This vignette will guide you through the more advanced uses of gmoviz
, such
as the incremental apporach to generating plots and
making finer modifications. It is highly recommended
that you have read the basic overview of gmoviz
before this vignette.
As well as high-level functions functions,
gmoviz
contains many lower-level functions that can be used to construct a
plot track-by-track for more flexibility.
This section will use the rBiocpkg("pasillaBamSubset")
package for example
data, so please ensure you have it installed before proceeding:
if (!require("pasillaBamSubset")) {
if (!require("BiocManager"))
install.packages("BiocManager")
BiocManager::install("GenomicAlignments")
}
library(pasillaBamSubset)
The first step in creating a circular plot is to initialise it. This involves creating the ideogram (the rectangles that represent each sequence), which lays out the sectors for data to be plotted into. To do this, we need some ideogram data, in one of the following formats:
GRanges
, with one range for each sector you’d like to plot.data.frame
, with three columns: chr
(sector’s name), start
and
end
.For example, the following two ideogram data are equivalent:
ideogram_1 <- GRanges(seqnames = c("chrA", "chrB", "chrC"),
ranges = IRanges(start = rep(0, 3), end = rep(1000, 3)))
ideogram_2 <- data.frame(chr = c("chrA", "chrB", "chrC"),
start = rep(0, 3),
end = rep(1000, 3))
print(ideogram_1)
#> GRanges object with 3 ranges and 0 metadata columns:
#> seqnames ranges strand
#> <Rle> <IRanges> <Rle>
#> [1] chrA 0-1000 *
#> [2] chrB 0-1000 *
#> [3] chrC 0-1000 *
#> -------
#> seqinfo: 3 sequences from an unspecified genome; no seqlengths
print(ideogram_2)
#> chr start end
#> 1 chrA 0 1000
#> 2 chrB 0 1000
#> 3 chrC 0 1000
Both of the higher level functions featureDiagram
and insertionDiagram
do
this as their first step.
Of course, typing this manually each time is troublesome. gmoviz
provides the
function getIdeogramData
which creates a GRanges
of the ideogram data
from either a .bam file, single .fasta file or a folder containing many
.fasta files.1 Note that reading in from a .bam file is significantly faster than from
a .fasta file. This function can be used as follows:
## from a .bam file
fly_ideogram <- getIdeogramData(bam_file = pasillaBamSubset::untreated3_chr4())
## from a single .fasta file
fly_ideogram_chr4_only <- getIdeogramData(
fasta_file = pasillaBamSubset::dm3_chr4())
But what if we wanted to read in just the chr3L? Luckily getIdeogramData
has
filters to select the specific sequences you want.
When reading in the ideogram data from file, there are often sequences in the
.bam file or .fasta file folder that are not necessary for the plot. Thus,
the getIdeogramData
function provides three filters to allow you to only
read in the sequences you want.2 These filters only work on the bam_file
and fasta_folder
input
methods. Using a fasta_file
means that filtering is not possible (although
you can of course edit the ideogram GRanges after it is generated).
If we want only a single chromosome/sequence, we can supply it to
wanted_chr
:
getIdeogramData(bam_file = pasillaBamSubset::untreated3_chr4(),
wanted_chr = "chr4")
#> GRanges object with 1 range and 0 metadata columns:
#> seqnames ranges strand
#> <Rle> <IRanges> <Rle>
#> [1] chr4 0-1351857 *
#> -------
#> seqinfo: 1 sequence from an unspecified genome; no seqlengths
Alternatively, if we want all chromosomes/sequences expect one, we can supply
it to unwanted_chr
:
getIdeogramData(bam_file = pasillaBamSubset::untreated3_chr4(),
unwanted_chr = "chrM")
#> GRanges object with 7 ranges and 0 metadata columns:
#> seqnames ranges strand
#> <Rle> <IRanges> <Rle>
#> [1] chr2L 0-23011544 *
#> [2] chr2R 0-21146708 *
#> [3] chr3L 0-24543557 *
#> [4] chr3R 0-27905053 *
#> [5] chr4 0-1351857 *
#> [6] chrX 0-22422827 *
#> [7] chrYHet 0-347038 *
#> -------
#> seqinfo: 7 sequences from an unspecified genome; no seqlengths
Finally, you can supply any regex pattern to just_pattern
to create your own
custom filter:
getIdeogramData(bam_file = pasillaBamSubset::untreated3_chr4(),
just_pattern = "R$")
#> GRanges object with 2 ranges and 0 metadata columns:
#> seqnames ranges strand
#> <Rle> <IRanges> <Rle>
#> [1] chr2R 0-21146708 *
#> [2] chr3R 0-27905053 *
#> -------
#> seqinfo: 2 sequences from an unspecified genome; no seqlengths
Of course, for these filters to work the spelling of the filter must exactly match the spelling of the .fasta file names or the sequences in the .bam file.
Now that we have the ideogram data, we can initialise the graph. For this example, we will just focus on chromosome 4.
gmovizInitialise(fly_ideogram_chr4_only, track_height = 0.15)
We can see that a rectangle has been plotted and labelled to indicate chr4. Changing a few visual settings, we can create a better looking ideogram:
gmovizInitialise(fly_ideogram_chr4_only,
space_between_sectors = 25, # bigger space between start & end
start_degree = 78, # rotate the circle
sector_label_size = 1, # bigger label
track_height = 0.15, # thicker rectangle
xaxis_spacing = 30) # label every 30 degrees on the x axis
However, these small tweaks are not the only way we can enhance the appearance
of our plot. gmovizInitialise
can also display
coverage data and labels, as well as
supporting zooming and alteration of sector widths.
As demonstrated with the insertionDiagram
and featureDiagram
functions, we
can supply some coverage_data
to enhance the ideogram and change the regular
rectangles into line graphs which display the coverage (‘coverage
rectangles’). This then allows the easy identification of deletions,
duplications and other events which alter the coverage.
To do this, we must first read in the coverage information from the .bam
file. This can be done with the getCoverage
function:
chr4_coverage <- getCoverage(
regions_of_interest = "chr4",
bam_file = pasillaBamSubset::untreated3_chr4(),
window_size = 350, smoothing_window_size = 400)
Here, we get the smoothed and windowed coverage for chr4.3 See below the section on smoothing and windowing
for the effect of each of these arguments As we wanted the
coverage for the entire chr4, we could simply make
regions_of_interest = "chr4"
. However, we could also have supplied a GRanges
describing that area instead. Whichever input is used, it is really important
that the sequence names match exactly. For example, the following will t
hrow an error, because there is no sequence named “4” or “Chr4” in the .bam
file:
getCoverage(regions_of_interest = "4",
bam_file = pasillaBamSubset::untreated3_chr4(),
window_size = 300, smoothing_window_size = 400)
#> Error in getCoverage(regions_of_interest = "4", bam_file = pasillaBamSubset::untreated3_chr4(), : Make sure all of the chromsomes in regions_of_interest are in
#> the bam file and spelled exactly the same as in the bam
getCoverage(regions_of_interest = "Chr4",
bam_file = pasillaBamSubset::untreated3_chr4(),
window_size = 300, smoothing_window_size = 400)
#> Error in getCoverage(regions_of_interest = "Chr4", bam_file = pasillaBamSubset::untreated3_chr4(), : Make sure all of the chromsomes in regions_of_interest are in
#> the bam file and spelled exactly the same as in the bam
Now that we have the coverage data, we can plot the ideogram again using this
information. To draw a ‘coverage rectangle’ we need to firstly specifiy the
coverage_data
to be used (as either a GRanges or a data frame) and then also
supply to coverage_rectangle
a vector of the sector names to plot the
coverage data for.4 This means that you can have the coverage of multiple sequences/regions
in the same GRanges but choose to plot only some of them.
gmovizInitialise(ideogram_data = fly_ideogram_chr4_only,
coverage_rectangle = "chr4",
coverage_data = chr4_coverage,
xaxis_spacing = 30)
As you can see, the chr4 ideogram rectangle is replaced with a line graph showing the coverage over the entire chromosome. The coloured area represents the coverage, allowing easy identification of high and low coverage areas.
When reading in the coverage data, there are two additional parameters
window_size
and smoothing_window_size
that modify the values.
window_size
controls the window size over which coverage is calculated
(where a window size of 1 is per base coverage. A larger window size will
reduce the time taken to read in, smooth and plot the coverage. It will also
remove some of the variation in the coverage, although this is not its primary
aim. If you have more than 10-15,000 points, it is highly recommended to
use a larger window size, as this will take a long time to plot.
smoothing_window_size
controls the window used for moving average
smoothing, as carried out by the pracma package. It does not
reduce the number of points and so offers no speed improvement (in fact, it
increases the time taken to read in the coverage data). It does, however,
reduce the variation to produce a smoother, more attractive plot.
For example, try running the following:
# default window size (per base coverage)
system.time({getCoverage(regions_of_interest = "chr4",
bam_file = pasillaBamSubset::untreated3_chr4())})
# window size 100
system.time({getCoverage(regions_of_interest = "chr4",
bam_file = pasillaBamSubset::untreated3_chr4(),
window_size = 100)})
# window size 500
system.time({getCoverage(regions_of_interest = "chr4",
bam_file = pasillaBamSubset::untreated3_chr4(),
window_size = 500)})
Notice how going from the default window size of 1 (per base coverage) to a relatively modest window size of 100 dramatically reduces the time needed to read in the coverage data.
In terms of the appearance of the plot: (note: for speed, we will plot only a subset of the chromosome: from 70000-72000bp)
# without smoothing
chr4_region <- GRanges("chr4", IRanges(70000, 72000))
chr4_region_coverage <- getCoverage(regions_of_interest = chr4_region,
bam_file = pasillaBamSubset::untreated3_chr4())
gmovizInitialise(ideogram_data = chr4_region, coverage_rectangle = "chr4",
coverage_data = chr4_region_coverage, custom_ylim = c(0,4))
# with moderate smoothing
chr4_region_coverage <- getCoverage(regions_of_interest = chr4_region,
bam_file = pasillaBamSubset::untreated3_chr4(),
smoothing_window_size = 10)
gmovizInitialise(ideogram_data = chr4_region, coverage_rectangle = "chr4",
coverage_data = chr4_region_coverage, custom_ylim = c(0,4))
# with strong smoothing
chr4_region_coverage <- getCoverage(regions_of_interest = chr4_region,
bam_file = pasillaBamSubset::untreated3_chr4(),
smoothing_window_size = 75)
gmovizInitialise(ideogram_data = chr4_region, coverage_rectangle = "chr4",
coverage_data = chr4_region_coverage, custom_ylim = c(0,4))
Notice how adding smoothing dramatically improves the appearance of the plot.
It also slightly reduces the time taken, because there are less extreme points.
However, it does result in the loss of the finer detail of the coverage data.
Thus, it is recommended that you play around with the values of
smoothing_window_size
and window_size
and choose a value that is best
suited to your own data.
One more functionality of gmovizInitialise
is the ability to add labels to
the outside of the plot. These can be used to identify regions of interest,
such as genes or exons. The format of this should be:
A GRanges
, with one range for each label & the label’s text as a metadata
column label
A data.frame
, with columns: chr
(sector’s name), start
and end
that represent the position of the label and label
that contains the label’s
text
For example:
label <- GRanges(seqnames = "chr4",
ranges = IRanges(start = 240000, end = 280000),
label = "region A")
gmovizInitialise(fly_ideogram_chr4_only, label_data = label,
space_between_sectors = 25, start_degree = 78,
sector_label_size = 1, xaxis_spacing = 30)
This is the same as how the labels in insertionDiagram
and featureDiagram
are implemented.
These labels can be manually specified as above, or read in from a .gff file,
which also gives the option of colour coding the labels.5 This works simply by supplying a vector of colours (with the same length
as the number of labels) to label_colour
rather than just a single colour.
You don’t have to have the colours as a part of the label data, it’s just a
bit easier to keep track of that way. :
labels_from_file <- getLabels(
gff_file = system.file("extdata", "example.gff3", package = "gmoviz"),
colour_code = TRUE)
gmovizInitialise(fly_ideogram_chr4_only,
label_data = labels_from_file,
label_colour = labels_from_file$colour,
space_between_sectors = 25, start_degree = 78,
sector_label_size = 1, xaxis_spacing = 30)
#### Changing sector sizes {#changing_sector_widths}
By default, when using gmovizInitialise
, each sector is sized to match its
length relative to all of the other sectors on the plot to faciliate accurate
representation of the scale. However, when a plot includes sectors that differ
greatly in size, this can lead to problems. For example:
fly_ideogram <- getIdeogramData(bam_file = pasillaBamSubset::untreated3_chr4(),
unwanted_chr = "chrM")
gmovizInitialise(fly_ideogram)
Notice that chr4 and chrYHet are much shorter than the other chromosomes. Thus, when we try to plot it, those three shorter sectors are so small that they are barely visible and their labels overlap leading to confusion.
We can deal with this in one of two ways: firstly by manually specifying the width (size) of each sector and secondly by zooming.
One way to manipulate the width/size of the sectors is to specify a
custom_sector_width
(custom sector width) vector. This vector should be the
same length as the number of sectors. For example:
gmovizInitialise(fly_ideogram,
custom_sector_width = c(0.2, 0.2, 0.2, 0.2, 0.2, 0.1, 0.1))
Notice that the custom_sector_width
vector had length 7, because this is how
many sectors there are. custom_sector_width
can also be used for the
insertionDiagram
and featureDiagram
functions in the same way.
Whilst it is quite easy to set custom sector widths when there are only a few sectors, it can be quite troublesome for entire genomes. Also, using this method loses the relative sizing of all sectors, potentially leading to misinterpretation.
We can solve this problem by using the zooming functionality of
gmovizInitialise
. Doing this is relatively easy, all we need to do is
supply the names of sector(s) to zoom to the zoom_sectors
argument:
gmovizInitialise(fly_ideogram, zoom_sectors = c("chr4", "chrYHet"),
zoom_prefix = "z_")
Now, chr4 and chrYHet are clearly visible alongside the rest of the sectors.
Notice that chrYHet is still around 1/4 of the size of chr4, as is expected
from their relative sizes (347038bp and 1351857bp, respectively). Also, all of
the other chromosomes are still proportional. Another advantage of using the
zooming is that the zoom_prefix
applied to the start of the zoomed sector
label makes it clear which sectors have been zoomed and which have not.
After initialising the graph, the next step is to add tracks containing data. The two main types of track are the feature track and the numeric tracks, which can be combined as desired to create a customised plot.
The ‘feature’ track, plots regions of interest just like the
featureDiagram function (in fact,
featureDiagram
is just a convenient combination of gmovizInitialise
and
drawFeatureTrack
). If you only want to plot features, then using
featureDiagram
is probably easier, but taking a track-by-track approach with
drawFeatureTrack
allows the combination of feature tracks with numeric data
(see here for an example).
Just like featureDiagram
, drawFeatureTrack
requires feature_data. See
here for an explanation of the format.
Feature data can be read in from a .gff file using the getFeatures
function.
features <- getFeatures(
gff_file = system.file("extdata", "example.gff3", package = "gmoviz"),
colours = gmoviz::rich_colours)
Here, we have set the colours
parameter to rich_colours
, one of the five
colour sets provided by gmoviz
(see here for a description of
each colour set) This means that the features will be allocated a colour from
this set based on the ‘type’ field of the .gff file.
Once the feature data is read in, it is highly recommended to take a look and tweak it, if necessary.
Once we have the feature data, we can add a feature track to our plot. As we
are only adding one track, increasing track_height
to 0.18 gives us a bit
more room to draw the features.
## remember to initialise first
gmovizInitialise(fly_ideogram_chr4_only, space_between_sectors = 25,
start_degree = 78, xaxis_spacing = 30, sector_label_size = 1)
drawFeatureTrack(features, feature_label_cutoff = 80000, track_height = 0.18)
Notice that the geneY label was drawn inside the arrow whilst the others were
drawn further into the circle. This is because we set feature_label_cutoff
to
80000, so any features less than 80000bp long have their labels drawn outside,
so that the label isn’t hanging off the end of the feature. See below for a
detailed discussion of this concept.
When using the featureDiagram
and drawFeatureTrack
functions, you
may have noticed that the position of the labels changes based on the size of
the feature being plotted. For example, in the following plot, the second ‘ins’
label is drawn outside the feature, further towards the centre of the circle.
This is because the size of the feature is less than the
feature_label_cutoff
.
## the data
plasmid_ideogram <- GRanges("plasmid", IRanges(start = 0, end = 3000))
plasmid_features <- getFeatures(
gff_file = system.file("extdata", "plasmid.gff3", package="gmoviz"),
colour_by_type = FALSE, # colour by name rather than type of feature
colours = gmoviz::rich_colours) # choose colours from rich_colours (see ?colourSets)
## the plot
featureDiagram(plasmid_ideogram, plasmid_features, track_height = 0.17)
Of course, you can specify your own cutoff. At 1, all labels will be plotted inside their respective features.
## smallest label cutoff
featureDiagram(plasmid_ideogram, plasmid_features, track_height = 0.17,
feature_label_cutoff = 1)
As well as the feature track, gmoviz
also contains more traditional numeric
data tracks: the scatterplot and the line graph.
To showcase these tracks, we will generate some example data:
numeric_data <- GRanges(seqnames = rep("chr4", 50),
ranges = IRanges(start = sample(0:1320000, 50),
width = 1),
value = runif(50, 0, 25))
Scatterplot tracks can be plotted with drawScatterplotTrack
and line graphs
with drawLinegraphTrack
:
## remember to initialise first
gmovizInitialise(fly_ideogram_chr4_only,
space_between_sectors = 25, start_degree = 78,
sector_label_size = 1, xaxis_spacing = 30)
## scatterplot
drawScatterplotTrack(numeric_data)
## line graph
drawLinegraphTrack(sort(numeric_data), gridline_colour = NULL)
Note that for the line graph track, the data should be sorted in ascending order before plotting.
These numeric tracks can then be combined with feature tracks, as desired:
gmovizInitialise(fly_ideogram_chr4_only, space_between_sectors = 25,
start_degree = 78, xaxis_spacing = 30, sector_label_size = 1)
drawScatterplotTrack(numeric_data, track_height = 0.14, yaxis_increment = 12)
drawFeatureTrack(features, feature_label_cutoff = 80000, track_height = 0.15)
Like circlize, gmoviz
relies on the package
ComplexHeatmap (Gu, Eils, and Schlesner 2016) to generate its legends. More
information about how this works can be found
here, but for
simplicity, gmoviz
provides the makeLegends
function to create legend
objects without requiring an understanding of how the ComplexHeatmap
package
works.
Here, we will make a legend for the plot shown just previously.
legend <- makeLegends(
feature_legend = TRUE, feature_data = features,
feature_legend_title = "Regions of interest", scatterplot_legend = TRUE,
scatterplot_legend_title = "Numeric data",
scatterplot_legend_labels = "value")
legend
is a legend object that can be plotted alongside a circos plot using
the gmovizPlot
function:
As explained here
the legends of ComplexHeatmap
are generated using grid graphics whilst the
circular plots of circlize
use base graphics. Thus, combining the two
requires the use of the gridBase package. More information can
be found at the aforementioned link, but gmoviz
provides the gmovizPlot
function to conveniently combine these two elements.
The gmovizPlot
function generates a plot based on the code supplied to the
plotting_functions
parameter and saves it as an image, alongside and optional
title and legend.6 The legend object can be either one generated using makeLegends
or
directly made using the functionality of the ComplexHeatmap
package.
gmovizPlot(file_name = "example.svg", file_type = "svg",
plotting_functions = {
gmovizInitialise(
fly_ideogram_chr4_only, space_between_sectors = 25, start_degree = 78,
xaxis_spacing = 30, sector_label_size = 1)
drawScatterplotTrack(
numeric_data, track_height = 0.14, yaxis_increment = 12)
drawFeatureTrack(
features, feature_label_cutoff = 80000, track_height = 0.15)
}, legends = legend, title = "Chromosome 4", background_colour = "white",
width = 8, height = 5.33, units = "in")
#> pdf
#> 2
gmovizPlot
also supports .svg and .ps outputs, as well as .png. Using a
vectorised output (.svg or .ps) is recommended as it allows you to easily edit
the plot in Illustrator or similar software.
gmoviz
colour setsOften 20+ sectors will be plotted during the initialisation of an entire
genome. Thus, gmoviz
includes five different colour sets each containing 34
colours in order to make it easier to give each of these sectors a unique,
beautiful colour. Many of the colours in these sets are from or are heavily
inspired by
colorBrewer.
The colour sets are:
nice_colours
: The default colour set. Medium brightness, light colours
designed for use on a white background.
pastel_colours
: A set of subdued/pastel colours (a less saturated version
of the nice_colours
set), designed for use on a white backgorund.
rich_colours
: A set of bright, vibrant colours (though not neon, like the
bright_colours_transparent
) designed for use on both white and black
backgrounds.
bright_colours_transparent
: A set of very bright/neon colours
with slight transparency designed for use on a black background.
bright_colours_opaque
: A set of very bright/neon colours
without transparency designed for use on a black background.
Using bright_colours_transparent
as the fill and bright_colours_opaque
as
the outline gives a nice effect on black backgrounds.
As mentioned, gmoviz
is based on the circlize (Gu et al. 2014)
package by Zuguang Gu. Thus, circlize
functions can be used alongside those
from gmoviz
to further customise plots.
Internally, gmoviz
calls circos.clear()
when initialising plots (at the
beginning of the gmovizInitialise
, featureDiagram
and insertionDiagram
functions) not at the end of functions. This means that, after you have run a
gmoviz
plotting function, you can use any circlize
function to make
further additions to the plot.
For an example, we will further annotate the insertionDiagram
plot produced
in the basic overview vignette here:
## the data
example_insertion <- GRanges(seqnames = "chr12",
ranges = IRanges(start = 70905597, end = 70917885),
name = "plasmid", colour = "#7270ea", length = 12000,
in_tandem = 11, shape = "forward_arrow")
## the original plot
insertionDiagram(example_insertion, either_side = c(70855503, 71398284),
start_degree = 45, space_between_sectors = 20)
## annotate with text
circos.text(x = 81000, y = 0.25, sector.index = "plasmid", track.index = 1,
facing = "bending.inside", labels = "(blah)", cex = 0.75)
## annotate with a box
circos.rect(xleft = 0, xright = 12000, ytop = 1, ybottom = 0,
track.index = 2, sector.index = "plasmid", border = "red")
Of course, use of circlize
functions is not just limited to small
annotations. Functions such as circos.trackPlotRegion()
and circos.track()
can be used to add additional tracks to plots generated with gmoviz
and
likewise the gmoviz
track functions (e.g. drawFeatureTrack
) can be used
to add to plots previously generated with circlize
. For more information
about using circlize
, see the comprehensive book
here
Warning: this also means that if you want to use circlize
to generate a
new plot after using gmoviz
, you will need to use circos.clear()
to reset.
This vignette was rendered in the following environment:
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#> [21] dbplyr_2.3.2 XVector_0.40.0
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#> [25] rmarkdown_2.21 pracma_2.4.2
#> [27] bit_4.0.5 xfun_0.39
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#> [71] hms_1.1.3 glue_1.6.2
#> [73] tools_4.3.0 BiocIO_1.10.0
#> [75] GenomicAlignments_1.36.0 XML_3.99-0.14
#> [77] grid_4.3.0 AnnotationDbi_1.62.0
#> [79] colorspace_2.1-0 GenomeInfoDbData_1.2.10
#> [81] restfulr_0.0.15 cli_3.6.1
#> [83] rappdirs_0.3.3 fansi_1.0.4
#> [85] ComplexHeatmap_2.16.0 dplyr_1.1.2
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#> [95] bit64_4.0.5
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Gu, Zuguang, Lei Gu, Roland Eils, Matthias Schlesner, and Benedikt Brors. 2014. “Circlize Implements and Enhances Circular Visualization in R.” Bioinformatics 30 (19): 2811–2. https://doi.org/10.1093/bioinformatics/btu393.