In ETHZ-INS/epiwraps: epiwraps: Wrappers for plotting and dealing with epigenomics data

library(BiocStyle)

Introduction

Since reading data from many regions is typically longer than plotting it, we split plotting and acquiring the data. The latter is done through function specific to this package, while the former wraps around the r BiocStyle::Biocpkg("EnrichedHeatmap") package. The interface here has been simplified, but for full functionality and customization it is recommended to have a look at the r BiocStyle::Biocpkg("EnrichedHeatmap") documentation.

Reading signal in/around a set of regions

The signal2Matrix function reads genomic signals around the centers of a set of regions. It can read from bam and BigWig files, although reading from bam files is considerably slower and we strongly recommend using bigwig files. For generating bigwig files that show the kind of signal you want to visualize, see the vignette to this effect.

suppressPackageStartupMessages(library(epiwraps))
# we fetch the path to the example bigwig file:
bwf <- system.file("extdata/example_atac.bw", package="epiwraps")
# we load example regions (could be a GRanges or a path to a bed-like file):
regions <- system.file("extdata/example_peaks.bed", package="epiwraps")
# we obtain the matrix of the signal around the regions. For the purpose of this
# example, we'll read twice from the same:
m <- signal2Matrix(c(atac1=bwf, atac2=bwf), regions, extend=1000L)
m

The result is an object of class EnrichmentSE, which inherits from a r BiocStyle::Biocpkg("SummarizedExperiment","RangedSummarizedExperiment"), and therefore affords all the manipulations that the latter offers. Each region is stored as a row, and each sample or signal track as a column of the object. So we can see that we have signal for r nrow(m) rows/regions from two tracks.

We could subset to the first 50 regions as follows:

m[1:50,]

or obtain the coordinates of the queried regions :

rowRanges(m)

One can further obtain more detailed information about the bins saved in the object:

showTrackInfo(m)

This means that each signal track is a matrix of 200 columns, because we asked to extend 1000bp on either side, and the default bin size is 10bp, making 100 bins/windows on each side.

Extracting and manipulating signal matrices

It is possible to extract the list of signal matrix for manipulations, e.g. for transformation:

# square-root transform
m2 <- lapply(getSignalMatrices(m), sqrt)

See ?addAssayToESE for adding a list of signal matrices (such as m2 here) to an existing EnrichmentSE object. In addition, signal matrices can be combined, either manually or using ?mergeSignalMatrices.

Normalization

By default, bigwig files generated by epiwraps are normalized for library size, but this is not always sufficient. EnrichmentSE can further be normalized using various methods. For an overview of these methods, see the normalization vignette.

Plotting heatmaps

Once the signal has been read and the object prepared, (and eventually normalized, see the section below), we can plot heatmaps based on them as follows:

plotEnrichedHeatmaps(m)

We can use most arguments that are supported by r BiocStyle::Biocpkg("EnrichedHeatmap") (and thus, by extension, by r BiocStyle::Biocpkg("ComplexHeatmap")), for example:

plotEnrichedHeatmaps(m, colors=c("white","darkred"), cluster_rows=TRUE,
                     show_row_dend=TRUE, top_annotation=FALSE, 
                     row_title="My list of cool regions")

It is often useful to subset to regions with a high enrichment, which we can do with the score function:

plotEnrichedHeatmaps(m[head(order(-rowMeans(score(m))),50),])

Color-scale trimming

By default, the colorscale is trimmed to prevent most of it being driven by rare extreme values. This can be controlled via the trim argument (which indicates up to which quantile of data points to keep to establish the colorscale). Compare for instance the following two heatmaps:

plotEnrichedHeatmaps(m[,1], trim=1, scale_title="trim=1", column_title="trim=1 (no trim)",
                     top_annotation=FALSE) +
  plotEnrichedHeatmaps(m[,1], trim=0.99, scale_title="trim=0.99",
                       column_title="trim=0.99", top_annotation=FALSE) +
  plotEnrichedHeatmaps(m[,1], trim=0.9, column_title="trim=0.9",
                       scale_title="trim=0.9", top_annotation=FALSE)

The underlying data is exactly the same, only the color-mapping changes. In the left one, which has no trimming, a single very high value at the top forces the colorscale to extend to high values, even though most of the data is in the very low range, resulting in a very dark heatmap. In the one on the right, it's the opposite: so much is trimmed that many points reach the top of the colorscale, resulting in a an 'over-exposed' heatmap. In practice, it is advisable to use minimal trimming (e.g. the default is c(0.02,0.98)).

Different colorscales for different tracks

It is also possible to have different colorscales for different tracks, which is especially useful when comparing very different signals. To illustrate this, let's load an example with different tracks:

data(exampleESE)
exampleESE

We can put each of the three tracks on its own color scale:

plotEnrichedHeatmaps(exampleESE, multiScale=TRUE)

One could also specify colors separately by providing them as a list:

plotEnrichedHeatmaps(exampleESE,
                     colors=list(c("white","darkblue"), "darkgreen", "darkred"))

This information can also be stored in the object, rather than specified everytime:

exampleESE$hmcolors <- list(viridisLite::inferno(100),  "darkgreen", "darkred")
plotEnrichedHeatmaps(exampleESE)

Scaled regions

By default, signal2Matrix looks at a pre-defined interval (defined by the extend argument) around the center of the provided regions. This means that the width of the input regions is ignored. In some circumstances, however, it can be useful to scale regions to the same width, which can be done using the type="scaled" argument. Consider the following example:

ese <- cbind(
  signal2Matrix(c(center=bwf), regions, extend=1000L),
  signal2Matrix(c(scaled=bwf), regions, extend=1000L, type="scaled")
)
plotEnrichedHeatmaps(ese)

For some purposes, such as when plotting signal over transcripts, it can be useful for the target region to consist of multiple regions (e.g. exons) stitched together. This can be done by supplying a GRangesList as regions:

# we make a dummy GRangesList:
a <- sort(rtracklayer::import(regions))
dummy.grl <- GRangesList(split(a, rep(LETTERS[1:15],each=10)))
sm <- signal2Matrix(c(scaled=bwf), dummy.grl, extend=1000L, type="scaled")
plotEnrichedHeatmaps(sm)

Heatmap rasterization

When plotting more regions that there are pixels available, several regions have to be summarized in one pixel, and doing this before generating the heatmap makes the plot much less heavy.

By default, EnrichedHeatmap performs rasterization using the magick package when it is installed, and falls back to a very suboptimal method when not. It is therefore recommended to install the magick package.

Depending on your settings, if the heatmap is very big you might hit the preset limits of 'magick' base rasterization, which could result in an error such as 'Image must have at least 1 frame to write a bitmap'. In such cases, you might have to degrade to a lower-quality rasterization using the additional arguments raster_by_magick=FALSE, raster_device="CairoJPEG".

Finally, on some systems, the rasterization sometimes encounters an 'UnableToOpenBlob' error. At the moment, the only workaround this has been to disable rasterization using use_raster=FALSE.

Sorting and clustering

The traditional ranking by decreasing overall enrichment can easily hide patterns in the data, which are instead revealed by clustering. One approach is to use hierarchical clustering of the rows:

plotEnrichedHeatmaps(exampleESE, cluster_rows=TRUE)

In this example, this already reveals important patterns in the data, namely the fact that p300 binding is associated with H3K27ac and tends to be mutually exclusive with the promoter-associated H3K4me3 mark.

The hierarchical clustering is based on the whole enrichment profile, can easily be led astray by patterns in individual signals, and seldom provides good results in practice. An alternative is to use enrichment score weighted by distance to the center, eventually row-normalized, to cluster the regions. We provide a function to this end:

# we cluster the regions using 2 clusters, and store the cluster labels in the 
# rowData of the object: 
rowData(exampleESE)$cluster <- clusterSignalMatrices(exampleESE, k=2, scaleRows=TRUE)
# we additionally label the clusters with colors:
plotEnrichedHeatmaps(exampleESE, row_split="cluster",
                     mean_color=c("1"="red", "2"="blue"))

Note that here we are splitting into 3 clusters, you can also provide a range of values (e.g. k=2:8) and the function will also return cluster quality metrics for each.

Users can of course use their own algorithms to cluster regions. To this end, it can be useful to summarize each region in each sample using a single score which weights the signal according to the distance to the center of the target (see ?EnrichedHeatmap::enriched_score). This can be done for the default assay with the score method:

head(score(exampleESE))

Plotting aggregated signals

It is also possible to plot only the average signals across regions. To do this, we first melt the signal matrices and then use r CRANpkg("ggplot2"). The meltSignals function will return a data.frame showing the mean, standard deviation, standard error and median at each position relative to the center, for each sample/matrix:

d <- meltSignals(exampleESE)
head(d)

This can then be used for plotting, simply with ggplot:

library(ggplot2)
ggplot(d, aes(position, mean, colour=sample)) +
  geom_vline(xintercept=0, linetype="dashed") +
  geom_ribbon(aes(position, ymin=mean-SE, ymax=mean+SE, fill=sample), alpha=0.4, colour=NA) + 
  geom_line(linewidth=1.2) + 
  theme_bw() + labs(x="relative position", y="mean RPKM")

We could also include cluster information:

d <- meltSignals(exampleESE, splitBy = "cluster")
ggplot(d, aes(position, mean, colour=sample)) +
  geom_vline(xintercept=0, linetype="dashed") +
  geom_ribbon(aes(position, ymin=mean-SE, ymax=mean+SE, fill=sample), alpha=0.4, colour=NA) + 
  geom_line(linewidth=1.2) + facet_wrap(~split) +
  theme_bw() + labs(x="relative position", y="mean RPKM")

Visualizing DNAme and sparse signals

Nucleotide-resolution DNA methylation (as obtained from bisulfite sequencing) signal differs from the signals used throughout this vignette in that it is not continuous across the genome, but specifically at C or CpG nucleotides which have a variable density throughout the genome. As a consequence, it is likely that some of the plotting bins do not contain a CpG, in which case they get assigned a value of 0, even though they could be in a completely methylated region. For this reason, it is advisable to smooth DNA methylation signals for the purpose of visualization.

As an example, let's look at the gene bodies of some active genes from chr8 of the A549 cell lines:

data("exampleDNAme")
head(exampleDNAme)

As is typical of DNAme data, the object is a GRanges object (or more specifically a GPos object, since all ranges have a width of 1 nucleotide) with, in the score column, the percentage of DNA methylation. Let's see what happens if we plot a heatmap of this signal, with and without smoothing (we use type="scaled" to scale the gene bodies to the same size, since these can have very different sizes) :

o1 <- signal2Matrix(list(noSmooth=exampleDNAme), geneBodies, type="scaled")
o2 <- signal2Matrix(list(smoothed=exampleDNAme), geneBodies, type="scaled", 
                    smooth=TRUE)
o <- cbind(o1,o2)
plotEnrichedHeatmaps(o, scale_title="%\nmethylation", axis_name=c("TSS","TES"))

Both heatmaps show a very clear absence of DNA methylation at the promoter of these genes (upstream of the TSS) an predominantly methylated gene bodies. However they disagree substantially on the methylation levels upstream the promoter and downstream the transcription end sites (TES). This is because of the density of these regions in (covered) CpG nucleotides. Since most genes are rather long, most of the bins in the heatmap contain a CpG, leading to an actual methylation signal. In the flanking regions, however, this is not necessarily the case, and the non-smoothed heatmap does not distinguish bins that are unmethylated form bins for which there is no information. Instead, the smoothed heatmap on the right uses neighborhing bins to estimate the methylation status of each bin, effectively filling out the gaps. In doing so it provides the truthful representation, i.e. that the regions downstream of the genes and upstream of the promoters are, most of the time, as methylated as the gene bodies.

Smoothing is performed by r BiocStyle::Biocpkg("EnrichedHeatmap"); see ?EnrichedHeatmap::normalizeToMatrix for more information/customization.

Session information {-}

sessionInfo()

ETHZ-INS/epiwraps documentation built on July 14, 2024, 6:50 p.m.