,circleFun(c(smc$x[3],smc$y[3]),0.06,npoints = 100)) PieOv<-rbind(PieOv,circleFun(c(smc$x[4],smc$y[4]),0.06,npoints = 100)) PieOv$id<-rep(1:4,each=100) smc$id<-1:4 UPOv<-rbind(smc[1,],PieOv[1:33,],smc[1,],smc[2,],PieOv[100:133,],smc[2,],smc[3,],PieOv[200:233,],smc[3,],smc[4,],PieOv[300:333,],smc[4,]) Change<-rbind(smc[1,],PieOv[33:66,],smc[1,],smc[2,],PieOv[133:166,],smc[2,],smc[3,],PieOv[233:266,],smc[3,],smc[4,],PieOv[333:366,],smc[4,]) DownOv<-rbind(smc[1,],PieOv[66:100,],smc[1,],smc[2,],PieOv[166:200,],smc[2,],smc[3,],PieOv[266:300,],smc[3,],smc[4,],PieOv[366:400,],smc[4,]) }else{ PieOv<-data.frame(x=numeric(),y=numeric(),id=numeric()) PieOv<-rbind(PieOv,circleFun(c(0.5,0.5),0.08,npoints = 100)) PieOv$id<-rep(1,100) center<-data.frame(x=0.5, y=0.5, id=1) UPOv<-rbind(center[1,],PieOv[1:33,]) Change<-rbind(center[1,],PieOv[33:66,]) DownOv<-rbind(center[1,],PieOv[66:100,]) } ### calc overlap pie text ### if (three){ x<-c();y<-c() for (i in unique(PieOv$id)){ x<-c(x,subset(UPOv,id==i)$x[1]+0.0115,subset(DownOv,id==i)$x[1]-0.018,subset(Change,id==i)$x[1]+0.01) y<-c(y,subset(UPOv,id==i)$y[1]+0.01,subset(DownOv,id==i)$y[1],subset(Change,id==i)$y[1]-0.013) } small.pieT<-data.frame(x=x,y=y,label=c(venn_df$UP[5],venn_df$Change[5],venn_df$DOWN[5],venn_df$UP[6],venn_df$Change[6],venn_df$DOWN[6],venn_df$UP[4],venn_df$Change[4],venn_df$DOWN[4],venn_df$UP[7],venn_df$Change[7],venn_df$DOWN[7])) }else{ x<-c(subset(UPOv,id==1)$x[1]+0.015,subset(DownOv,id==1)$x[1]-0.018,subset(Change,id==1)$x[1]+0.01) y<-c(subset(UPOv,id==1)$y[1]+0.015,subset(DownOv,id==1)$y[1],subset(Change,id==1)$y[1]-0.013) small.pieT<-data.frame(x=x,y=y,label=c(venn_df$UP[3],venn_df$Change[3],venn_df$DOWN[3])) } g<- ggplot()+ geom_polygon(data=outerCircle, aes(x,y, group=id, fill=id) ,alpha=0.5,color='black')+ scale_fill_manual(values=circle.col)+ guides(fill=guide_legend(title=''))+ geom_polygon(data=UP, aes(x,y,group=id),fill=lfc.col[1],color='white')+ geom_polygon(data=Down, aes(x,y,group=id),fill=lfc.col[3],color='white')+ geom_text(data=pieText, aes(x=x,y=y,label=label),size=5)+ geom_polygon(data=UPOv, aes(x,y,group=id),fill=lfc.col[1],color='white')+ geom_polygon(data=DownOv, aes(x,y,group=id),fill=lfc.col[3],color='white')+ geom_polygon(data=Change, aes(x,y,group=id),fill=lfc.col[2],color='white')+ geom_text(data=small.pieT,aes(x=x,y=y,label=label),size=4)+ theme_blank+ labs(title=title) if (plot) return(g) else return(list(plot=g,table=table)) } GOplot/R/GOCluster.R0000644000175100001440000004217012676706421013706 0ustar hornikusers#' #' @name GOCluster #' @title Circular dendrogram. #' @description GOCluster generates a circular dendrogram of the \code{data} #' clustering using by default euclidean distance and average linkage.The #' inner ring displays the color coded logFC while the outside one encodes the #' assigned terms to each gene. #' @param data A data frame which should be the result of #' \code{\link{circle_dat}} in case the data contains only one logFC column. #' Otherwise \code{data} is a data frame whereas the first column contains the #' genes, the second the term and the following columns the logFCs of the #' different contrasts. #' @param process A character vector of selected processes (ID or term #' description) #' @param metric A character vector specifying the distance measure to be used #' (default='euclidean'), see \code{dist} #' @param clust A character vector specifying the agglomeration method to be #' used (default='average'), see \code{hclust} #' @param clust.by A character vector specifying if the clustering should be #' done for gene expression pattern or functional categories. By default the #' clustering is done based on the functional categories. #' @param nlfc If TRUE \code{data} contains multiple logFC columns (default= #' FALSE) #' @param lfc.col Character vector to define the color scale for the logFC of #' the form c(high, midpoint,low) #' @param lfc.min Specifies the minimium value of the logFC scale (default = -3) #' @param lfc.max Specifies the maximum value of the logFC scale (default = 3) #' @param lfc.space The space between the leafs of the dendrogram and the ring #' for the logFC #' @param lfc.width The width of the logFC ring #' @param term.col A character vector specifying the colors of the term bands #' @param term.space The space between the logFC ring and the term ring #' @param term.width The width of the term ring #' @details The inner ring can be split into smaller rings to display multiply #' logFC values resulting from various comparisons. #' @import ggplot2 #' @import ggdendro #' @import RColorBrewer #' @import stats #' @examples #' \dontrun{ #' #Load the included dataset #' data(EC) #' #' #Generating the circ object #' circ<-circular_dat(EC$david, EC$genelist) #' #' #Creating the cluster plot #' GOCluster(circ, EC$process) #' #' #Cluster the data according to gene expression and assigning a different color scale for the logFC #' GOCluster(circ,EC$process,clust.by='logFC',lfc.col=c('darkgoldenrod1','black','cyan1')) #' } #' @export #' GOCluster<-function(data, process, metric, clust, clust.by, nlfc, lfc.col, lfc.min, lfc.max, lfc.space, lfc.width, term.col, term.space, term.width){ x <- y <- xend <- yend <- width <- space <- logFC <- NULL if (missing(metric)) metric<-'euclidean' if (missing(clust)) clust<-'average' if (missing(clust.by)) clust.by<-'term' if (missing(nlfc)) nlfc <- 0 if (missing(lfc.col)) lfc.col<-c('firebrick1','white','dodgerblue') if (missing(lfc.min)) lfc.min <- -3 if (missing(lfc.max)) lfc.max <- 3 if (missing(lfc.space)) lfc.space<- (-0.5) else lfc.space<-lfc.space*(-1) if (missing(lfc.width)) lfc.width<- (-1.6) else lfc.width<-lfc.space-lfc.width-0.1 if (missing(term.col)) term.col<-brewer.pal(length(process), 'Set3') if (missing(term.space)) term.space<- lfc.space+lfc.width else term.space<-term.space*(-1)+lfc.width if (missing(term.width)) term.width<- 2*lfc.width+term.space else term.width<-term.width*(-1)+term.space if (clust.by=='logFC') distance <- stats::dist(chord[,dim(chord)[2]], method=metric) if (clust.by=='term') distance <- stats::dist(chord, method=metric) cluster <- stats::hclust(distance, method=clust) dendr <- dendro_data(cluster) y_range <- range(dendr$segments$y) x_pos <- data.frame(x=dendr$label$x, label=as.character(dendr$label$label)) chord <- as.data.frame(chord) chord$label <- as.character(rownames(chord)) all <- merge(x_pos, chord, by='label') all$label <- as.character(all$label) if (nlfc){ lfc_rect <- all[,c(2, dim(all)[2])] for (l in 4:dim(data)[2]) lfc_rect <- cbind(lfc_rect, sapply(all$label, function(x) data[match(x, data$genes), l])) num <- dim(data)[2]-1 tmp <- seq(lfc.space, lfc.width, length = num) lfc<-data.frame(x=numeric(),width=numeric(),space=numeric(),logFC=numeric()) for (l in 1:(length(tmp)-1)){ tmp_df<-data.frame(x=lfc_rect[,1],width=tmp[l+1],space=tmp[l],logFC=lfc_rect[,l+1]) lfc<-rbind(lfc,tmp_df) } }else{ lfc <- all[,c(2, dim(all)[2])] lfc$space <- lfc.space lfc$width <- lfc.width } term <- all[,c(2:(length(process)+2))] color<-NULL;termx<-NULL;tspace<-NULL;twidth<-NULL for (row in 1:dim(term)[1]){ idx <- which(term[row,-1] != 0) if(length(idx) != 0){ termx<-c(termx,rep(term[row,1],length(idx))) color<-c(color,term.col[idx]) tmp<-seq(term.space,term.width,length=length(idx)+1) tspace<-c(tspace,tmp[1:(length(tmp)-1)]) twidth<-c(twidth,tmp[2:length(tmp)]) } } tmp <- sapply(lfc$logFC, function(x) ifelse(x > lfc.max, lfc.max, x)) logFC <- sapply(tmp, function(x) ifelse(x < lfc.min, lfc.min, x)) lfc$logFC <- logFC term_rect <- data.frame(x = termx, width = twidth, space = tspace, col = color) legend <- data.frame(x = 1:length(process),label = process) ggplot()+ geom_segment(data=segment(dendr), aes(x=x, y=y, xend=xend, yend=yend))+ geom_rect(data=lfc,aes(xmin=x-0.5,xmax=x+0.5,ymin=width,ymax=space,fill=logFC))+ scale_fill_gradient2('logFC', space = 'Lab', low=lfc.col[3],mid=lfc.col[2],high=lfc.col[1],guide=guide_colorbar(title.position='top',title.hjust=0.5),breaks=c(min(lfc$logFC),max(lfc$logFC)),labels=c(round(min(lfc$logFC)),round(max(lfc$logFC))))+ geom_rect(data=term_rect,aes(xmin=x-0.5,xmax=x+0.5,ymin=width,ymax=space),fill=term_rect$col)+ geom_point(data=legend,aes(x=x,y=0.1,size=factor(label,levels=label),shape=NA))+ guides(size=guide_legend("GO Terms",ncol=4,byrow=T,override.aes=list(shape=22,fill=term.col,size = 8)))+ coord_polar()+ scale_y_reverse()+ theme(legend.position='bottom',legend.background = element_rect(fill='transparent'),legend.box='horizontal',legend.direction='horizontal')+ theme_blank } #' #' @name GOChord #' @title Displays the relationship between genes and terms. #' @description The GOChord function generates a circularly composited overview #' of selected/specific genes and their assigned processes or terms. More #' generally, it joins genes and processes via ribbons in an intersection-like #' graph. The input can be generated with the \code{\link{chord_dat}} #' function. #' @param data The matrix represents the binary relation (1= is related to, 0= #' is not related to) between a set of genes (rows) and processes (columns); a #' column for the logFC of the genes is optional #' @param title The title (on top) of the plot #' @param space The space between the chord segments of the plot #' @param gene.order A character vector defining the order of the displayed gene #' labels #' @param gene.size The size of the gene labels #' @param gene.space The space between the gene labels and the segement of the #' logFC #' @param nlfc Defines the number of logFC columns (default=1) #' @param lfc.col The fill color for the logFC specified in the following form: #' c(color for low values, color for the mid point, color for the high values) #' @param lfc.min Specifies the minimium value of the logFC scale (default = -3) #' @param lfc.max Specifies the maximum value of the logFC scale (default = 3) #' @param ribbon.col The background color of the ribbons #' @param border.size Defines the size of the ribbon borders #' @param process.label The size of the legend entries #' @param limit A vector with two cutoff values (default= c(0,0)). The first #' value defines the minimum number of terms a gene has to be assigned to. The #' second the minimum number of genes assigned to a selected term. #' @details The \code{gene.order} argument has three possible options: "logFC", #' "alphabetical", "none", which are quite self- explanatory. #' #' Maybe the most important argument of the function is \code{nlfc}.If your #' \code{data} does not contain a column of logFC values you have to set #' \code{nlfc = 0}. Differential expression analysis can be performed for #' multiple conditions and/or batches. Therefore, the data frame might contain #' more than one logFC value per gene. To adjust to this situation the #' \code{nlfc} argument is used as well. It is a numeric value and it defines #' the number of logFC columns of your \code{data}. The default is "1" #' assuming that most of the time only one contrast is considered. #' #' To represent the data more useful it might be necessary to reduce the #' dimension of \code{data}. This can be achieved with \code{limit}. The first #' value of the vector defines the threshold for the minimum number of terms a #' gene has to be assigned to in order to be represented in the plot. Most of #' the time it is more meaningful to represent genes with various functions. A #' value of 3 excludes all genes with less than three term assignments. #' Whereas the second value of the parameter restricts the number of terms #' according to the number of assigned genes. All terms with a count smaller #' or equal to the threshold are excluded. #' @seealso \code{\link{chord_dat}} #' @import ggplot2 #' @import grDevices #' @examples #' \dontrun{ #' # Load the included dataset #' data(EC) #' #' # Generating the binary matrix #' chord<-chord_dat(circ,EC$genes,EC$process) #' #' # Creating the chord plot #' GOChord(chord) #' #' # Excluding process with less than 5 assigned genes #' GOChord(chord, limit = c(0,5)) #' #' # Creating the chord plot genes ordered by logFC and a different logFC color scale #' GOChord(chord,space=0.02,gene.order='logFC',lfc.col=c('red','black','cyan')) #' } #' @export GOChord <- function(data, title, space, gene.order, gene.size, gene.space, nlfc = 1, lfc.col, lfc.min, lfc.max, ribbon.col, border.size, process.label, limit){ y <- id <- xpro <- ypro <- xgen <- ygen <- lx <- ly <- ID <- logFC <- NULL Ncol <- dim(data)[2] if (missing(title)) title <- '' if (missing(space)) space = 0 if (missing(gene.order)) gene.order <- 'none' if (missing(gene.size)) gene.size <- 3 if (missing(gene.space)) gene.space <- 0.2 if (missing(lfc.col)) lfc.col <- c('brown1', 'azure', 'cornflowerblue') if (missing(lfc.min)) lfc.min <- -3 if (missing(lfc.max)) lfc.max <- 3 if (missing(border.size)) border.size <- 0.5 if (missing (process.label)) process.label <- 11 if (missing(limit)) limit <- c(0, 0) if (gene.order == 'logFC') data <- data[order(data[, Ncol], decreasing = T), ] if (gene.order == 'alphabetical') data <- data[order(rownames(data)), ] if (sum(!is.na(match(colnames(data), 'logFC'))) > 0){ if (nlfc == 1){ cdata <- check_chord(data[, 1:(Ncol - 1)], limit) lfc <- sapply(rownames(cdata), function(x) data[match(x,rownames(data)), Ncol]) }else{ cdata <- check_chord(data[, 1:(Ncol - nlfc)], limit) lfc <- sapply(rownames(cdata), function(x) data[, (Ncol - nlfc + 1)]) } }else{ cdata <- check_chord(data, limit) lfc <- 0 } if (missing(ribbon.col)) colRib <- grDevices::rainbow(dim(cdata)[2]) else colRib <- ribbon.col nrib <- colSums(cdata) ngen <- rowSums(cdata) Ncol <- dim(cdata)[2] Nrow <- dim(cdata)[1] colRibb <- c() for (b in 1:length(nrib)) colRibb <- c(colRibb, rep(colRib[b], 202 * nrib[b])) r1 <- 1; r2 <- r1 + 0.1 xmax <- c(); x <- 0 for (r in 1:length(nrib)){ perc <- nrib[r] / sum(nrib) xmax <- c(xmax, (pi * perc) - space) if (length(x) <= Ncol - 1) x <- c(x, x[r] + pi * perc) } xp <- c(); yp <- c() l <- 50 for (s in 1:Ncol){ xh <- seq(x[s], x[s] + xmax[s], length = l) xp <- c(xp, r1 * sin(x[s]), r1 * sin(xh), r1 * sin(x[s] + xmax[s]), r2 * sin(x[s] + xmax[s]), r2 * sin(rev(xh)), r2 * sin(x[s])) yp <- c(yp, r1 * cos(x[s]), r1 * cos(xh), r1 * cos(x[s] + xmax[s]), r2 * cos(x[s] + xmax[s]), r2 * cos(rev(xh)), r2 * cos(x[s])) } df_process <- data.frame(x = xp, y = yp, id = rep(c(1:Ncol), each = 4 + 2 * l)) xp <- c(); yp <- c(); logs <- NULL x2 <- seq(0 - space, -pi - (-pi / Nrow) - space, length = Nrow) xmax2 <- rep(-pi / Nrow + space, length = Nrow) for (s in 1:Nrow){ xh <- seq(x2[s], x2[s] + xmax2[s], length = l) if (nlfc <= 1){ xp <- c(xp, (r1 + 0.05) * sin(x2[s]), (r1 + 0.05) * sin(xh), (r1 + 0.05) * sin(x2[s] + xmax2[s]), r2 * sin(x2[s] + xmax2[s]), r2 * sin(rev(xh)), r2 * sin(x2[s])) yp <- c(yp, (r1 + 0.05) * cos(x2[s]), (r1 + 0.05) * cos(xh), (r1 + 0.05) * cos(x2[s] + xmax2[s]), r2 * cos(x2[s] + xmax2[s]), r2 * cos(rev(xh)), r2 * cos(x2[s])) }else{ tmp <- seq(r1, r2, length = nlfc + 1) for (t in 1:nlfc){ logs <- c(logs, data[s, (dim(data)[2] + 1 - t)]) xp <- c(xp, (tmp[t]) * sin(x2[s]), (tmp[t]) * sin(xh), (tmp[t]) * sin(x2[s] + xmax2[s]), tmp[t + 1] * sin(x2[s] + xmax2[s]), tmp[t + 1] * sin(rev(xh)), tmp[t + 1] * sin(x2[s])) yp <- c(yp, (tmp[t]) * cos(x2[s]), (tmp[t]) * cos(xh), (tmp[t]) * cos(x2[s] + xmax2[s]), tmp[t + 1] * cos(x2[s] + xmax2[s]), tmp[t + 1] * cos(rev(xh)), tmp[t + 1] * cos(x2[s])) }}} if(lfc[1] != 0){ if (nlfc == 1){ df_genes <- data.frame(x = xp, y = yp, id = rep(c(1:Nrow), each = 4 + 2 * l), logFC = rep(lfc, each = 4 + 2 * l)) }else{ df_genes <- data.frame(x = xp, y = yp, id = rep(c(1:(nlfc*Nrow)), each = 4 + 2 * l), logFC = rep(logs, each = 4 + 2 * l)) } }else{ df_genes <- data.frame(x = xp, y = yp, id = rep(c(1:Nrow), each = 4 + 2 * l)) } aseq <- seq(0, 180, length = length(x2)); angle <- c() for (o in aseq) if((o + 270) <= 360) angle <- c(angle, o + 270) else angle <- c(angle, o - 90) df_texg <- data.frame(xgen = (r1 + gene.space) * sin(x2 + xmax2/2),ygen = (r1 + gene.space) * cos(x2 + xmax2 / 2),labels = rownames(cdata), angle = angle) df_texp <- data.frame(xpro = (r1 + 0.15) * sin(x + xmax / 2),ypro = (r1 + 0.15) * cos(x + xmax / 2), labels = colnames(cdata), stringsAsFactors = FALSE) cols <- rep(colRib, each = 4 + 2 * l) x.end <- c(); y.end <- c(); processID <- c() for (gs in 1:length(x2)){ val <- seq(x2[gs], x2[gs] + xmax2[gs], length = ngen[gs] + 1) pros <- which((cdata[gs, ] != 0) == T) for (v in 1:(length(val) - 1)){ x.end <- c(x.end, sin(val[v]), sin(val[v + 1])) y.end <- c(y.end, cos(val[v]), cos(val[v + 1])) processID <- c(processID, rep(pros[v], 2)) } } df_bezier <- data.frame(x.end = x.end, y.end = y.end, processID = processID) df_bezier <- df_bezier[order(df_bezier$processID,-df_bezier$y.end),] x.start <- c(); y.start <- c() for (rs in 1:length(x)){ val<-seq(x[rs], x[rs] + xmax[rs], length = nrib[rs] + 1) for (v in 1:(length(val) - 1)){ x.start <- c(x.start, sin(val[v]), sin(val[v + 1])) y.start <- c(y.start, cos(val[v]), cos(val[v + 1])) } } df_bezier$x.start <- x.start df_bezier$y.start <- y.start df_path <- bezier(df_bezier, colRib) if(length(df_genes$logFC) != 0){ tmp <- sapply(df_genes$logFC, function(x) ifelse(x > lfc.max, lfc.max, x)) logFC <- sapply(tmp, function(x) ifelse(x < lfc.min, lfc.min, x)) df_genes$logFC <- logFC } g<- ggplot() + geom_polygon(data = df_process, aes(x, y, group=id), fill='gray70', inherit.aes = F,color='black') + geom_polygon(data = df_process, aes(x, y, group=id), fill=cols, inherit.aes = F,alpha=0.6,color='black') + geom_point(aes(x = xpro, y = ypro, size = factor(labels, levels = labels), shape = NA), data = df_texp) + guides(size = guide_legend("GO Terms", ncol = 4, byrow = T, override.aes = list(shape = 22, fill = unique(cols), size = 8))) + theme(legend.text = element_text(size = process.label)) + geom_text(aes(xgen, ygen, label = labels, angle = angle), data = df_texg, size = gene.size) + geom_polygon(aes(x = lx, y = ly, group = ID), data = df_path, fill = colRibb, color = 'black', size = border.size, inherit.aes = F) + labs(title = title) + theme_blank if (nlfc >= 1){ g + geom_polygon(data = df_genes, aes(x, y, group = id, fill = logFC), inherit.aes = F, color = 'black') + scale_fill_gradient2('logFC', space = 'Lab', low = lfc.col[3], mid = lfc.col[2], high = lfc.col[1], guide = guide_colorbar(title.position = "top", title.hjust = 0.5), breaks = c(min(df_genes$logFC), max(df_genes$logFC)), labels = c(round(min(df_genes$logFC)), round(max(df_genes$logFC)))) + theme(legend.position = 'bottom', legend.background = element_rect(fill = 'transparent'), legend.box = 'horizontal', legend.direction = 'horizontal') }else{ g + geom_polygon(data = df_genes, aes(x, y, group = id), fill = 'gray50', inherit.aes = F, color = 'black')+ theme(legend.position = 'bottom', legend.background = element_rect(fill = 'transparent'), legend.box = 'horizontal', legend.direction = 'horizontal') } }GOplot/R/GOHeat.R0000644000175100001440000000577412676706421013157 0ustar hornikusers#' #' @name GOHeat #' @title Displays heatmap of the relationship between genes and terms. #' @description The GOHeat function generates a heatmap of the relationship #' between genes and terms. Biological processes are displayed in rows and #' genes in columns. In addition genes are clustered to highlight groups of #' genes with similar annotated functions. The input can be generated with the #' \code{\link{chord_dat}} function. #' @param data The matrix represents the binary relation (1= is related to, 0= #' is not related to) between a set of genes (rows) and processes (columns) #' @param nlfc Defines the number of logFC columns (default = 0) #' @param fill.col Defines the color scale break points #' @details The heatmap has in general two modes which depend on the \code{nlfc} #' argument. If \code{nlfc = 0}, so no logFC values are available, the #' coloring encodes for the overall number of processes the respective gene is #' assigned to. In case of \code{nlfc = 1} the color corresponds to the logFC #' of the gene. #' @import ggplot2 #' @examples #' \dontrun{ #' # Load the included dataset #' data(EC) #' #' # Generate the circ object #' circ <- circle_dat(EC$david, EC$genelist) #' #' # Generate the chord object #' chord <- chord_dat(circ, EC$genes, EC$process) #' #' # Create the plot with user-defined colors #' GOHeat(chord, nlfc = 1, fill.col = c('red', 'yellow', 'green')) #' } #' @export #' GOHeat <- function(data, nlfc, fill.col){ x <- y <- z <- NULL if(missing(nlfc)) nlfc <- 0 else nlfc <- nlfc if(missing(fill.col)) fill.col <- c('firebrick', 'white', 'dodgerblue') else fill.col <- fill.col distance <- dist(data) cluster <- hclust(distance) M <- dim(data)[2] nterm <- M - nlfc if(nlfc == 0){ s <- rowSums(data[,1:nterm]) tmp <- NULL for(r in 1:nrow(data)){ tmp <- c(tmp, as.numeric(gsub(1, s[r], data[r, 1:nterm]))) } }else{ tmp <- NULL for(r in 1:nrow(data)){ tmp <- c(tmp, as.numeric(gsub(1, data[r, (nterm + 1)], data[r, 1:nterm]))) } } df <- data.frame(x = rep(cluster$order, each = nterm), y = rep(colnames(data[,1:nterm]), length(rownames(data))), z = tmp, lab = rep(rownames(data), each = nterm)) df_o <- df[order(df$x),] g <- ggplot() + geom_tile(data = df_o, aes(x = x, y = y, fill = z))+ scale_x_discrete(breaks = 1:length(unique(df_o$x)), labels = unique(df_o$lab)) + theme(axis.text.x = element_text(angle = 90, vjust = 0.5), axis.title.x=element_blank(), axis.title.y=element_blank(), axis.text.y = element_text(size = 14), panel.background=element_blank(), panel.grid.major=element_blank(), panel.grid.minor=element_blank()) if(nlfc == 0){ g + scale_fill_gradient2('Count', space = 'Lab', low=fill.col[2], mid=fill.col[3], high=fill.col[1]) }else{ g + scale_fill_gradient2('logFC', space = 'Lab', low=fill.col[3], mid=fill.col[2], high=fill.col[1]) } } GOplot/R/GOCore.R0000644000175100001440000006357512676706421013171 0ustar hornikusers#' Transcriptomic information of endothelial cells. #' #' The data set contains the transcriptomic information of endothelial cells #' from two steady state tissues (brain and heart). More detailed information #' can be found in the paper by Nolan et al. 2013. The data was normalized and a #' statistical analysis was performed to determine differentially expressed #' genes. DAVID functional annotation tool was used to perform a gene- #' annotation enrichment analysis of the set of differentially expressed genes #' (adjusted p-value < 0.05). #' #' @docType data #' @keywords datasets #' @name EC #' @usage data(EC) #' @format A list containing 5 items #' @source \url{http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47067} "EC" #' #' @name circle_dat #' @title Creates a plotting object. #' @description The function takes the results from a functional analysis (for #' example DAVID) and combines it with a list of selected genes and their #' logFC. The resulting data frame can be used as an input for various ploting #' functions. #' @param terms A data frame with columns for 'category', 'ID', 'term', adjusted #' p-value ('adj_pval') and 'genes' #' @param genes A data frame with columns for 'ID', 'logFC' #' @details Since most of the gene- annotation enrichment analysis are based on #' the gene ontology database the package was build with this structure in #' mind, but is not restricted to it. Gene ontology is structured as an #' acyclic graph and it provides terms covering different areas. These terms #' are grouped into three independent \code{categories}: BP (biological #' process), CC (cellular component) or MF (molecular function). #' #' The "ID" and "term" columns of the \code{terms} data frame refer to the ID #' and term description, whereas the ID is optional. #' #' The "ID" column of the \code{genes} data frame can contain any unique #' identifier. Nevertheless, the identifier has to be the same as in "genes" #' from \code{terms}. #' @examples #' \dontrun{ #' #Load the included dataset #' data(EC) #' #' #Building the circ object #' circ<-circular_dat(EC$david, EC$genelist) #' } #' @export circle_dat <- function(terms, genes){ colnames(terms) <- tolower(colnames(terms)) terms$genes <- toupper(terms$genes) genes$ID <- toupper(genes$ID) tgenes <- strsplit(as.vector(terms$genes), ', ') if (length(tgenes[[1]]) == 1) tgenes <- strsplit(as.vector(terms$genes), ',') count <- sapply(1:length(tgenes), function(x) length(tgenes[[x]])) logFC <- sapply(unlist(tgenes), function(x) genes$logFC[match(x, genes$ID)]) if(class(logFC) == 'factor'){ logFC <- gsub(",", ".", gsub("\\.", "", logFC)) logFC <- as.numeric(logFC) } s <- 1; zsc <- c() for (c in 1:length(count)){ value <- 0 e <- s + count[c] - 1 value <- sapply(logFC[s:e], function(x) ifelse(x > 0, 1, -1)) zsc <- c(zsc, sum(value) / sqrt(count[c])) s <- e + 1 } if (is.null(terms$id)){ df <- data.frame(category = rep(as.character(terms$category), count), term = rep(as.character(terms$term), count), count = rep(count, count), genes = as.character(unlist(tgenes)), logFC = logFC, adj_pval = rep(terms$adj_pval, count), zscore = rep(zsc, count), stringsAsFactors = FALSE) }else{ df <- data.frame(category = rep(as.character(terms$category), count), ID = rep(as.character(terms$id), count), term = rep(as.character(terms$term), count), count = rep(count, count), genes = as.character(unlist(tgenes)), logFC = logFC, adj_pval = rep(terms$adj_pval, count), zscore = rep(zsc, count), stringsAsFactors = FALSE) } return(df) } #' #' @name chord_dat #' @title Creates a binary matrix. #' @description The function creates a matrix which represents the binary #' relation (1= is related to, 0= is not related to) between selected genes #' (row) and processes (column). The resulting matrix can be visualized with #' the \code{\link{GOChord}} function. #' @param data A data frame with at least two coloumns: GO ID|term and genes. #' Each row contains exactly one GO ID|term and one gene. A column containing #' logFC values is optional and might be used if \code{genes} is missing. #' @param genes A character vector of selected genes OR data frame with coloumns #' for gene ID and logFC. #' @details If more than one logFC value for each gene is at disposal, only one #' should be used to create the binary matrix. The other values have to be #' added manually later. #' @param process A character vector of selected processes #' @return A binary matrix #' @seealso \code{\link{GOChord}} #' @examples #' \dontrun{ #' # Load the included dataset #' data(EC) #' #' # Building the circ object #' circ <- circle_dat(EC$david, EC$genelist) #' #' # Building the binary matrix #' chord <- chord_dat(circ, EC$genes, EC$process) #' #' } #' @export chord_dat <- function(data, genes, process){ id <- term <- logFC <- BPprocess <- NULL colnames(data) <- tolower(colnames(data)) if (missing(genes)){ if (is.null(data$logFC)){ genes <- as.character(unique(data$genes)) }else{ genes <- subset(data, !duplicated(genes), c(genes, logFC)) } }else{ if(is.vector(genes)){ genes <- as.character(genes) }else{ if(class(genes[, 2]) != 'numeric') genes[, 2] <- as.numeric(levels(genes[, 2]))[genes[, 2]] genes[, 1] <- as.character(genes[, 1]) colnames(genes) <- c('genes', 'logFC') } } if (missing(process)){ process <- as.character(unique(data$term)) }else{ if(class(process) != 'character') process <- as.character(process) } if (strsplit(process[1],':')[[1]][1] == 'GO'){ subData <- subset(data, id%in%process) colnames(subData)[which(colnames(subData) == 'id')] <- 'BPprocess' }else{ subData <- subset(data, term%in%process) colnames(subData)[which(colnames(subData) == 'term')] <- 'BPprocess' } if(is.vector(genes)){ M <- genes[genes%in%unique(subData$genes)] mat <- matrix(0, ncol = length(process), nrow = length(M)) rownames(mat) <- M colnames(mat) <- process for (p in 1:length(process)){ sub2 <- subset(subData, BPprocess == process[p]) for (g in 1:length(M)) mat[g, p] <- ifelse(M[g]%in%sub2$genes, 1, 0) } }else{ genes <- subset(genes, genes %in% unique(subData$genes)) N <- length(process) + 1 M <- genes[,1] mat <- matrix(0, ncol = N, nrow = length(M)) rownames(mat) <- M colnames(mat) <- c(process, 'logFC') mat[,N] <- genes[,2] for (p in 1:(N-1)){ sub2 <- subset(subData, BPprocess == process[p]) for (g in 1:length(M)) mat[g, p] <- ifelse(M[g]%in%sub2$genes, 1, 0) } } return(mat) } #' #' @name reduce_overlap #' @title Eliminates redundant terms. #' @description The function eliminates all terms with a gene overlap >= set #' threshold (\code{overlap}) The reduced dataset can be used to improve the #' readability of plots such as \code{GOBubble} and \code{GOBar} #' @param data A data frame created with \code{circle_dat}. #' @param overlap Skalar indicating the threshold for gene overlap (default = 0.75). #' @details The function is currently very slow. #' @examples #' \dontrun{ #' # Load the included dataset #' data(EC) #' #' # Building the circ object #' circ <- circle_dat(EC$david, EC$genelist) #' #' # Eliminate redundant terms #' reduced_circ <- reduce_overlap(circ) #' #' # Plot reduced data #' GOBubble(reduced_circ) #' #' } #' @export reduce_overlap <- function(data, overlap){ term <- genes <- NULL if (missing(overlap)) overlap <- 0.75 terms <- unique(data$term) FUN <- function(x,y) round(sum(x$genes %in% y$genes)/nrow(x), digits = 2) tmp <- matrix(0, ncol = length(terms), nrow = length(terms), dimnames = list(terms, terms)) for (row in 1:nrow(tmp)){ for (col in 1:ncol(tmp)){ tmp[row, col] <- FUN(subset(data, term == terms[row], genes), subset(data, term == terms[col], genes)) } } tmp[base::upper.tri(tmp)] <- 0 for(col in 1:ncol(tmp)){ idx <- which(tmp[,col] >= overlap) sel_col <- idx[which(idx != col)] tmp[,sel_col] <- 0 } sel_terms <- colnames(tmp)[colSums(tmp) != 0] dat <- subset(data, term %in% sel_terms) data <- dat[!duplicated(dat$term), ] return(data) } #' #' @name GOBubble #' @title Bubble plot. #' @description The function creates a bubble plot of the input \code{data}. The #' input \code{data} can be created with the help of the #' \code{\link{circle_dat}} function. #' @param data A data frame with coloumns for category, GO ID, term, adjusted #' p-value, z-score, count(num of genes) #' @param display A character vector. Indicates whether it should be a single #' plot ('single') or a facet plot with panels for each category #' (default='single') #' @param title The title (on top) of the plot #' @param colour A character vector which defines the colour of the bubbles for #' each category #' @param labels Sets a threshold for the displayed labels. The threshold refers #' to the -log(adjusted p-value) (default=5) #' @param ID If TRUE then labels are IDs else terms #' @param table.legend Defines whether a table of GO ID and GO term should be #' displayed on the right side of the plot or not (default = TRUE) #' @param table.col If TRUE then the table entries are coloured according to #' their category, if FALSE then entries are black #' @param bg.col Should only be used in case of a facet plot. If TRUE then the #' panel backgrounds are coloured according to the displayed category #' @details The x- axis of the plot represents the z-score. The negative #' logarithm of the adjusted p-value (corresponding to the significance of the #' term) is displayed on the y-axis. The area of the plotted circles is #' proportional to the number of genes assigned to the term. Each circle is #' coloured according to its category and labeled alternatively with the ID or #' term name.If static is set to FALSE the mouse hover effect will be enabled. #' @import ggplot2 #' @import gridExtra #' @import graphics #' @examples #' \dontrun{ #' #Load the included dataset #' data(EC) #' #' #Building the circ object #' circ <- circular_dat(EC$david, EC$genelist) #' #' #Creating the bubble plot colouring the table entries according to the category #' GOBubble(circ, table.col = T) #' #' #Creating the bubble plot displaying the term instead of the ID and without the table #' GOBubble(circ, ID = F, table.legend = F) #' #' #Faceting the plot #' GOBubble(circ, display = 'multiple') #' } #' @export GOBubble <- function(data, display, title, colour, labels, ID = T, table.legend = T, table.col = T, bg.col = F){ zscore <- adj_pval <- category <- count <- id <- term <- NULL if (missing(display)) display <- 'single' if (missing(title)) title <- '' if (missing(colour)) cols <- c("chartreuse4", "brown2", "cornflowerblue") else cols <- colour if (missing(labels)) labels <- 5 if (bg.col == T & display == 'single') cat("Parameter bg.col will be ignored. To use the parameter change display to 'multiple'") colnames(data) <- tolower(colnames(data)) if(!'count'%in%colnames(data)){ rang <- c(5, 5) data$count <- rep(1, dim(data)[1]) }else {rang <- c(1, 30)} data$adj_pval <- -log(data$adj_pval, 10) sub <- data[!duplicated(data$term), ] g <- ggplot(sub, aes(zscore, adj_pval, fill = category, size = count))+ labs(title = title, x = 'z-score', y = '-log (adj p-value)')+ geom_point(shape = 21, col = 'black', alpha = 1 / 2)+ geom_hline(yintercept = 1.3, col = 'orange')+ scale_size(range = rang, guide = 'none') if (!is.character(labels)) sub2 <- subset(sub, subset = sub$adj_pval >= labels) else sub2 <- subset(sub, sub$id%in%labels | sub$term%in%labels) if (display == 'single'){ g <- g + scale_fill_manual('Category', values = cols, labels = c('Biological Process', 'Cellular Component', 'Molecular Function'))+ theme(legend.position = 'bottom')+ annotate ("text", x = min(sub$zscore)+0.2, y = 1.4, label = "Threshold", colour = "orange", size = 4) if (ID) g <- g+ geom_text(data = sub2, aes(x = zscore, y = adj_pval, label = id), size = 5) else g <- g + geom_text(data = sub2, aes(x = zscore, y = adj_pval, label = term), size = 4) if (table.legend){ if (table.col) table <- draw_table(sub2, col = cols) else table <- draw_table(sub2) g <- g + theme(axis.text = element_text(size = 14), axis.line = element_line(colour = 'grey80'), axis.ticks = element_line(colour = 'grey80'), axis.title = element_text(size = 14, face = 'bold'), panel.background = element_blank(), panel.grid.minor = element_blank(), panel.grid.major = element_line(colour = 'grey80'), plot.background = element_blank()) graphics::par(mar = c(0.1, 0.1, 0.1, 0.1)) grid.arrange(g, table, ncol = 2) }else{ g + theme(axis.text = element_text(size = 14), axis.line = element_line(colour = 'grey80'), axis.ticks = element_line(colour = 'grey80'), axis.title = element_text(size = 14, face = 'bold'), panel.background = element_blank(), panel.grid.minor = element_blank(), panel.grid.major = element_line(colour = 'grey80'), plot.background = element_blank()) } }else{ if(bg.col){ dummy_col <- data.frame(category = c('BP', 'CC', 'MF'), adj_pval = sub$adj_pval[1:3], zscore = sub$zscore[1:3], size = 1:3, count = 1:3) g <- g + geom_rect(data = dummy_col, aes(fill = category), xmin = -Inf, xmax = Inf, ymin = -Inf, ymax = Inf, alpha = 0.1)+ facet_grid(.~category, space = 'free_x', scales = 'free_x')+ scale_fill_manual(values = cols, guide ='none') }else{ g <- g + facet_grid(.~category, space = 'free_x', scales = 'free_x')+ scale_fill_manual(values = cols, guide ='none') } if (ID) { g + geom_text(data = sub2, aes(x = zscore, y = adj_pval, label = id), size = 5) + theme(axis.title = element_text(size = 14, face = 'bold'), axis.text = element_text(size = 14), axis.line = element_line(colour = 'grey80'), axis.ticks = element_line(colour = 'grey80'), panel.border = element_rect(fill = 'transparent', colour = 'grey80'), panel.background = element_blank(), panel.grid = element_blank(), plot.background = element_blank()) }else{ g + geom_text(data = sub2, aes(x = zscore, y = adj_pval, label = term), size = 5) + theme(axis.title = element_text(size = 14, face = 'bold'), axis.text = element_text(size = 14), axis.line = element_line(colour = 'grey80'), axis.ticks = element_line(colour = 'grey80'), panel.border = element_rect(fill = 'transparent', colour = 'grey80'), panel.background = element_blank(), panel.grid = element_blank(), plot.background = element_blank()) } } } #' #' @name GOBar #' @title Z-score coloured barplot. #' @description Z-score coloured barplot of terms ordered alternatively by #' z-score or the negative logarithm of the adjusted p-value #' @param data A data frame containing at least the term ID and/or term, the #' adjusted p-value and the z-score. A possible input can be generated with #' the \code{circle_dat} function #' @param display A character vector indicating whether a single plot ('single') #' or a facet plot with panels for each category should be drawn #' (default='single') #' @param order.by.zscore Defines the order of the bars. If TRUE the bars are #' ordered according to the z-scores of the processes. Otherwise the bars are #' ordered by the negative logarithm of the adjusted p-value #' @param title The title of the plot #' @param zsc.col Character vector to define the colour scale for the z-score of #' the form c(high, midpoint,low) #' @details If \code{display} is used to facet the plot the width of the panels #' will be proportional to the length of the x scale. #' @import ggplot2 #' @import gridExtra #' @import stats #' @examples #' \dontrun{ #' #Load the included dataset #' data(EC) #' #' #Building the circ object #' circ<-circular_dat(EC$david, EC$genelist) #' #' #Creating the bar plot #' GOBar(circ) #' #' #Faceting the plot #' GOBar(circ, display='multiple') #' } #' @export GOBar <- function(data, display, order.by.zscore = T, title, zsc.col){ id <- adj_pval <- zscore <- NULL if (missing(display)) display <- 'single' if (missing(title)) title <- '' if (missing(zsc.col)) zsc.col <- c('firebrick1', 'white', 'dodgerblue1') colnames(data) <- tolower(colnames(data)) data$adj_pval <- -log(data$adj_pval, 10) sub <- data[!duplicated(data$term), ] if (order.by.zscore == T) { sub <- sub[order(sub$zscore, decreasing = T), ] leg <- theme(legend.position = 'bottom') g <- ggplot(sub, aes(x = factor(id, levels = stats::reorder(id, adj_pval)), y = adj_pval, fill = zscore)) + geom_bar(stat = 'identity', colour = 'black') + scale_fill_gradient2('z-score', space = 'Lab', low = zsc.col[3], mid = zsc.col[2], high = zsc.col[1], guide = guide_colourbar(title.position = "top", title.hjust = 0.5), breaks = c(min(sub$zscore), max(sub$zscore)), labels = c('decreasing', 'increasing')) + labs(title = title, x = '', y = '-log (adj p-value)') + leg }else{ sub <- sub[order(sub$adj_pval, decreasing = T), ] leg <- theme(legend.justification = c(1, 1), legend.position = c(0.98, 0.995), legend.background = element_rect(fill = 'transparent'), legend.box = 'vertical', legend.direction = 'horizontal') g <- ggplot(sub, aes( x = factor(id, levels = reorder(id, adj_pval)), y = zscore, fill = adj_pval)) + geom_bar(stat = 'identity', colour = 'black') + scale_fill_gradient2('Significance', space = 'Lab', low = zsc.col[3], mid = zsc.col[2], high = zsc.col[1], guide = guide_colourbar(title.position = "top", title.hjust = 0.5), breaks = c(min(sub$adj_pval), max(sub$adj_pval)), labels = c('low', 'high')) + labs(title = title, x = '', y = 'z-score') + leg } if (display == 'single'){ g + theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1), axis.line = element_line(colour = 'grey80'), axis.ticks = element_line(colour = 'grey80'), axis.title = element_text(size = 14, face = 'bold'), axis.text = element_text(size = 14), panel.background = element_blank(), panel.border = element_blank(), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), plot.background = element_blank()) }else{ g + facet_grid(.~category, space = 'free_x', scales = 'free_x')+ theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1), axis.line = element_line(colour = 'grey80'), axis.ticks = element_line(colour = 'grey80'), axis.title = element_text(size = 14, face = 'bold'), axis.text = element_text(size = 14), panel.background = element_blank(), panel.border = element_blank(), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), plot.background = element_blank()) } } #' #' @name GOCircle #' @title Circular visualization of the results of a functional analysis. #' @description The circular plot combines gene expression and gene- annotation #' enrichment data. A subset of terms is displayed like the \code{GOBar} plot #' in combination with a scatterplot of the gene expression data. The whole #' plot is drawn on a specific coordinate system to achieve the circular #' layout.The segments are labeled with the term ID. #' @param data A special data frame which should be the result of #' \code{circle_dat} #' @param title The title of the plot #' @param nsub A numeric or character vector. If it's numeric then the number #' defines how many processes are displayed (starting from the first row of #' \code{data}). If it's a character string of processes then these processes #' are displayed #' @param rad1 The radius of the inner circle (default=2) #' @param rad2 The radius of the outer circle (default=3) #' @param table.legend Shall a table be displayd or not? (default=TRUE) #' @param zsc.col Character vector to define the colour scale for the z-score of #' the form c(high, midpoint,low) #' @param lfc.col A character vector specifying the colour for up- and #' down-regulated genes #' @param label.size Size of the segment labels (default=5) #' @param label.fontface Font style of the segment labels (default='bold') #' @details The outer circle shows a scatter plot for each term of the logFC of #' the assigned genes. The colours can be changed with the argument #' \code{lfc.col}. #' #' The \code{nsub} argument needs a bit more explanation to be used wisely. First of #' all, it can be a numeric or a character vector. If it is a character vector #' then it contains the IDs or term descriptions of the displayed processes.If #' \code{nsub} is a numeric vector then the number defines how many terms are #' displayed. It starts with the first row of the input data frame. #' @import ggplot2 #' @import gridExtra #' @import stats #' @import graphics #' @seealso \code{\link{circle_dat}}, \code{\link{GOBar}} #' @examples #' \dontrun{ #' # Load the included dataset #' data(EC) #' #' # Building the circ object #' circ <- circle_dat(EC$david, EC$genelist) #' #' # Creating the circular plot #' GOCircle(circ) #' #' # Creating the circular plot with a different colour scale for the logFC #' GOCircle(circ, lfc.col = c('purple', 'orange')) #' #' # Creating the circular plot with a different colour scale for the z-score #' GOCircle(circ, zsc.col = c('yellow', 'black', 'cyan')) #' #' # Creating the circular plot with different font style #' GOCircle(circ, label.size = 5, label.fontface = 'italic') #' } #' @export GOCircle <- function(data, title, nsub, rad1, rad2, table.legend = T, zsc.col, lfc.col, label.size, label.fontface){ xmax <- y1<- zscore <- y2 <- ID <- logx <- logy2 <- logy <- logFC <- NULL if (missing(title)) title <- '' if (missing(nsub)) if (dim(data)[1] > 10) nsub <- 10 else nsub <- dim(data)[1] if (missing(rad1)) rad1 <- 2 if (missing(rad2)) rad2 <- 3 if (missing(zsc.col)) zsc.col <- c('red', 'white', 'blue') if (missing(lfc.col)) lfc.col <- c('cornflowerblue', 'firebrick1') else lfc.col <- rev(lfc.col) if (missing(label.size)) label.size = 5 if (missing(label.fontface)) label.fontface = 'bold' data$adj_pval <- -log(data$adj_pval, 10) suby <- data[!duplicated(data$term), ] if (is.numeric(nsub) == T){ suby <- suby[1:nsub, ] }else{ if (strsplit(nsub[1], ':')[[1]][1] == 'GO'){ suby <- suby[suby$ID%in%nsub, ] }else{ suby <- suby[suby$term%in%nsub, ] } nsub <- length(nsub)} N <- dim(suby)[1] r_pval <- round(range(suby$adj_pval), 0) + c(-2, 2) ymax <- c() for (i in 1:length(suby$adj_pval)){ val <- (suby$adj_pval[i] - r_pval[1]) / (r_pval[2] - r_pval[1]) ymax <- c(ymax, val)} df <- data.frame(x = seq(0, 10 - (10 / N), length = N), xmax = rep(10 / N - 0.2, N), y1 = rep(rad1, N), y2 = rep(rad2, N), ymax = ymax, zscore = suby$zscore, ID = suby$ID) scount <- data[!duplicated(data$term), which(colnames(data) == 'count')][1:nsub] idx_term <- which(!duplicated(data$term) == T) xm <- c(); logs <- c() for (sc in 1:length(scount)){ idx <- c(idx_term[sc], idx_term[sc] + scount[sc] -1) val <- stats::runif(scount[sc], df$x[sc] + 0.06, (df$x[sc] + df$xmax[sc] - 0.06)) xm <- c(xm, val) r_logFC <- round(range(data$logFC[idx[1]:idx[2]]), 0) + c(-1, 1) for (lfc in idx[1]:idx[2]){ val <- (data$logFC[lfc] - r_logFC[1]) / (r_logFC[2] - r_logFC[1]) logs <- c(logs, val)} } cols <- c() for (ys in 1:length(logs)) cols <- c(cols, ifelse(data$logFC[ys] > 0, 'upregulated', 'downregulated')) dfp <- data.frame(logx = xm, logy = logs, logFC = factor(cols), logy2 = rep(rad2, length(logs))) c <- ggplot()+ geom_rect(data = df, aes(xmin = x, xmax = x + xmax, ymin = y1, ymax = y1 + ymax, fill = zscore), colour = 'black') + geom_rect(data = df, aes(xmin = x, xmax = x + xmax, ymin = y2, ymax = y2 + 1), fill = 'gray70') + geom_rect(data = df, aes(xmin = x, xmax = x + xmax, ymin = y2 + 0.5, ymax = y2 + 0.5), colour = 'white') + geom_rect(data = df, aes(xmin = x, xmax = x + xmax, ymin = y2 + 0.25, ymax = y2 + 0.25), colour = 'white') + geom_rect(data = df, aes(xmin = x, xmax = x + xmax, ymin = y2 + 0.75, ymax = y2 + 0.75), colour = 'white') + geom_text(data = df, aes(x = x + (xmax / 2), y = y2 + 1.3, label = ID, angle = 360 - (x = x + (xmax / 2)) / (10 / 360)), size = label.size, fontface = label.fontface) + coord_polar() + labs(title = title) + ylim(1, rad2 + 1.6) + xlim(0, 10) + theme_blank + scale_fill_gradient2('z-score', space = 'Lab', low = zsc.col[3], mid = zsc.col[2], high = zsc.col[1], guide = guide_colourbar(title.position = "top", title.hjust = 0.5), breaks = c(min(df$zscore), max(df$zscore)),labels = c('decreasing', 'increasing')) + theme(legend.position = 'bottom', legend.background = element_rect(fill = 'transparent'), legend.box = 'horizontal', legend.direction = 'horizontal') + geom_point(data = dfp, aes(x = logx, y = logy2 + logy), pch = 21, fill = 'transparent', colour = 'black', size = 3)+ geom_point(data = dfp, aes(x = logx, y = logy2 + logy, colour = logFC), size = 2.5)+ scale_colour_manual(values = lfc.col, guide = guide_legend(title.position = "top", title.hjust = 0.5)) if (table.legend){ table <- draw_table(suby) graphics::par(mar = c(0.1, 0.1, 0.1, 0.1)) grid.arrange(c, table, ncol = 2) }else{ c + theme(plot.background = element_rect(fill = 'aliceblue'), panel.background = element_rect(fill = 'white')) } } GOplot/vignettes/0000755000175100001440000000000012676706463013525 5ustar hornikusersGOplot/vignettes/GOBubble2.png0000644000175100001440000030265712676706421015745 0ustar hornikusersPNG  IHDRZϡѳ IDATxy\kAV *䞨1KhR&sfҖӧ{yxz:y|yy2`HMMK>A}Ìb)--ZyyyӳL&|2Lz<y_3utd~tǓe VRRCPznsssnwBtq>X0<F!>{uKMM-//z<EQ17@uᤏ[TTTtȻ /l63q]p:: F.w=߰a~ʕ+^tBFEQ<=(.[dZeDWut\>up8233EHf|=❃L;Dw*=<]!sI/uX,e˖Qm,@`,,@`+++X _G?;#Bjt P,87o^xSO=Yp0bjj6oC@7qSO=yf? 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LMII9~տ,[[G:lʼ:# ݾjW&2shhoq=$ǀ0ٽZ #/rv o۷oR'ԡ3g9rD"m[҈͋-9js\%JiӴhkaN[*33Pp!׾y{zxxL sj:///))Ƶʞmf}~Nڥi'J%ʱN+U~t)MicWW)$DTTT=z4;;9,H< "Н: xk]bHIDAT7zOZ9%s֕]HU[2$<>>>22F33oR4+++--,hOX9iPB>;'vcQ(ě!F䓜뾴(Jv`zqqql6mG:4 }xV2v2γutY{^5MĶV쮛=΁ܚ[W^iӦ(\RwlSٽd+[t;KjLa~aukkb왬P[I]DVPPx<^}cݜKYX>\Sd6cͪ6ΩJ 3J=Ÿ.Qޮ収 #JaUGCy{cf˘4,,L,kJD...xEEEeee k e3e&C4ےi\wsuT5N6_K>چQ(F~{cN{sHG#+DQR(|>B*Ĝ̜n4a)͔BwgZ,!F˚F#"%ۜiM٦s\"EpLIH}_s羰G$ $l6;WSٷψMԴv.VN63ml-Y'U V6w*E=nX|L s`Eu90hw^v˥>ɠv`aaa Ad>Z[[b\.T*dX,www1c/ޔd2L, Ls8# %\VignetteIndexEntry{GOplot_0.2} %\VignetteEngine{knitr::rmarkdown} \usepackage[utf8]{inputenc} --- A manual to exploit the possibilities and limitations of the R package GOplot. ##Introduction The GOplot package concentrates on the visualization of biological data. More precisely, the package will help combine and integrate expression data with the results of a functional analysis. The package cannot be used to perform any of these analyses. It is for visualization purpose only. In all the scientific fields we visualize information to meet a basic need- to tell a story. Attributable to space restrictions and a general need to present everything neat and tidy most of the times it is simply not possible to actually tell a story. Therefore, we use vision to communicate information. A well designed and elaborated figure provides the beholder with high-dimensional information in a much smaller space than for example a table. The idea of the package is to provide the user with functions that allow a quick examination of large amounts of data, expose trends and find patterns & correlations within the data. Effective data visualization is an important tool in the decision making process and helps to find further pieces of the puzzle picturing the answer of your biological question. Based on that you will be able to confirm or falsify your hypotheses. You might even start to look in a different direction to investigate your topic relying on the insight a new visualization provides. The plotting functions of the package were developed with a hierarchical structure in mind; starting with a general overview and closing with definite subsets of selected genes and terms. To explain the idea let us use an example. ##The toy example GOplot comes with a manually compiled data set. Selected samples were downloaded from gene expression omnibus (accession number: *[GSE47067](http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47067)*). As a brief summary, the data set contains the transcriptomic information of endothelial cells from two steady state tissues (brain and heart). More detailed information can be found in the paper by *[Nolan et al. 2013](http://www.ncbi.nlm.nih.gov/pubmed/23871589)*. The data was normalized and a statistical analysis was performed to determine differentially expressed genes. *DAVID* functional annotation tool was used to perform a gene- annotation enrichment analysis of the set of differentially expressed genes (adjusted p-value < 0.05). The data set contains the five following items: ```{r table1, echo = FALSE, results = 'asis'} toy<-data.frame(Name=c('EC$eset','EC$genelist','EC$david','EC$genes','EC$process'),Description=c('Data frame of normalized expression values of brain and heart endothelial cells (3 replicates)','Data frame of differentially expressed genes (adjusted p-value < 0.05)','Data frame of results from a functional analysis of the differentially expressed genes performed with DAVID','Data frame of selected genes with logFC','Character vector of selected enriched biological processes'),Dimension=c('20644 x 7','2039 x 7','174 x 5','37 x 2','7')) knitr::kable(toy, colnames=c('Name','Description','Dimension (row, col)')) ``` ##Getting started As a first step we want to get an overview of the enriched GO terms of our differentially expressed genes. But before we start plotting we need to bring the data in the right format for the plotting functions. In general, the data object of the plotting functions can be created manually, but the package includes a function that does the job for you. The *circle_dat* function combines the result of the functional analysis with a list of selected genes and their logFC. Most likely a list of differentially expressed genes. *circle_dat* takes two data frames as an input. The first one contains the results of the functional analysis and should have at least four columns (category, term, genes, adjusted p-value). Additionally, a data frame of the selected genes and their logFC is needed. This data frame can be, for example, the result from a statistical analysis performed with *limma*. Let us have a look at the mentioned data frames. ```{r glimpse, warning = FALSE, message = FALSE} library(GOplot) # Load the dataset data(EC) # Get a glimpse of the data format of the results of the functional analysis... head(EC$david) # ...and of the data frame of selected genes head(EC$genelist) ``` Now, that we know what the input data looks like it's time to use the *cirlce_dat* function to create the plotting object. ```{r circ_object, warning = FALSE, message = FALSE} # Generate the plotting object circ <- circle_dat(EC$david, EC$genelist) ``` The **circ** object has eight columns with the following names: * category * ID * term * count * gene * logFC * adj_pval * zscore Since most of the gene- annotation enrichment analysis are based on the gene ontology database the package was build with this structure in mind, but is not restricted to it. As explained by *[Ashburner et al.](http://www.ncbi.nlm.nih.gov/pubmed/10802651)* in their paper from the year 2000, gene ontology is structured as an acyclic graph and it provides terms covering different areas. These terms are grouped into three independent categories: BP (biological process), CC (cellular component) or MF (molecular function). The first column of the **circ** object contains this information, which was already given in the input. For more information on the structure of gene ontology, have a look at the documentation section of the gene ontology consortium [website](http://geneontology.org/page/ontology-documentation). All the terms from inside the gene ontology database come with a GO **ID** and a GO **term** description. The **ID** column of the circ object is optional. So in case you want to use a functional analysis tool that is not based on gene ontology you won't have an **ID** column. The term description column does contain just that: a description of the term and the performance of the implemented functions does not depend on possible resemblance with gene ontology terms. **Count** is the number of genes assigned to a term. **Gene** names and their **logFC** are taken from the input list of selected genes. The significance of a term is indicated by the adjusted p-value (**adj_pval**). Terms with an adjusted p-value < 0.05 are considered as significantly enriched and are more likely to provide reliable information. The last column contains the **zscore**, an easy to calculate value to give you a hint if the biological process (/molecular function/cellular components) is more likely to be decreased (negative value) or increased (positive value). $$zscore=\frac{(up-down)}{\sqrt{count}}$$ Whereas *up* and *down* are the number of assigned genes up-regulated (logFC>0) in the data or down- regulated (logFC<0), respectively. ##The plots ###The modified barplot (GOBar) Since we do not really know what to expect from our data the aim of the first figure should be to display as many terms as possible without going too much into details. Nevertheless, the figure shall help us to pick the interesting and valuable terms. Therefore, we need to have some parameters to quantify the importance. Since the majority of scientific sampled data is plotted using bar charts a modified version of the normal barplot function, named *GOBar*, is included. The *GOBar* function allows the user to quickly create an appealing barplot. ```{r GOBar, warning = FALSE, message = FALSE, fig.width = 8.3, fig.height = 6} # Generate a simple barplot GOBar(subset(circ, category == 'BP')) ``` On the y-axis the significance of the terms is shown and the bars are ordered according to their z-score. If you want, you can change the order by setting the argument *order.by.zscore* to FALSE. In this case the bars are ordered based on their significance. Additionally, the barplot can be easily faceted according to the categories of the terms using the argument *display* of the plotting function (output not shown). ```{r GOBar2, eval = FALSE, warning = FALSE, message = FALSE} # Facet the barplot according to the categories of the terms GOBar(circ, display = 'multiple') ``` To add a title use *title* and to change the colour scale of the z-score use the argument *zsc.col* (output not shown). ```{r GOBar3, eval = FALSE, warning = FALSE, message = FALSE} # Facet the barplot, add a title and change the colour scale for the z-score GOBar(circ, display = 'multiple', title = 'Z-score coloured barplot', zsc.col = c('yellow', 'black', 'cyan')) ``` Barplots are common and very easy to read, but they might not be the absolute solution. Another possibility to display an overview for high- dimensional data is the bubble plot. ###The bubble plot (GOBubble) The bubble plot is another possibility to get an overview of the enriched terms. The z-score is assigned to the x-axis and the negative logarithm of the adjusted p-value to the y-axis, as in the barplot (the higher the more significant). The area of the displayed circles is proportional to the number of genes (circ$count) assigned to the term and the colour corresponds to the category. The help page of the plotting function (?GOBubble) lists all the arguments to change the layout of the plot. As a default the circles are labeled with the term ID. Therefore, a table connecting the IDs and terms is displayed on the right side by default. You can hide it by setting the argument *table.legend* to FALSE. If you want to display the term description instead set the argument *ID* to FALSE. Not all the circles are labeld due to the limited space and the overlap of the circles. A threshold for the labeling is set (default=5) based on the negative logarithm of the adjusted p-value. ```{r GOBubble1, warning = FALSE, message = FALSE, fig.keep = 'none'} # Generate the bubble plot with a label threshold of 3 GOBubble(circ, labels = 3) ``` ![GOBubble1.](GOBubble1.png) To add a title, change the colour of the circles, facet the plot and to change the label threshold use the following arguments: ```{r GOBubble2, warning = FALSE, message = FALSE, fig.keep = 'none'} # Add a title, change the colour of the circles, facet the plot according to the categories and change the label threshold GOBubble(circ, title = 'Bubble plot', colour = c('orange', 'darkred', 'gold'), display = 'multiple', labels = 3) ``` ![GOBubble2.](GOBubble2.png) For the facet plot it is also possible to colour the background of the panels according to the displayed category by setting *bg.col* to TRUE. ```{r GOBubble3, warning = FALSE, message = FALSE, fig.keep = 'none'} # Colour the background according to the category GOBubble(circ, title = 'Bubble plot with background colour', display = 'multiple', bg.col = T, labels = 3) ``` ![GOBubble3.](GOBubble3.png) A new function, *reduce_overlap*, was included in the updated version of the package to reduce the number of redundant terms. So far, the implemented method is very simple + slow and needs further refinement. Nevertheless, by reducing the number of redundant terms the readability of plots, like the bubble plot, improves significantly. The function deletes all terms that have a gene overlap greater than or equal to a set threshold. The function keeps one term per group as a representative without taking into consideration the GO hierarchy. ```{r GOBubble4, warning = FALSE, message = FALSE, fig.keep = 'none', eval = FALSE} # Reduce redundant terms with a gene overlap >= 0.75... reduced_circ <- reduce_overlap(circ, overlap = 0.75) # ...and plot it GOBubble(reduced_circ, labels = 2.8) ``` ![GOBubble4.](GOBubble4.png) ### Circular visualization of the results of gene- annotation enrichment analysis (GOCircle) The overview plots shall help to decide which of the terms are the most interesting to us. Of course, this decision depends although on the hypothesis and ideas you want to confirm with your data. Not always are the most significant terms the ones you are interested in. So, after manually selecting a set of valuable terms (EC$process) the next figure should provide us with more details on this specific terms. One of the major issues we figured out by presenting the plots was: it was sometimes difficult to interpret the information the z-score provides. Since the measure is not that common. As shown above it is simply the number of up- regulated genes minus the number of down- regulated genes divided by the square root of the count. The *GOCircle* plot emphasizes this fact. ```{r GOCircle1, warning = FALSE, message = FALSE, fig.keep = 'none'} # Generate a circular visualization of the results of gene- annotation enrichment analysis GOCircle(circ) ``` ![Circle plot.](GOCirc.png) The outer circle shows a scatter plot for each term of the logFC of the assigned genes. Red circles display up- regulation and blue ones down- regulation by default. The colours can be changed with the argument *lfc.col*. Therefore, it is easier to understand, why in some cases highly significant terms have a z-score close to zero. A z-score of zero does not mean that the term is not important. At least not as long as it is significantly enriched. It just shows that the z-score is a crude measure, because obviously the score does not take into account the functional level and activation dependencies of the single genes within a process. You can change the layout of the plot with various arguments, see ?GOCirlce.The *nsub* argument needs a little bit more explanation to be used wisely. First of all, it can be a numeric or a character vector. If it is a character vector then it contains the IDs or term descriptions of the processes you want to display (output not shown). ```{r GOCircle2, eval = FALSE} # Generate a circular visualization of selected terms IDs <- c('GO:0007507', 'GO:0001568', 'GO:0001944', 'GO:0048729', 'GO:0048514', 'GO:0005886', 'GO:0008092', 'GO:0008047') GOCircle(circ, nsub = IDs) ``` If *nsub* is a numeric vector then the number defines how many terms are displayed. It starts with the first row of the input data frame (output not shown). ```{r GOCircle3, eval = FALSE} # Generate a circular visualization for 10 terms GOCircle(circ, nsub = 10) ``` This kind of visualization is only useful for a smaller set of terms. The maximum number of terms lies around 12. While the number of terms decreases the amount of displayed information increases. ### Display of the relationship between genes and terms (GOChord) Based on the **[Circos](http://circos.ca/)** plots designed by *[Martin Krzywinski](http://mkweb.bcgsc.ca/)* the *GOChord* plotting function was implemented. It displays the relationship between a list of selected genes and terms, as well as the logFC of the genes. As an input a binary membership matrix is necessary. You can build the matrix on your own or you use the implemented function *chord_dat* which does the job for you. The function takes three arguments: *data*, *genes* and *process*, of which from the last two only one is mandatory. So, the *circle_dat* combined your expression data with the results from the functional analysis. The bar and bubble plot allowed you to get a first impression of your data and now, you selected a list of genes and processes you think are valuable. *GOCircle* adds a layer to display the expression values of the genes assigned to the terms, but it lacks the information of the relationship between the genes and the terms. It is not easy to figure out if some of the genes are linked to multiple processes. The chord plot fills the void left by *GOCircle*. ```{r GOChord1, warning = FALSE, message = FALSE} # Define a list of genes which you think are interesting to look at. The item EC$genes of the toy # sample contains the data frame of selected genes and their logFC. Have a look... head(EC$genes) # Since we have a lot of significantly enriched processes we selected some specific ones (EC$process) EC$process # Now it is time to generate the binary matrix chord <- chord_dat(circ, EC$genes, EC$process) head(chord) ``` Rows are genes and columns are terms. A '0' indicates that the gene is not assigned to the term; a '1' the opposite. As mentioned before it is possible to leave either the *genes* or the *process* argument out. If you pass on the *process* argument the binary matrix is build for the list of selected genes and all the processes with at least one assigned gene. On the other hand, if you just provide a set of processes without limiting the list of genes, the binary matrix is generated for all the genes which are assigned to at least one of the processes from your list (output not shown). ```{r GOChord2, eval=FALSE, warning = FALSE, message = FALSE} # Generate the matrix with a list of selected genes chord <- chord_dat(data = circ, genes = EC$genes) # Generate the matrix with selected processes chord <- chord_dat(data = circ, process = EC$process) ``` Be aware that a pass on either *genes* or *process* might lead to a large binary matrix which results in a confusing visualization. The chart was designed for smaller subsets of high-dimensional data. Like the other plotting functions *GOChord* provides the user with a lot of arguments to change the layout of the plot, see ?GOChord. Most of the arguments address the adjustment of the font size of the labels, the space between them, the colour scale for the logFC and the colour of the ribbons. Despite the asthetics there are two other arguments: *gene.order* and *nlfc*. The first argument defines the order of the genes with the three possible options: 'logFC', 'alphabetical', 'none'. Actually the options are quite self- explanatory. Sometimes you are performing the differential expression analysis for multiple conditions and/or batches. Therefore, you want to include more than one logFC value per gene. To adjust to this situation you should use the *nlfc* argument. It is a numeric value and it defines the number of logFC columns within your binary membership matrix. The default is '1' assuming that most of the time you just have one contrast and one logFC value per gene. ```{r GOChord3, warning = FALSE, message = FALSE, fig.keep = 'none'} # Create the plot GOChord(chord, space = 0.02, gene.order = 'logFC', gene.space = 0.25, gene.size = 5) ``` ![Chord1.](GOChord1.png) The *space* argument defines the space between the coloured rectangles representing the logFC. Also the font size of the gene labels (*gene.size*) and the space (*gene.space*) between them was changed. The genes were ordered according to their logFC values setting *gene.order* to 'logFC'. Sometimes the plot gets a bit crowded and you would like to reduce the number of displayed genes or processes. You can do this automatically by making use of the *limit* argument. Limit is a vector with two cutoff values (default = c(0, 0)). The first value defines the minimum (>=) number of terms a gene has to be assigned to. The second value determines the number of genes assigned to a selected term. For example, to display only genes which are assigned to at least three processes you would use the following line of code (output not shown): ```{r GOChord4, warning = FALSE, message = FALSE, fig.keep = 'none'} # Display only genes which are assigned to at least three processes GOChord(chord, limit = c(3, 0), gene.order = 'logFC') ``` ### Heatmap of genes and terms (GOHeat) Thanks to a very nice suggestion from *[Maureen Sartor, Ph.D.](http://sartorlab.ccmb.med.umich.edu/)* I implemented *GOHeat*. The *GOHeat* function generates a heatmap of the relationship between genes and terms similar to *GOChord*. Biological processes are displayed in rows and genes in columns. Each column is divided into smaller rectangles and the colouring of the tiles depends on the presence or abscence of logFC values. In addition genes are clustered to highlight groups of genes with similar annotated functions. Basically the function has two modes depending on the *nlfc* argument. If *nlfc = 0*, so no logFC values are available, the colouring encodes for the overall number of processes the respective gene is assigned to. Let's have a look at an example... ```{r GOHeat1, warning = FALSE, message = FALSE, fig.keep = 'none'} # First, we use the chord object without logFC column to create the heatmap GOHeat(chord[,-8], nlfc = 0) ``` ![Heat1.](GOHeat_nolfc.png) In case of *nlfc = 1* the colour corresponds to the logFC of the gene... ```{r GOHeat2, warning = FALSE, message = FALSE, fig.keep = 'none'} # First, we use the chord object without logFC column to create the heatmap GOHeat(chord, nlfc = 1, fill.col = c('red', 'yellow', 'green')) ``` ![Heat2.](GOHeat_lfc.png) ### Golden eye (GOCluster) The idea behind the *GOCluster* function is to visualize as much information as possible. Here is an example: ```{r GOCluster, warning=FALSE, eval=FALSE, message=FALSE, fig.keep='none'} GOCluster(circ, EC$process, clust.by = 'logFC', term.width = 2) ``` ![GOCluster.](GOCluster.png) Hierarchical clustering is a popular method for gene expression analysis due to its unsupervised nature assuring an unbiased result. Genes are grouped together based on their expression patterns, thus clusters are likely to contain sets of co-regulated or functionally related genes. *GOCluster* performs the hierarchical clustering of the gene expression profiles using the *hclust* method in core R. If you want to change the distance metric or the clustering algorithm use the arguments *metric* and *clust*, respectively. The resulting dendrogram is transformed with the help of *ggdendro* to be suitable for a visualization with *ggplot2*. As before a circular layout was chosen, because it is not only effective but also visually appealing. The first ring next to the dendrogram represents the logFC of the genes, which are actually the leaves of the clustering tree. In case you are interested in more than one contrast the *nlfc* argument is also available for this function. By default it is set to '1', so only one ring is drawn. Like always the logFC values are colour- coded with an user- definable colour scale (*lfc.col*). The next ring represents the terms assigned to the genes. For aesthetic reasons the terms should be reduced to a reasonable number with the argument *process*. The terms are colour- coded as well and you can change the default colours by using the argument *term.col*. Once again, the plotting function provides you with a bunch of arguments to change the layout of the plot and you can check them out on the help page, ?GOCluster. Probably the most important argument of the function is *clust.by*. It expects a character vector specifying if the clustering should be done for gene expression pattern ('logFC', as in the figure above) or functional categories ('terms'). ```{r GOCluster2, warning=FALSE, eval=FALSE, message=FALSE, fig.keep='none'} GOCluster(circ, EC$process, clust.by = 'term', lfc.col = c('darkgoldenrod1', 'black', 'cyan1')) ``` ![GOCluster2.](GOCluster2.png) ### Venn diagram (GOVenn) In this biological context we implemented a Venn diagram that can be used to detect relations between various lists of differentially expressed genes or to explore the intersection of genes of multiple terms from the functional analysis. The Venn diagram does not only display the number of overlap genes, but it also displays the information about the gene expression patterns (commonly up- regulated, commonly down- regulated or contra- regulated). At the moment, maximal three datasets are aloud as an input. The input data frame contains at least two columns: one for the gene names and one for the logFC value. ```{r GOVenn, warning=FALSE, message=FALSE, fig.keep='none'} l1 <- subset(circ, term == 'heart development', c(genes,logFC)) l2 <- subset(circ, term == 'plasma membrane', c(genes,logFC)) l3 <- subset(circ, term == 'tissue morphogenesis', c(genes,logFC)) GOVenn(l1,l2,l3, label = c('heart development', 'plasma membrane', 'tissue morphogenesis')) ``` ![Venn diagram.](GOVenn.png) For example, heart development and tissue morphogenesis share a set of 22 genes, whereas 5 are commonly up-regulated and 17 are commonly down-regulated. The important thing to notice is, that the pie charts don't display redundant information. Thus, if you compare three datasets the genes which are shared by all datasets (pie chart in the middle) are not included in the other pie charts. The following [link](https://wwalter.shinyapps.io/Venn/) refers to the shinyapp of this tool. 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GOplot takes the output of any general enrichment analysis and generates plots at different levels of detail: from a general overview to identify the most enriched categories (bar plot, bubble plot) to a more detailed view displaying different types of information for molecules in a given set of categories (circle plot, chord plot, cluster plot). The package provides a deeper insight into omics data and allows scientists to generate insightful plots with only a few lines of code to easily communicate the findings. ## Installation GOplot is available via CRAN: http://cran.r-project.org/web/packages/GOplot * the latest released version: `install.packages("GOplot")` * the latest development version: `install_github("wencke/wencke.github.io")` ## Available functions For preprocessing: circle_dat(), chord_dat() and reduce_overlap() For plotting: GOBubble(), GOBar(), GOChord(), GOCluster(), GOCircle(), GOVenn(), GOHeat() A manual can be found on the website https://wencke.github.io/ GOplot/MD50000644000175100001440000000414412677016126012017 0ustar hornikusers1715bfe67bec477dd3cb8d7602e4fa20 *DESCRIPTION e61dd1ca8c29cbb323e13d8517d9c02a *NAMESPACE 4b28a97ef1acd9ed6c62896137721df8 *NEWS.md 23abcbfe83b5aba778ee015295f137aa *R/GOCluster.R e572f5815342fade1478975babe13886 *R/GOCore.R dfc4d334a0d33f9a93b570eff3e9e80f *R/GOHeat.R 42cbd333583bc85dd011a233cb3f2f74 *R/GOVenn.R c5f5d6bd7353ce68cefdc2be652b5960 *R/Helper.R 584ae11d874b64d6e5c4ff9c5b575d22 *README.md 0a9da26cff7c27c4304cd6c957e04af8 *build/vignette.rds 322babedca883c827c36456263b2a8ec *data/EC.rda b7bcdd8ea7db60feca771ffc0332b250 *inst/CITATION fcd68b105afd0eeb7427bbf4fd9d6841 *inst/doc/GOplot_vignette.R bb17de94d29ab2e4eb59b63b01accfb6 *inst/doc/GOplot_vignette.Rmd 452ddf9993f7c63c2ee59ce2ece6f673 *inst/doc/GOplot_vignette.html 4251e378a6add0b3ae7a596abdcbb2a6 *man/EC.Rd e90a8e3541703f5e5883bb3266e24a19 *man/GOBar.Rd c5bb4945d9ff65d94135ce4efaaf5459 *man/GOBubble.Rd 44149f89ac4b3e6198648742e408de8c *man/GOChord.Rd ee2fcf3f06f78085787602c7f8c6093b *man/GOCircle.Rd ce63714fd31d2075dc19890283c90c32 *man/GOCluster.Rd 0e75b8f0a887a4b435f3101e75977931 *man/GOHeat.Rd 12b7a3dc5e14f387675f436209b22346 *man/GOVenn.Rd 5c0c6435c09dab8eede3bb7bc3c91be0 *man/chord_dat.Rd 8c9fc2bea8e29d8e69191d14876c4177 *man/circle_dat.Rd e571685ac3bd31d377069f5340b3902f *man/reduce_overlap.Rd da9c6b713c73d7cf668dab80ac28f2de *vignettes/GOBar.png 9d64131438541b889d07e8e2b7f44d30 *vignettes/GOBubble1.png 0ea657fee85af6e5dc3c8f43e2ec9cbd *vignettes/GOBubble2.png fbf7808d3c7235e04feac329eadc9295 *vignettes/GOBubble3.png cdb1824e3bf1830599ffe85eb60bbcd4 *vignettes/GOBubble4.png e5787103b84b40771253e4e76beaf5b3 *vignettes/GOChord1.png ea9374ef982d235dcbcc1f8b2158a804 *vignettes/GOCirc.png 8e332df56b7dd05c8c81bbda25811926 *vignettes/GOCluster.png 93aa247b84c448148ecbc7edf4e94c50 *vignettes/GOCluster2.png d6fb473e4aaa993d3532bc78fa32b5c9 *vignettes/GOHeat_lfc.png 140ee05839dbcb04483ab1735cc18f62 *vignettes/GOHeat_nolfc.png 0514ae4a1ca467974d4197efddba0639 *vignettes/GOVenn.png 5f57576c51fe172d93f3622cc433f762 *vignettes/GOplot.css bb17de94d29ab2e4eb59b63b01accfb6 *vignettes/GOplot_vignette.Rmd f9eb01d9aba255d27107fd69cc246ba5 *vignettes/Titel.png GOplot/build/0000755000175100001440000000000012676706463012614 5ustar hornikusersGOplot/build/vignette.rds0000644000175100001440000000032012676706463015146 0ustar hornikusersb```b`fba`b2 1# 'v///LK-)I MASUbg $7M @G @ ּb4S]R RR@g;<E T [fN*ސ89 d Bw(,/׃ @?{49'ݣ\)%ziE@ w&GOplot/DESCRIPTION0000644000175100001440000000270612677016126013217 0ustar hornikusersPackage: GOplot Type: Package Title: Visualization of Functional Analysis Data Version: 1.0.2 Date: 2016-03-30 Authors@R: c( person("Wencke", "Walter", , email = "wencke.walter@arcor.de", role = c("aut", "cre")), person("Fatima", "Sanchez-Cabo", , role = "aut") ) URL: https://github.com/wencke/wencke.github.io BugReports: https://github.com/wencke/wencke.github.io/issues Description: Implementation of multilayered visualizations for enhanced graphical representation of functional analysis data. It combines and integrates omics data derived from expression and functional annotation enrichment analyses. Its plotting functions have been developed with an hierarchical structure in mind: starting from a general overview to identify the most enriched categories (modified bar plot, bubble plot) to a more detailed one displaying different types of relevant information for the molecules in a given set of categories (circle plot, chord plot, cluster plot, Venn diagram, heatmap). Depends: ggplot2 (>= 2.0.0), ggdendro (>= 0.1-17), gridExtra (>= 2.0.0), RColorBrewer (>= 1.1.2), R (>= 3.2.3) License: GPL-2 Suggests: knitr, rmarkdown VignetteBuilder: knitr LazyData: TRUE RoxygenNote: 5.0.1 NeedsCompilation: no Packaged: 2016-03-30 08:24:21 UTC; BioinfoNerd Author: Wencke Walter [aut, cre], Fatima Sanchez-Cabo [aut] Maintainer: Wencke Walter Repository: CRAN Date/Publication: 2016-03-30 20:35:02 GOplot/man/0000755000175100001440000000000012676706421012262 5ustar hornikusersGOplot/man/chord_dat.Rd0000644000175100001440000000252112676706421014500 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \name{chord_dat} \alias{chord_dat} \title{Creates a binary matrix.} \usage{ chord_dat(data, genes, process) } \arguments{ \item{data}{A data frame with at least two coloumns: GO ID|term and genes. Each row contains exactly one GO ID|term and one gene. A column containing logFC values is optional and might be used if \code{genes} is missing.} \item{genes}{A character vector of selected genes OR data frame with coloumns for gene ID and logFC.} \item{process}{A character vector of selected processes} } \value{ A binary matrix } \description{ The function creates a matrix which represents the binary relation (1= is related to, 0= is not related to) between selected genes (row) and processes (column). The resulting matrix can be visualized with the \code{\link{GOChord}} function. } \details{ If more than one logFC value for each gene is at disposal, only one should be used to create the binary matrix. The other values have to be added manually later. } \examples{ \dontrun{ # Load the included dataset data(EC) # Building the circ object circ <- circle_dat(EC$david, EC$genelist) # Building the binary matrix chord <- chord_dat(circ, EC$genes, EC$process) } } \seealso{ \code{\link{GOChord}} } GOplot/man/EC.Rd0000644000175100001440000000151112676706421013036 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \docType{data} \name{EC} \alias{EC} \title{Transcriptomic information of endothelial cells.} \format{A list containing 5 items} \source{ \url{http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47067} } \usage{ data(EC) } \description{ The data set contains the transcriptomic information of endothelial cells from two steady state tissues (brain and heart). More detailed information can be found in the paper by Nolan et al. 2013. The data was normalized and a statistical analysis was performed to determine differentially expressed genes. DAVID functional annotation tool was used to perform a gene- annotation enrichment analysis of the set of differentially expressed genes (adjusted p-value < 0.05). } \keyword{datasets} GOplot/man/GOCluster.Rd0000644000175100001440000000516712676706421014431 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCluster.R \name{GOCluster} \alias{GOCluster} \title{Circular dendrogram.} \usage{ GOCluster(data, process, metric, clust, clust.by, nlfc, lfc.col, lfc.min, lfc.max, lfc.space, lfc.width, term.col, term.space, term.width) } \arguments{ \item{data}{A data frame which should be the result of \code{\link{circle_dat}} in case the data contains only one logFC column. Otherwise \code{data} is a data frame whereas the first column contains the genes, the second the term and the following columns the logFCs of the different contrasts.} \item{process}{A character vector of selected processes (ID or term description)} \item{metric}{A character vector specifying the distance measure to be used (default='euclidean'), see \code{dist}} \item{clust}{A character vector specifying the agglomeration method to be used (default='average'), see \code{hclust}} \item{clust.by}{A character vector specifying if the clustering should be done for gene expression pattern or functional categories. By default the clustering is done based on the functional categories.} \item{nlfc}{If TRUE \code{data} contains multiple logFC columns (default= FALSE)} \item{lfc.col}{Character vector to define the color scale for the logFC of the form c(high, midpoint,low)} \item{lfc.min}{Specifies the minimium value of the logFC scale (default = -3)} \item{lfc.max}{Specifies the maximum value of the logFC scale (default = 3)} \item{lfc.space}{The space between the leafs of the dendrogram and the ring for the logFC} \item{lfc.width}{The width of the logFC ring} \item{term.col}{A character vector specifying the colors of the term bands} \item{term.space}{The space between the logFC ring and the term ring} \item{term.width}{The width of the term ring} } \description{ GOCluster generates a circular dendrogram of the \code{data} clustering using by default euclidean distance and average linkage.The inner ring displays the color coded logFC while the outside one encodes the assigned terms to each gene. } \details{ The inner ring can be split into smaller rings to display multiply logFC values resulting from various comparisons. } \examples{ \dontrun{ #Load the included dataset data(EC) #Generating the circ object circ<-circular_dat(EC$david, EC$genelist) #Creating the cluster plot GOCluster(circ, EC$process) #Cluster the data according to gene expression and assigning a different color scale for the logFC GOCluster(circ,EC$process,clust.by='logFC',lfc.col=c('darkgoldenrod1','black','cyan1')) } } GOplot/man/GOCircle.Rd0000644000175100001440000000536412676706421014210 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \name{GOCircle} \alias{GOCircle} \title{Circular visualization of the results of a functional analysis.} \usage{ GOCircle(data, title, nsub, rad1, rad2, table.legend = T, zsc.col, lfc.col, label.size, label.fontface) } \arguments{ \item{data}{A special data frame which should be the result of \code{circle_dat}} \item{title}{The title of the plot} \item{nsub}{A numeric or character vector. If it's numeric then the number defines how many processes are displayed (starting from the first row of \code{data}). If it's a character string of processes then these processes are displayed} \item{rad1}{The radius of the inner circle (default=2)} \item{rad2}{The radius of the outer circle (default=3)} \item{table.legend}{Shall a table be displayd or not? (default=TRUE)} \item{zsc.col}{Character vector to define the colour scale for the z-score of the form c(high, midpoint,low)} \item{lfc.col}{A character vector specifying the colour for up- and down-regulated genes} \item{label.size}{Size of the segment labels (default=5)} \item{label.fontface}{Font style of the segment labels (default='bold')} } \description{ The circular plot combines gene expression and gene- annotation enrichment data. A subset of terms is displayed like the \code{GOBar} plot in combination with a scatterplot of the gene expression data. The whole plot is drawn on a specific coordinate system to achieve the circular layout.The segments are labeled with the term ID. } \details{ The outer circle shows a scatter plot for each term of the logFC of the assigned genes. The colours can be changed with the argument \code{lfc.col}. The \code{nsub} argument needs a bit more explanation to be used wisely. First of all, it can be a numeric or a character vector. If it is a character vector then it contains the IDs or term descriptions of the displayed processes.If \code{nsub} is a numeric vector then the number defines how many terms are displayed. It starts with the first row of the input data frame. } \examples{ \dontrun{ # Load the included dataset data(EC) # Building the circ object circ <- circle_dat(EC$david, EC$genelist) # Creating the circular plot GOCircle(circ) # Creating the circular plot with a different colour scale for the logFC GOCircle(circ, lfc.col = c('purple', 'orange')) # Creating the circular plot with a different colour scale for the z-score GOCircle(circ, zsc.col = c('yellow', 'black', 'cyan')) # Creating the circular plot with different font style GOCircle(circ, label.size = 5, label.fontface = 'italic') } } \seealso{ \code{\link{circle_dat}}, \code{\link{GOBar}} } GOplot/man/GOHeat.Rd0000644000175100001440000000277712676706421013675 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOHeat.R \name{GOHeat} \alias{GOHeat} \title{Displays heatmap of the relationship between genes and terms.} \usage{ GOHeat(data, nlfc, fill.col) } \arguments{ \item{data}{The matrix represents the binary relation (1= is related to, 0= is not related to) between a set of genes (rows) and processes (columns)} \item{nlfc}{Defines the number of logFC columns (default = 0)} \item{fill.col}{Defines the color scale break points} } \description{ The GOHeat function generates a heatmap of the relationship between genes and terms. Biological processes are displayed in rows and genes in columns. In addition genes are clustered to highlight groups of genes with similar annotated functions. The input can be generated with the \code{\link{chord_dat}} function. } \details{ The heatmap has in general two modes which depend on the \code{nlfc} argument. If \code{nlfc = 0}, so no logFC values are available, the coloring encodes for the overall number of processes the respective gene is assigned to. In case of \code{nlfc = 1} the color corresponds to the logFC of the gene. } \examples{ \dontrun{ # Load the included dataset data(EC) # Generate the circ object circ <- circle_dat(EC$david, EC$genelist) # Generate the chord object chord <- chord_dat(circ, EC$genes, EC$process) # Create the plot with user-defined colors GOHeat(chord, nlfc = 1, fill.col = c('red', 'yellow', 'green')) } } GOplot/man/reduce_overlap.Rd0000644000175100001440000000162012676706421015547 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \name{reduce_overlap} \alias{reduce_overlap} \title{Eliminates redundant terms.} \usage{ reduce_overlap(data, overlap) } \arguments{ \item{data}{A data frame created with \code{circle_dat}.} \item{overlap}{Skalar indicating the threshold for gene overlap (default = 0.75).} } \description{ The function eliminates all terms with a gene overlap >= set threshold (\code{overlap}) The reduced dataset can be used to improve the readability of plots such as \code{GOBubble} and \code{GOBar} } \details{ The function is currently very slow. } \examples{ \dontrun{ # Load the included dataset data(EC) # Building the circ object circ <- circle_dat(EC$david, EC$genelist) # Eliminate redundant terms reduced_circ <- reduce_overlap(circ) # Plot reduced data GOBubble(reduced_circ) } } GOplot/man/circle_dat.Rd0000644000175100001440000000301412676706421014640 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \name{circle_dat} \alias{circle_dat} \title{Creates a plotting object.} \usage{ circle_dat(terms, genes) } \arguments{ \item{terms}{A data frame with columns for 'category', 'ID', 'term', adjusted p-value ('adj_pval') and 'genes'} \item{genes}{A data frame with columns for 'ID', 'logFC'} } \description{ The function takes the results from a functional analysis (for example DAVID) and combines it with a list of selected genes and their logFC. The resulting data frame can be used as an input for various ploting functions. } \details{ Since most of the gene- annotation enrichment analysis are based on the gene ontology database the package was build with this structure in mind, but is not restricted to it. Gene ontology is structured as an acyclic graph and it provides terms covering different areas. These terms are grouped into three independent \code{categories}: BP (biological process), CC (cellular component) or MF (molecular function). The "ID" and "term" columns of the \code{terms} data frame refer to the ID and term description, whereas the ID is optional. The "ID" column of the \code{genes} data frame can contain any unique identifier. Nevertheless, the identifier has to be the same as in "genes" from \code{terms}. } \examples{ \dontrun{ #Load the included dataset data(EC) #Building the circ object circ<-circular_dat(EC$david, EC$genelist) } } GOplot/man/GOBar.Rd0000644000175100001440000000270112676706421013503 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \name{GOBar} \alias{GOBar} \title{Z-score coloured barplot.} \usage{ GOBar(data, display, order.by.zscore = T, title, zsc.col) } \arguments{ \item{data}{A data frame containing at least the term ID and/or term, the adjusted p-value and the z-score. A possible input can be generated with the \code{circle_dat} function} \item{display}{A character vector indicating whether a single plot ('single') or a facet plot with panels for each category should be drawn (default='single')} \item{order.by.zscore}{Defines the order of the bars. If TRUE the bars are ordered according to the z-scores of the processes. Otherwise the bars are ordered by the negative logarithm of the adjusted p-value} \item{title}{The title of the plot} \item{zsc.col}{Character vector to define the colour scale for the z-score of the form c(high, midpoint,low)} } \description{ Z-score coloured barplot of terms ordered alternatively by z-score or the negative logarithm of the adjusted p-value } \details{ If \code{display} is used to facet the plot the width of the panels will be proportional to the length of the x scale. } \examples{ \dontrun{ #Load the included dataset data(EC) #Building the circ object circ<-circular_dat(EC$david, EC$genelist) #Creating the bar plot GOBar(circ) #Faceting the plot GOBar(circ, display='multiple') } } GOplot/man/GOBubble.Rd0000644000175100001440000000453412676706421014200 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCore.R \name{GOBubble} \alias{GOBubble} \title{Bubble plot.} \usage{ GOBubble(data, display, title, colour, labels, ID = T, table.legend = T, table.col = T, bg.col = F) } \arguments{ \item{data}{A data frame with coloumns for category, GO ID, term, adjusted p-value, z-score, count(num of genes)} \item{display}{A character vector. Indicates whether it should be a single plot ('single') or a facet plot with panels for each category (default='single')} \item{title}{The title (on top) of the plot} \item{colour}{A character vector which defines the colour of the bubbles for each category} \item{labels}{Sets a threshold for the displayed labels. The threshold refers to the -log(adjusted p-value) (default=5)} \item{ID}{If TRUE then labels are IDs else terms} \item{table.legend}{Defines whether a table of GO ID and GO term should be displayed on the right side of the plot or not (default = TRUE)} \item{table.col}{If TRUE then the table entries are coloured according to their category, if FALSE then entries are black} \item{bg.col}{Should only be used in case of a facet plot. If TRUE then the panel backgrounds are coloured according to the displayed category} } \description{ The function creates a bubble plot of the input \code{data}. The input \code{data} can be created with the help of the \code{\link{circle_dat}} function. } \details{ The x- axis of the plot represents the z-score. The negative logarithm of the adjusted p-value (corresponding to the significance of the term) is displayed on the y-axis. The area of the plotted circles is proportional to the number of genes assigned to the term. Each circle is coloured according to its category and labeled alternatively with the ID or term name.If static is set to FALSE the mouse hover effect will be enabled. } \examples{ \dontrun{ #Load the included dataset data(EC) #Building the circ object circ <- circular_dat(EC$david, EC$genelist) #Creating the bubble plot colouring the table entries according to the category GOBubble(circ, table.col = T) #Creating the bubble plot displaying the term instead of the ID and without the table GOBubble(circ, ID = F, table.legend = F) #Faceting the plot GOBubble(circ, display = 'multiple') } } GOplot/man/GOChord.Rd0000644000175100001440000000747312676706421014051 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOCluster.R \name{GOChord} \alias{GOChord} \title{Displays the relationship between genes and terms.} \usage{ GOChord(data, title, space, gene.order, gene.size, gene.space, nlfc = 1, lfc.col, lfc.min, lfc.max, ribbon.col, border.size, process.label, limit) } \arguments{ \item{data}{The matrix represents the binary relation (1= is related to, 0= is not related to) between a set of genes (rows) and processes (columns); a column for the logFC of the genes is optional} \item{title}{The title (on top) of the plot} \item{space}{The space between the chord segments of the plot} \item{gene.order}{A character vector defining the order of the displayed gene labels} \item{gene.size}{The size of the gene labels} \item{gene.space}{The space between the gene labels and the segement of the logFC} \item{nlfc}{Defines the number of logFC columns (default=1)} \item{lfc.col}{The fill color for the logFC specified in the following form: c(color for low values, color for the mid point, color for the high values)} \item{lfc.min}{Specifies the minimium value of the logFC scale (default = -3)} \item{lfc.max}{Specifies the maximum value of the logFC scale (default = 3)} \item{ribbon.col}{The background color of the ribbons} \item{border.size}{Defines the size of the ribbon borders} \item{process.label}{The size of the legend entries} \item{limit}{A vector with two cutoff values (default= c(0,0)). The first value defines the minimum number of terms a gene has to be assigned to. The second the minimum number of genes assigned to a selected term.} } \description{ The GOChord function generates a circularly composited overview of selected/specific genes and their assigned processes or terms. More generally, it joins genes and processes via ribbons in an intersection-like graph. The input can be generated with the \code{\link{chord_dat}} function. } \details{ The \code{gene.order} argument has three possible options: "logFC", "alphabetical", "none", which are quite self- explanatory. Maybe the most important argument of the function is \code{nlfc}.If your \code{data} does not contain a column of logFC values you have to set \code{nlfc = 0}. Differential expression analysis can be performed for multiple conditions and/or batches. Therefore, the data frame might contain more than one logFC value per gene. To adjust to this situation the \code{nlfc} argument is used as well. It is a numeric value and it defines the number of logFC columns of your \code{data}. The default is "1" assuming that most of the time only one contrast is considered. To represent the data more useful it might be necessary to reduce the dimension of \code{data}. This can be achieved with \code{limit}. The first value of the vector defines the threshold for the minimum number of terms a gene has to be assigned to in order to be represented in the plot. Most of the time it is more meaningful to represent genes with various functions. A value of 3 excludes all genes with less than three term assignments. Whereas the second value of the parameter restricts the number of terms according to the number of assigned genes. All terms with a count smaller or equal to the threshold are excluded. } \examples{ \dontrun{ # Load the included dataset data(EC) # Generating the binary matrix chord<-chord_dat(circ,EC$genes,EC$process) # Creating the chord plot GOChord(chord) # Excluding process with less than 5 assigned genes GOChord(chord, limit = c(0,5)) # Creating the chord plot genes ordered by logFC and a different logFC color scale GOChord(chord,space=0.02,gene.order='logFC',lfc.col=c('red','black','cyan')) } } \seealso{ \code{\link{chord_dat}} } GOplot/man/GOVenn.Rd0000644000175100001440000000456412676706421013716 0ustar hornikusers% Generated by roxygen2: do not edit by hand % Please edit documentation in R/GOVenn.R \name{GOVenn} \alias{GOVenn} \title{Venn diagram of differentially expressed genes.} \usage{ GOVenn(data1, data2, data3, title, label, lfc.col, circle.col, plot = T) } \arguments{ \item{data1}{A data frame consisting of two columns: ID, logFC} \item{data2}{A data frame consisting of two columns: ID, logFC} \item{data3}{A data frame consisting of two columns: ID, logFC} \item{title}{The title of the plot} \item{label}{A character vector to define the legend keys} \item{lfc.col}{A character vector determining the background colors of the pie segments representing up- and down- regulated genes} \item{circle.col}{A character vector to assign clockwise colors for the circles} \item{plot}{If TRUE only the venn diagram is plotted. Otherwise the function returns a list with two items: the actual plot and a list containing the overlap entries (default= TRUE)} } \description{ The function compares lists of differentially expressed genes and illustrates possible relations.Additionally it represents the variety of gene expression patterns within the intersection in small pie charts with three segements. Clockwise are shown the number of commonly up- regulated, commonly down- regulated and contra- regulated genes. } \details{ The \code{plot} argument can be used to adjust the amount of information that is returned by calling the function. If you are only interested in the actual plot of the venn diagram, \code{plot} should be set to TRUE. Sometimes you also want to know the elements of the intersections. In this case \code{plot} should be set to FALSE and the function call will return a list of two items. The first item, that can be accessed by $plot, contains the plotting information. Additionally, a list ($table) will be returned containing the elements of the various overlaps. } \examples{ \dontrun{ #Load the included dataset data(EC) #Generating the circ object circ<-circular_dat(EC$david, EC$genelist) #Selecting terms of interest l1<-subset(circ,term=='heart development',c(genes,logFC)) l2<-subset(circ,term=='plasma membrane',c(genes,logFC)) l3<-subset(circ,term=='tissue morphogenesis',c(genes,logFC)) GOVenn(l1,l2,l3, label=c('heart development','plasma membrane','tissue morphogenesis')) } }