2.1 The epivizr app

The connection to Epiviz is managed through a session manager object of class EpivizApp. We can create this object and open Epiviz using the startEpiviz function.

app <- startEpiviz(workspace="qyOTB6vVnff", gists="2caf8e891201130c7daa")

This opens a websocket connection between the interactive R session and the browser client. This will allow us to visualize data stored in the Epiviz server along with data in the interactive R session.

Windows users: In Windows platforms we need to use the service function to let the interactive R session connect to the epiviz web app and serve data requests. We then escape (using ctl-c or esc depending on your environment) to continue with the interactive R session. This is required anytime you want epivizr to serve data to the web app, for example, when interacting with the UI. (We are actively developing support for non-blocking sessions in Windows platforms).

app$server$service()

2.2 Listing available chart types

Once the browser is open and is connected to an active epivizr session, the epivizr session registers available chart types supported in the epiviz JS app. To list available chart types we use method list_chart_types. In this vignette, we only show three chart types available for use in the epiviz JS app. The production epiviz JS app has a larger number of chart types available. Chart types can also be added dynamically as described in the epiviz JS documentation: http://epiviz.cbcb.umd.edu/help. Chart types that are added dynamically are listed and usable within an epivizr session.

app$chart_mgr$list_chart_types()

##          type                          js_class num_settings
## 1 BlocksTrack epiviz.plugins.charts.BlocksTrack            6
## 2   LineTrack   epiviz.plugins.charts.LineTrack           15
## 3 ScatterPlot epiviz.plugins.charts.ScatterPlot           11
##                                                                              settings
## 1                title,marginTop,marginBottom,marginLeft,marginRight,minBlockDistance
## 2 title,marginTop,marginBottom,marginLeft,marginRight,measurementGroupsAggregator,...
## 3 title,marginTop,marginBottom,marginLeft,marginRight,measurementGroupsAggregator,...
##   num_colors
## 1         10
## 2          8
## 3         10

Chart type settings, e.g., line widths, colors, margins etc. can be modified dynamically. Settings available for each chart type are briefly listed in this call. See below for further detail on customizing charts through settings and colors.

3.1 Adding block region tracks

Once the browser is open we can visualize the tcga_colon_blocks object containing blocks of methylation modifications in colon cancer. We use the plot method to do so.

blocks_chart <- app$plot(tcga_colon_blocks, datasource_name="450k colon_blocks")

Windows users: We need the service function to let the interactive R session serve data requests from the browser client as you interact with the UI. Escape (using ctl-c or esc depending on your environment) to continue with the interactive R session.

app$server$service()

You should now see that a new track is added to the Epiviz web app. You can think of this track as an interactive display device in R. As you navigate on the browser, data is requested from the R session through the websocket connection. Remember to escape to continue with your interactive R session if you are not running “non-blocking” mode. The blocks_chart object inherits from class EpivizChart, which we can use to get information about the data being displayed and the chart used to display it. Note that the “brushing” effect we implement in Epiviz works for epivizr tracks as well.

Now that we have interactive data connections to Epiviz we may want to iteratively compute and visualize our data. For example, we may want to only display methylation blocks inferred at a certain statistical significance level. In this case, we will filter by block size.

# subset to those with length > 250Kbp
keep <- width(tcga_colon_blocks) > 250000

# get the data object for chart
ms_obj <- app$get_ms_object(blocks_chart)
app$update_measurements(ms_obj, tcga_colon_blocks[keep,])

Now, only this subset of blocks will be displayed in the already existing track.

3.2 Modifying chart settings and colors

To modify default chart colors or settings, we need to know what settings can be applied to the chart. Again, as these are defined dynamically in the epiviz JS app we use a method, print_chart_type_info on the EpivizChartMgr class to list settings that can be applied to specific chart type. For example, to list chart settings available for a BlocksTrack chart we use the following:

app$chart_mgr$print_chart_type_info("BlocksTrack")

## Settings for chart type  BlocksTrack 
##                 id                  label default_value possible_values
## 1            title                  Title                              
## 2        marginTop             Top margin            25                
## 3     marginBottom          Bottom margin            23                
## 4       marginLeft            Left margin            20                
## 5      marginRight           Right margin            10                
## 6 minBlockDistance Minimum block distance             5                
##     type
## 1 string
## 2 number
## 3 number
## 4 number
## 5 number
## 6 number
## Colors: #1f77b4, #ff7f0e, #2ca02c, #d62728, #9467bd, #8c564b, #e377c2, #7f7f7f, #bcbd22, #17becf

There are two ways to set settings and colors to a chart: when the chart is initially created using the plot method, or after the chart is created using the set method in class EpivizChart. We will illustrate both ways of doing this:

If using the plot method, use the list_chart_settings method as above to list settings that can be applied to a chart type. For example, for a BlocksTrack chart, we can set minBlockDistance as a setting for the plot function which controls how close genomic regions can be before they are merged into a single rectangle in the chart. We can also change colors used in the chart this way.

settings <- list(minBlockDistance=50)
colors <- c("#d15014", "#5e97eb", "#e81ccd")
blocks_chart <- app$plot(tcga_colon_blocks, datasource_name="450k colon_blocks", settings=settings, colors=colors)

This will create a second blocks track using a different color map.

On the other hand, if the BlockChart is already added to the epiviz JS application session, use the set_chart_settings method to update settings and colors.

# create a list of settings and colors to update the plot
settings <- list(minBlockDistance=100)
colors <- c("#5e97eb", "#d15014", "#e81ccd")
app$chart_mgr$set_chart_settings(blocks_chart, settings=settings, colors=colors)

This changes the color map for the second blocks track added. Use the print_chart_info method to list settings and colors currently used in the chart.

# to list applied chart settings
app$chart_mgr$print_chart_info(blocks_chart)

## Chart settings for chart id  epivizChart_2 :
##                 id                  label default_value possible_values
## 1            title                  Title                              
## 2        marginTop             Top margin            25                
## 3     marginBottom          Bottom margin            23                
## 4       marginLeft            Left margin            20                
## 5      marginRight           Right margin            10                
## 6 minBlockDistance Minimum block distance           100                
##     type
## 1 string
## 2 number
## 3 number
## 4 number
## 5 number
## 6 number
## Colors: #5e97eb, #d15014, #e81ccd

Methods are also available directly on the EpivizChart objects. E.g., calls blocks_chart$set(settings=settings, colors=colors) and blocks_chart$print_info() yield the same result.

3.3 Printing charts

Charts can be printed as a pdf or png file through the epivizr session. To do so, use the print_chart method:

app$chart_mgr$print_chart(blocks_chart, file_name="blocks_chart", file_type="pdf")

This will create a file named blocks_chart.pdf which will be downloaded through your web browser. It will be found in the default file download location for your web browser.

3.4 Adding line plots along the genome

There are a number of different data types available to use through epivizr. You can see a list of R/BioC classes that are supported using ?register. In the previous section, we used the block data type to register a GenomicRanges object. To visualize methylation data at base-pair resolution from the tcga_colon_curves GenomicRanges object, we will use the bp type.

# add low-filter smoothed methylation estimates
means_track <- app$plot(tcga_colon_curves, datasource_name="450kMeth",type="bp",columns=c("cancerMean","normalMean"))

NOTE: You can adjust track settings to change how this new track looks like. For instance, to show all points in the window set the step parameter to 1, and to see a smooth interpolation of the data set the interpolation parameter to basis:

means_track$set(settings=list(step=1, interpolation="basis"))

Notice that we added two lines in this plot, one for mean methylation in cancer and another for mean methylation in normal. The columns argument specifies which columns in mcols(colon_curves) will be displayed.

We can also add a track containing the smooth methylation difference estimate used to define methylation blocks.

diff_chart <- app$plot(tcga_colon_curves, datasource_name="450kMethDiff",type="bp",columns=c("smooth"),ylim=matrix(c(-.5,.5),nc=1))

We pass limits for the y axis in this case. To see other arguments supported, you can use the help framework in R ?"EpivizApp". As before, we can specify settings for this new track.

5.1 Adding a scatterplot

Now we want to visualize the colon expression data in apColonData object as an MA plot in Epiviz. First, we add an "MA" assay to the ExpressionSet:

keep <- pData(apColonData)$SubType!="adenoma"
apColonData <- apColonData[,keep]
status <- pData(apColonData)$Status
Indexes <- split(seq(along=status),status)

exprMat <- exprs(apColonData)
mns <- sapply(Indexes, function(ind) rowMeans(exprMat[,ind]))
mat <- cbind(colonM=mns[,"1"]-mns[,"0"], colonA=0.5*(mns[,"1"]+mns[,"0"]))

pd <- data.frame(stat=c("M","A"))
rownames(pd) <- colnames(mat)

maEset <- ExpressionSet(
  assayData=mat,
  phenoData=AnnotatedDataFrame(pd),
  featureData=featureData(apColonData),
  annotation=annotation(apColonData)
)
show(maEset)

## ExpressionSet (storageMode: lockedEnvironment)
## assayData: 5339 features, 2 samples 
##   element names: exprs 
## protocolData: none
## phenoData
##   sampleNames: colonM colonA
##   varLabels: stat
##   varMetadata: labelDescription
## featureData: none
## experimentData: use 'experimentData(object)'
## Annotation: hgu133plus2

epivizr will use the annotation(maEset) annotation to determine genomic locations using the AnnotationDbi package so that only probesets inside the current browser window are displayed.

eset_chart <- app$plot(maEset, datasource_name="MAPlot", columns=c("colonA","colonM"))

## Loading required package: hgu133plus2.db

## Loading required package: AnnotationDbi

## Loading required package: org.Hs.eg.db

## 

## 

## 'select()' returned 1:many mapping between keys and columns

In this case, we specify which data is displayed in each axis of the scatter plot using the columns argument. The assay arguments indicates where data is obtained.

5.2 The RangedSummarizedExperiment Object

Epiviz is also able to display plots of data in the form of a RangedSummarizedExperiment object. After loading the tcga_colon_expression dataset in the epivizrData package, we can see that this object contains information on 239322 exons in 40 samples.

data(tcga_colon_expression)
show(tcga_colon_expression)

## class: RangedSummarizedExperiment 
## dim: 12800 40 
## metadata(0):
## assays(1): ''
## rownames(12800): 143686 149186 ... 149184 149185
## rowData names(2): exon_id gene_id
## colnames(40): TCGA-A6-5659-01A-01R-1653-07
##   TCGA-A6-5659-11A-01R-1653-07 ... TCGA-D5-5540-01A-01R-1653-07
##   TCGA-D5-5541-01A-01R-1653-07
## colData names(110): bcr_patient_barcode bcr_sample_uuid ...
##   Basename fullID

The assay slot holds a matrix of raw sequencing counts, so before we can plot a scatterplot showing expression, we must first normalize the count data. We use the geometric mean of each row as a reference sample to divide each column (sample) by, then use the median of each column as a scaling factor to divide each row (exon) by.

ref_sample <- 2 ^ rowMeans(log2(assay(tcga_colon_expression) + 1))
scaled <- (assay(tcga_colon_expression) + 1) / ref_sample
scaleFactor <- Biobase::rowMedians(t(scaled))
assay_normalized <- sweep(assay(tcga_colon_expression), 2, scaleFactor, "/")
assay(tcga_colon_expression) <- assay_normalized

Now, using the expression data in the assay slot and the sample data in the colData slot, we can compute mean exon expression by sample type.

status <- colData(tcga_colon_expression)$sample_type
index <- split(seq(along = status), status)
logCounts <- log2(assay(tcga_colon_expression) + 1)
means <- sapply(index, function(ind) rowMeans(logCounts[, ind]))
mat <- cbind(cancer = means[, "Primary Tumor"], normal = means[, "Solid Tissue Normal"])

Now, create a new RangedSummarizedExperiment object with the two column matrix, and all the information about the features of interest, in this case exons, are stored in the rowRanges slot to be queried by Epiviz.

sumexp <- SummarizedExperiment(mat, rowRanges=rowRanges(tcga_colon_expression))
se_chart <- app$plot(sumexp, datasource_name="Mean by Sample Type", columns=c("normal", "cancer"))

Again, the columns argument specifies what data will be displayed along which axis.

6.1 Slideshow

We can navigate to a location on the genome using the navigate method of the app object:

app$navigate("chr11", 110000000, 120000000)

There is a convenience function to quickly navigate to a series of locations in succession. We can use that to browse the genome along a ranked list of regions. Let’s navigate to the 5 most up-regulated exons in the colon exon expression data.

foldChange <- mat[,"cancer"]-mat[,"normal"]
ind <- order(foldChange,decreasing=TRUE)

# bounding 1Mb around each exon
slideshowRegions <- trim(rowRanges(sumexp)[ind] + 1000000L)
app$slideshow(slideshowRegions, n=5)

6.2 Printing the epivizr workspace

To print the current epiviz workspace as a pdf or png, we use the print_workspace method:

app$print_workspace(file_name="workspace", file_type="pdf")

This will create a file named workspace.pdf and will be downloaded through your web browser. It will be saved to the default file download location for your web browser.

6.3 Closing the session

To close the connection to Epiviz and remove all tracks added during the interactive session, we use the stop_app function.

app$stop_app()