Nothing
The **seqsetvis** package
In seqsetvis: Set Based Visualizations for Next-Gen Sequencing Data
knitr::opts_chunk$set(
collapse = TRUE,
dpi=60
)
Synopsis
seq - set - vis
r Githubpkg("jrboyd/seqsetvis") enables the visualization and analysis of
multiple genomic datasets. Although seqsetvis was designed for the comparison
of mulitple ChIP-seq datasets, this package is domain-agnostic and allows the
processing of multiple genomic coordinate files (bed-like files) and signal
files (bigwig files).
seqsetvis is ideal for the following tasks:
- Feature assessment and comparison.
- Differential enrichment or expression results assessment.
- Replicate comparison and reproducibility analysis.
- Chromatin state inspection.
- Clustering of histone modification and/or transcription factor enrichment
at features such as promoters or enhancers.
seqsetvis accepts and outputs the Bioconductor standard GRanges object while
leveraging the power and speed of the data.table package (an enhancement of
data.frame) with the flexibility of ggplot2 to provide users with
visualizations that are simultaneously publication ready while still being
highly customizable. By default, data.table is used entirely internally so
users unfamiliar with its use can safely ignore it. That said, fans of
data.table can use seqsetvis in a data.table exclusive mode if they wish.
Features
- Mostly it "just works" with minimal assumed knowledge of R or other
Bioconductor packages.
- All plots produced are
r CRANpkg("ggplot2") objects.
- easily customized according to user preferences by appending ggplot2
functions.
- powerful plot arrangement with with
r CRANpkg("cowplot"),
r CRANpkg("gridExtra"), etc.
- Although various plots are implemented, the underlying data are simple in
structure and easily accessible so you can do what you want with them.
- membership table for feature overlaps is just a logical matrix.
- signal data is stored in a tidy formatted
r CRANpkg("data.table"), ideal
for r CRANpkg("ggplot2") visualization.
Although initially designed to focus on peak call sets, seqsetvis accepts any
data in GRanges format and is therefore flexible enough to
work dowstream of many genomics tools and packages. Indeed, set comparison
visualizations are genomics agnostic and also accept generic data types defining
sets.
Functions
ssv* - most primary user facing functions are prefixed by ssv
ssvOverlapIntervalSets - derives sets from a list of GRangesssvMakeMembTable - coerces various ways of defining sets to a membership
tablessvFactorizeMembTable - converts a multi column membership table into
a single factor.ssvFetch* - retrieves properly formatted data from .bigwig and .bam
filesssvFeature* - visualizations of set counts and overlapsssvSignal* - visualizations of signal mapped to genomic coordinates in
sets context
- utility functions
easyLoad_* - loads files into a list of GRanges for input into ssv*
functions- miscellaneous functions such as wrappers for
data.table functions.
Installation and Loading
From Bioconductor
## try http:// if https:// URLs are not supported
if (!requireNamespace("BiocManager", quietly=TRUE))
install.packages("BiocManager")
BiocManager::install("seqsetvis")
Load the library
library(seqsetvis)
Load optional useful libraries
library(GenomicRanges)
library(data.table)
library(cowplot)
theme_set(cowplot::theme_cowplot())
Defining sets
seqsetvis has several ways of accepting set information. Since seqsetvis
was originally intended to focus on comparing sets of peak calls, a list of
GRanges or a GRangesList is directly supported but there are more
generic alternatives.
Overlapping a list of GRanges
Many functions in seqsetvis require some kind of sets definition
as input. Sets can be defined in one of several ways.
ssvOverlapIntervalSets() creates a single GRanges from a list of GRanges
with a logical membership table as metadata columns defining sets.
olaps = ssvOverlapIntervalSets(CTCF_in_10a_narrowPeak_grs)
head(olaps)
ssvOverlapIntervalSets() also accepts a GRangesList if that's more natural.
olaps_fromGRangesList = ssvOverlapIntervalSets(
GenomicRanges::GRangesList(CTCF_in_10a_narrowPeak_grs))
The membership data contained in the metadata columns of olaps is
the data structure seqsetvis set overlap functions use internally.
The S4 method ssvMakeMembTable() handles the necessary conversions and
can be extended
to accept a new class of data as long as it converts to an already accepted
data type and calls ssvMakeMembTable again.
For instance, ssvMakeMembTable(olaps) extracts the membership table from
the mcols of a single GRanges object.
head(ssvMakeMembTable(olaps))
Overlapping a list of more generic data
ssvMakeMembTable() can handle sets defined by a list of vectors.
These happen to be numeric but nearly anything that as.character() works on
should be acceptable.
my_set_list = list(1:3, 2:3, 3:6)
ssvMakeMembTable(my_set_list)
Supplying a named list is recommended
names(my_set_list) = c("first", "second", "third")
ssvMakeMembTable(my_set_list)
A list of character vectors works just as well.
my_set_list_char = lapply(my_set_list, function(x)letters[x])
ssvMakeMembTable(my_set_list_char)
Notice the rownames changed as the items changed.
Visualization of set overlaps, ssvFeature*
The ssvFeature* family of functions all accept a valid set definition and output
a ggplot.
Barplot
Barplots are useful for for conveying the size of each set/group.
ssvFeatureBars(olaps)
Pie chart
Pie charts are an alternative to barplots but are generally more difficult to
interpret.
ssvFeaturePie(olaps)
Venn diagram
Venn diagrams show the number of items falling into each overlap category.
At release, seqsetvis is limited to displaying 3 sets via venn diagram. This
will be increased in a future update.
ssvFeatureVenn(olaps)
Euler diagram
Often confused with Venn diagrams, Euler diagrams attempt to convey the size
of each set and the degree to which the overlap with circles or ellipses of
varying sizes. For data of any appreciable complexity, Euler diagrams are
approximations by neccessity.
There is no hard limit to the number of sets seqsetvis can show in an Euler
diagram but computation time increases rapidly along with the inaccuracy of
approximation.
ssvFeatureEuler(olaps)
Binary Heatmap
An alternative to Venn/Euler diagrams, this "heatmap" contains one column per
set and one row per item with item membership per set mapped to color.
In this example black indicates TRUE for membership hence the top 2/3 is black
for all three sets.
ssvFeatureBinaryHeatmap(olaps)
Visualization of genomic signal, ssvSignal*
The ssvSignal family of functions all take as input continuous signal data
mapped to the genome. This type of data is generally stored in bedGraph,
wiggle, or bigWig format. At this time, seqsetvis* directly supports the
bigwig format and creating pileups from indexed .bam files.
This is accomplished via the ssvFetchBigwig() and
ssvFetchBam() functions.
If you don't have .bigWig (or .bigwig or .bw) files you should be able to
easily generate some using r Biocpkg("rtracklayer") export.bw()
alongside import.bedGraph()
or import.wig() as appropriate.
Loading bigwig data
bigwig_files = c(
system.file("extdata", "MCF10A_CTCF_FE_random100.bw",
package = "seqsetvis"),
system.file("extdata", "MCF10AT1_CTCF_FE_random100.bw",
package = "seqsetvis"),
system.file("extdata", "MCF10CA1_CTCF_FE_random100.bw",
package = "seqsetvis")
)
names(bigwig_files) = sub("_FE_random100.bw", "", basename(bigwig_files))
# names(bigwig_files) = letters[1:3]
olap_gr = CTCF_in_10a_overlaps_gr
target_size = quantile(width(olap_gr), .75)
window_size = 50
target_size = round(target_size / window_size) * window_size
olap_gr = resize(olap_gr, target_size, fix = "center")
bw_gr = ssvFetchBigwig(bigwig_files, olap_gr, win_size = window_size)
bw_gr
olap_gr = CTCF_in_10a_overlaps_gr
bw_gr = CTCF_in_10a_profiles_gr
Overlap information must be transformed into a factor tied to id to be useful.
olap_groups = ssvFactorizeMembTable(mcols(olap_gr))
Line plots
Line plots are perhaps the most natural and common type of visualization for
this type of continuous genomic data. Many genome browsers and other BioC
packages support
this type of visualization. These other approaches are very good at show
lot's of different data of multiple types at single regions. In contrast,
seqsetvis is focused on bringing in data from multiple regions simultaneously.
Individual line plots
Basic line plot of a subset of regions
# facet labels will display better if split into multiple lines
bw_gr$facet_label = sub("_", "\n", bw_gr$sample)
ssvSignalLineplot(bw_data = subset(bw_gr, id %in% 1:12), facet_ = "facet_label")
Facetting by region id is less overwhelming
ssvSignalLineplot(bw_data = subset(bw_gr, id %in% 1:4), facet_ = "id")
Aggregated line plots
Aggregate regions by mean
ssvSignalLineplotAgg(bw_data = bw_gr)
Apply spline of 10 to make a prettier plot
ssvSignalLineplotAgg(bw_data = bw_gr, spline_n = 10)
By adding some info to bw_gr, tweaking the group_ parameter, and adding a
facet call we can apply the aggregation function by peak call set and plot them
separately.
# append set info, modify aggregation group_ and add facet
olap_2groups = ssvFactorizeMembTable(ssvMakeMembTable(olap_gr)[, 1:2])
grouped_gr = GRanges(merge(bw_gr, olap_2groups))
grouped_gr = subset(grouped_gr, sample %in% c("MCF10A_CTCF", "MCF10AT1_CTCF"))
ssvSignalLineplotAgg(bw_data = grouped_gr, spline_n = 10,
group_ = c("sample", "group")) +
facet_wrap("group", ncol = 2) +
labs(title = "Aggregated by peak call set", y = "FE", x = "bp from center")
Scatterplot
Scatterplots are a common way of viewing the relationship between two variables.
seqsetvis allows users to easily apply a summary function (mean by default) to
each region as measured in two samples and plot them on the x and y axis.
A basic scatterplot
ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF")
Using the result of olap_groups = ssvFactorizeMembTable(olap_gr) to add color
based on our peak calls.
ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF",
color_table = olap_groups)
More than a few colors gets confusing quickly. Let's limit the color groups to
the samples being plotted.
# by subsetting the matrix returned by ssvMakeMembTable() we have a lot of
# control over the coloring.
olap_2groups = ssvFactorizeMembTable(ssvMakeMembTable(olap_gr)[, 1:2])
ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF",
color_table = olap_2groups)
Coloring by the third sample's peak call might be interesting.
olap_OutGroups = ssvFactorizeMembTable(
ssvMakeMembTable(olap_gr)[, 3, drop = FALSE])
ssvSignalScatterplot(bw_gr,
x_name = "MCF10A_CTCF",
y_name = "MCF10AT1_CTCF",
color_table = olap_OutGroups)
We can also get clever with ggplot2's facetting.
#tweaking group description will clean up plot labels a lot
olap_groups$group = gsub("_CTCF", "", olap_groups$group)
olap_groups$group = gsub(" & ", "\n", olap_groups$group)
ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF",
color_table = olap_groups) +
facet_wrap("group") + guides(color = "none") +
theme_linedraw()
Banded quantiles
These quantile bands are a more sophisticated way to aggregate the same data
shown in the line plots.
ssvSignalBandedQuantiles(bw_gr, by_ = "sample", hsv_grayscale = TRUE,
hsv_symmetric = TRUE,
hsv_reverse = TRUE)
Heatmap
Heatmaps combined with clustering are a powerful way to find similarly marked
regions of the genome. Calling ssvSignalHeatmap will perform k-means clustering
automatically. k-means clusters sorted by similarity as determined by
hierarchical clustering.
ssvSignalHeatmap(bw_gr, nclust = 3, facet_ = "facet_label")
Performing clustering manually allows for use of cluster info.
bw_clust = ssvSignalClustering(bw_gr, nclust = 3)
bw_clust
Cluster selection:
subset(bw_clust, cluster_id == 3)
Plotting and pre-clustering (identical to just calling heatmap with same
parameters)
ssvSignalHeatmap(bw_clust, facet_ = "facet_label")
Use case : CTCF in breast cancer
This vignette runs using a small subset of datasets that are publicly available
via GEO. These data are fully described
in GSE98551 and
the associated paper:
Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer
In brief, the three cell lines MCF10A, MCF10A-at1, and MCF10A-CA1a are used as
a model of breast cancer progression from normal-like MCF10A to
metastatic MCF10A-CA1a. The ChIP-seq target CTCF is both a transcriptional
regulator and an insulator frequently associated with the boundaries of genomic
structural domains.
Therefore, regions where CTCF changes are interesting for follow-up to see if
they are also associated wtih regulatory or structural changes and perhaps
important to breast cancer progression.
To download the full dataset see: narrowPeak from GEO and
bigwig from GEO
We'll use r CRANpkg("cowplot") to arrange our plots in this section.
Setup
Set paths to bigwig files and narrowPeak files. There is some extra code here
to download the complete datasets using BiocFileCache IF the package
BiocFileCache is installed and cache_path already exists. If enabled, this
downloads ~3 GB of data.
pkgdata_path = system.file("extdata",
package = "seqsetvis")
cache_path = paste0(pkgdata_path, "/.cache")
# the next line is enough to initialize the cache
# BiocFileCache(cache = cache_path)
use_full_data = dir.exists(cache_path) & require(BiocFileCache)
use_full_data = FALSE
if(use_full_data){
library(BiocFileCache)
ssv_bfc = BiocFileCache(cache = cache_path)
bw_files = vapply(seq_along(CTCF_in_10a_bigWig_urls), function(i){
rname = paste(names(CTCF_in_10a_bigWig_urls)[i],
"bigwig",
sep = ",")
fpath = CTCF_in_10a_bigWig_urls[i]
#bfcrpath calls bfcadd() if necessary and returns file path
bfcrpath(ssv_bfc, rname = rname, fpath = fpath)
}, "char")
names(bw_files) = names(CTCF_in_10a_bigWig_urls)
np_files = vapply(seq_along(CTCF_in_10a_narrowPeak_urls), function(i){
rname = paste(names(CTCF_in_10a_narrowPeak_urls)[i],
"narrowPeak",
sep = ",")
fpath = CTCF_in_10a_narrowPeak_urls[i]
#bfcrpath calls bfcadd() if necessary and returns file path
bfcrpath(ssv_bfc, rname = rname, fpath = fpath)
}, "a")
names(np_files) = names(CTCF_in_10a_narrowPeak_urls)
}else{
bw_files = vapply(c("MCF10A_CTCF", "MCF10AT1_CTCF", "MCF10CA1_CTCF"),
function(x){
system.file("extdata", paste0(x, "_FE_random100.bw"),
package = "seqsetvis")
}, "char")
# set filepaths
np_files = c(
system.file("extdata", "MCF10A_CTCF_random100.narrowPeak",
package = "seqsetvis"),
system.file("extdata", "MCF10AT1_CTCF_random100.narrowPeak",
package = "seqsetvis"),
system.file("extdata", "MCF10CA1_CTCF_random100.narrowPeak",
package = "seqsetvis")
)
names(np_files) = sub("_random100.narrowPeak", "",
x = basename(np_files))
}
Loading narrowPeak files as GRanges
# load peak calls
np_grs = easyLoad_narrowPeak(np_files)
Perform overlapping
olaps = ssvOverlapIntervalSets(np_grs)
Peak set desciption
Here we use a barplot (A) to convey the total number of peaks in each
cell line. The Venn diagram (B) shows precisely how many peaks are in each
overlap set. The Euler diagram (C) lacks the numerical precision (who could
say how many peaks are only present in MCF10AT1?) but the basic trends are
more obvious (MCF10A has the most peaks and the majority of peaks are common to
all 3 cell lines).
p_bars = ssvFeatureBars(olaps, show_counts = FALSE) +
theme(legend.position = "left")
p_bars = p_bars + theme(axis.text.x = element_blank(),
axis.ticks.x = element_blank(),
legend.justification = "center") +
labs(fill = "cell line")
p_venn = ssvFeatureVenn(olaps, counts_txt_size = 4) +
guides(fill = "none", color = "none")
p_euler = ssvFeatureEuler(olaps) +
guides(fill = "none", color = "none")
cowplot::ggdraw() +
cowplot::draw_plot(p_bars, x = 0, y = 0, width = .4, height = .7) +
cowplot::draw_plot(p_venn, x = .4, y = .1, width = .3, height = .7) +
cowplot::draw_plot(p_euler, x = 0.7, y = .1, width = 0.3, height = .7) +
cowplot::draw_plot_label(c("CTCF binding in breast cancer cell lines",
"A", "B", "C"),
x = c(.04, .17, 0.4, .7),
y = c(.92, .75, .75, .75), size = 10, hjust = 0)
Plot colors can be modified via the ggplot objects without recalling plot
functions. (These colors aren't better per se.)
col_vals = c("MCF10A_CTCF" = 'red',
"MCF10AT1_CTCF" = "blue",
"MCF10CA1_CTCF" = "green")
sf = scale_fill_manual(values = col_vals)
sc = scale_color_manual(values = col_vals)
cowplot::ggdraw() +
cowplot::draw_plot(p_bars + sf,
x = 0, y = 0,
width = .4, height = .7) +
cowplot::draw_plot(p_venn + sf + sc,
x = .4, y = .1,
width = .3, height = .7) +
cowplot::draw_plot(p_euler + sf + sc,
x = 0.7, y = .1,
width = 0.3, height = .7) +
cowplot::draw_plot_label(c("CTCF binding in breast cancer cell lines",
"A", "B", "C"),
x = c(.04, .17, 0.4, .7),
y = c(.92, .75, .75, .75), size = 10, hjust = 0)
Annotation with ChIPpeakAnno
By using an annotation package like r Biocpkg("ChIPpeakAnno") we can
assign some biology to each peak.
library(ChIPpeakAnno)
data(TSS.human.GRCh38)
macs.anno <- annotatePeakInBatch(olaps, AnnotationData=TSS.human.GRCh38)
This gets us to easily selectable "gene lists" that everyone wants.
Apply a limit of 10 kb to reduce non-specific associations.
macs.anno = subset(macs.anno, distancetoFeature < 1000)
Gene ids associated with peaks present in MCF10A and MCF10AT1 but not
MCF10CA1.
subset(macs.anno, MCF10AT1_CTCF & MCF10A_CTCF & !MCF10CA1_CTCF)$feature
Gene ids associated with the peaks present in MCF10A and neither MCF10AT1 or
MCF10CA1.
subset(macs.anno, MCF10A_CTCF & !MCF10AT1_CTCF & !MCF10CA1_CTCF)$feature
With a meaningfully chosen set of substantial size we could then move on
to gene ontology or a gene set enrichment type of analysis.
... if we're confident that this peak call is picking up real biological
differences. Let's take a closer look at these peak calls in the next section.
Peak call validation
Digging into a new set of peak calls can be time-consuming and arbitrary. It's
easy to spend a lot of time with a genome browser checking genes of interest or
peaks at random and not come away with a clear idea how you well your peak call
is performing.
seqsetvis is ideal for systematically assessing just what qualifies as a peak
in each sample of an experiment.
Loading bigwig data
Before loading bigwigs, we need to decide on a uniform region size to retrieve.
In this case we'll use the 75th quantile of merged peak widths and round up to
the nearest multiple of window size. This is a bit arbitrary but a good fit in
practice. We'll fully capture 75% of peak regions
and not be influenced by outliers.
This is the same procedure ssvFetchBigwig() uses to automatically
set a fixed width.
window_size = 50
width_q75 = quantile(width(olaps), .75)
width_q75 = ceiling(width_q75 / window_size) * window_size
hist_res = hist(width(olaps))
lines(rep(width_q75, 2), c(0, max(hist_res$counts)), col = "red", lwd = 5)
text(width_q75, max(hist_res$counts), "fixedSize", adj = c(-.1, 1), col = "red")
## apply width
olaps_fixedSize = centerFixedSizeGRanges(olaps, width_q75)
Fetch profiles
Reading bigWig formatted files will not work at all on Windows OS.
If you are able to read bigwigs on your system,
there are few options.
Loading from the subsetted bigwigs inlcuded with seqsetvis.
This code chunk results in identical data to that stored in the
CTCF_in_10a_profiles_gr data object.
if(use_full_data){
bw_gr = ssvFetchBigwig(file_paths = bw_files,
qgr = olaps_fixedSize,
win_size = 50)
}
Inspecting peaks
A) This scatterplot makes it clear that the peaks that looked unique to MCF10A
have comparable fold-enrichment in MCF10AT1. This suggests that there may not
be a meaningful biological difference between peaks that are present in MCF10A
but absent in MCF10AT1.
B) Likewise for the comparison of MCF10A and MCF10CA1. In this case, there is a
systematic reduction in fold-enrichment in CA1 compared to 10A. While it's
possible there's some biology at play, it's far more likely that the
ChIP procedure for CA1 simply wasn't as successful.
# shortening colnames will make group names less cumbersome in plot legend
colnames(mcols(olaps_fixedSize)) = sub("_CTCF", "",
colnames(mcols(olaps_fixedSize)))
all_groups = levels(ssvFactorizeMembTable(
ssvMakeMembTable(olaps_fixedSize))$group)
all_colors = RColorBrewer::brewer.pal(length(all_groups), "Set1")
all_colors[5:7] = safeBrew(3, "Dark2")
names(all_colors) = all_groups
olap_groups_12 = ssvFactorizeMembTable(
ssvMakeMembTable(olaps_fixedSize)[, 1:2])
p_12 = ssvSignalScatterplot(bw_gr,
x_name = "MCF10A_CTCF",
y_name = "MCF10AT1_CTCF",
color_table = olap_groups_12) +
scale_color_manual(values = all_colors)
olap_groups_13 = ssvFactorizeMembTable(
ssvMakeMembTable(olaps_fixedSize)[, c(1,3)])
p_13 = ssvSignalScatterplot(bw_gr,
x_name = "MCF10A_CTCF",
y_name = "MCF10CA1_CTCF",
color_table = olap_groups_13) +
scale_color_manual(values = all_colors)
if(use_full_data){
tp_12 = p_12 + scale_size_continuous(range = .1) +
scale_alpha_continuous(range = .1) +
geom_density2d(aes(color = group), h = 40, bins = 3)
tp_13 = p_13 + scale_size_continuous(range = .1) +
scale_alpha_continuous(range = .1) +
geom_density2d(aes(color = group), h = 40, bins = 3)
cowplot::plot_grid(tp_12 + labs(title = ""),
tp_13 + labs(title = ""),
label_y = .85, labels = "AUTO")
}else{
cowplot::plot_grid(p_12 + labs(title = ""),
p_13 + labs(title = ""),
label_y = .85, labels = "AUTO")
}
These observations are reinforced by the heatmap.
bw_gr$facet_label = sub("_", "\n", bw_gr$sample)
clust_gr = ssvSignalClustering(bw_gr, nclust = 3, facet_ = "facet_label")
ssvSignalHeatmap(clust_gr, facet_ = "facet_label") + labs(fill = "FE",
y = "region",
x = "bp from center")
The heatmap can be improved by using centerAtMax() to allow each region to
be shifted such that the maximum signal is at the center. A couple parameter
notes:
+ view_size controls how far each region can be shifted.
Shifting will cause overhangs which are trimmed by default so the resulting
regions are smaller. You may need to extened the origial query GRanges
set and repeat the fetch if the resulting regions are too small.
+ The by_ parameter is particularly tricky. Its default value of "id"
is probably what we want. This causes each region to be individually shifted
according to the maximum across samples. One alternative,
by_ = c("id", "sample") would have shifted each region in each sample.
Another alternative, by_ = "" or by_ = NULL would shift every region in
every sample identically.
center_gr = centerAtMax(clust_gr, view_size = 150,
by_ = "id", check_by_dupes = FALSE)
p_center_hmap = ssvSignalHeatmap(center_gr, facet_ = "facet_label") +
labs(fill = "FE",
y = "region",
x = "bp from center")
## since center_gr still retains clustering information, clustering is not
## repeated by default, the following reclusters the data.
clust_center_gr = ssvSignalClustering(center_gr, nclust = 3)
p_center_hmap_reclust = ssvSignalHeatmap(clust_center_gr,
facet_ = "facet_label") +
labs(fill = "FE",
y = "region",
x = "bp from center")
cowplot::plot_grid(p_center_hmap + labs(title = "original clustering"),
p_center_hmap_reclust + labs(title = "reclustered"))
Cluster 1 and 2 seem to have the most robust enrichment. Let's extract them
and annotate.
clust_df = as.data.frame(mcols(clust_gr))
clust_df = unique(clust_df[,c("id", "cluster_id")])
olap_clust_annot = olap_gr
mcols(olap_clust_annot) = data.frame(id = seq_along(olap_clust_annot))
olap_clust_annot = GRanges(merge(olap_clust_annot, clust_df))
olap_clust_annot = subset(olap_clust_annot, cluster_id %in% 1:2)
olap_clust_annot <- annotatePeakInBatch(olap_clust_annot,
AnnotationData=TSS.human.GRCh38)
olap_clust_annot$feature
Use case: ChromHMM inspection
In this demonstration we'll use the same CTCF dataset in breast cancer but
bring in a chromHMM state file from an other dataset. This is meant to
highlight the flexibility of seqsetvis.
Setup
If the previous CTCF in breast cancer Use Case made use of BiocFileCache,
we'll continue caching external data sources. If not, we'll skip to a
pre-cooked version of the same data.
The pre-cooked version is quite a narrow slice and there's a significant
amount of data manipulation
target_data = "MCF10A_CTCF"
chmm_win = 200 #window size is an important chromHMM parameter.
# 200 is the default window size and matches the state segementation
if(use_full_data){
# set all file paths
chmm_bw_file = bfcrpath(ssv_bfc, rnames = paste(target_data, "bigwig",
sep = ","))
chmm_np_file = bfcrpath(ssv_bfc, rnames = paste(target_data, "narrowPeak",
sep = ","))
chmm_seg_file = bfcrpath(ssv_bfc, rnames = "MCF7,segmentation",
fpath = chromHMM_demo_segmentation_url)
query_chain = bfcquery(ssv_bfc, "hg19ToHg38,chain")
if(nrow(query_chain) == 0){
chain_hg19ToHg38_gz = bfcrpath(ssv_bfc, rnames = "hg19ToHg38,gz",
fpath = chromHMM_demo_chain_url)
ch_raw = readLines(gzfile(chain_hg19ToHg38_gz))
ch_file = bfcnew(ssv_bfc, rname = "hg19ToHg38,chain")
writeLines(ch_raw, con = ch_file)
}
chmm_chain_file = bfcrpath(ssv_bfc, rnames = "hg19ToHg38,chain")
ch = rtracklayer::import.chain(chmm_chain_file)
# load segmentation data
chmm_gr = rtracklayer::import.bed(chmm_seg_file)
#cleanup state names.
chmm_gr$name = gsub("\\+", " and ", chmm_gr$name)
chmm_gr$name = gsub("_", " ", chmm_gr$name)
#setup color to state mapping
colDF = unique(mcols(chmm_gr)[c("name", "itemRgb")])
state_colors = colDF$itemRgb
names(state_colors) = colDF$name
#liftover states from hg19 to hg38
ch = rtracklayer::import.chain(chmm_chain_file)
chmm_gr_hg38 = rtracklayer::liftOver(chmm_gr, ch)
chmm_gr_hg38 = unlist(chmm_gr_hg38)
chmm_grs_list = as.list(GenomicRanges::split(chmm_gr_hg38,
chmm_gr_hg38$name))
#transform narrowPeak ranges to summit positions
chmm_np_grs = easyLoad_narrowPeak(chmm_np_file, file_names = target_data)
chmm_summit_grs = lapply(chmm_np_grs, function(x){
start(x) = start(x) + x$relSummit
end(x) = start(x)
x
})
qlist = append(chmm_summit_grs[1], chmm_grs_list)
chmm_olaps = ssvOverlapIntervalSets(qlist, use_first = TRUE)
#discard the columns for peak call and no_hit, not informative here.
mcols(chmm_olaps)[[1]] = NULL
chmm_olaps$no_hit = NULL
#total width of genome assigned each state
state_total_widths = sapply(chmm_grs_list, function(my_gr){
sum(as.numeric(width(my_gr)))
})
#Expand state regions into 200 bp windows.
state_wingrs = lapply(chmm_grs_list, function(my_gr){
st = my_gr$name[1]
wgr = unlist(slidingWindows(my_gr, chmm_win, chmm_win))
wgr$state = st
wgr
})
state_wingrs = unlist(GRangesList(state_wingrs))
# fetch bw data for each state
# it probably isn't useful to grab every single window for each state
# so we can cap the number of each state carried forward
max_per_state = 5000
# flank size zooms out a bit from each chromHMM window
flank_size = 400
state_split = split(state_wingrs, state_wingrs$state)
state_split = lapply(state_split, function(my_gr){
samp_gr = sample(my_gr, min(length(my_gr), max_per_state))
samp_gr = sort(samp_gr)
names(samp_gr) = seq_along(samp_gr)
samp_gr
})
state_gr = unlist(GRangesList(state_split))
state_gr = resize(state_gr, width = chmm_win + 2 * flank_size,
fix = "center")
bw_states_gr = ssvFetchBigwig(file_paths = chmm_bw_file,
qgr = state_gr,
win_size = 50)
bw_states_gr$grp = sub("\\..+", "", bw_states_gr$id)
bw_states_gr$grp_id = sub(".+\\.", "", bw_states_gr$id)
}else{
max_per_state = 20
flank_size = 400
state_colors = chromHMM_demo_state_colors
bw_states_gr = chromHMM_demo_bw_states_gr
chmm_olaps = chromHMM_demo_overlaps_gr
state_total_widths = chromHMM_demo_state_total_widths
}
Peak overlaps with states, counts
Create barplot of raw count of peaks overlapping states.
olaps_df = as.data.frame(mcols(chmm_olaps))
colnames(olaps_df) = gsub("\\.", " ", colnames(olaps_df))
p_state_raw_count = ssvFeatureBars(olaps_df, show_counts = FALSE) +
labs(fill = "state", x = "") +
scale_fill_manual(values = state_colors) +
theme_cowplot() + guides(fill = "none") +
theme(axis.text.x = element_text(angle = 90, size = 8,
hjust = 1, vjust = .5))
p_state_raw_count
Peak overlaps with states, enrichment
Create barplot of enrichment of peaks overlapping states.
state_width_fraction = state_total_widths / sum(state_total_widths)
state_peak_hits = colSums(olaps_df)
state_peak_fraction = state_peak_hits / sum(state_peak_hits)
enrichment = state_peak_fraction /
state_width_fraction[names(state_peak_fraction)]
enrich_df = data.frame(state = names(enrichment), enrichment = enrichment)
p_state_enrichment = ggplot(enrich_df) +
geom_bar(aes(x = state, fill = state, y = enrichment), stat = "identity") +
labs(x = "") +
theme_cowplot() + guides(fill = "none") +
scale_fill_manual(values = state_colors) +
theme(axis.text.x = element_text(angle = 90, size = 8,
hjust = 1, vjust = .5))
p_state_enrichment
Aggregated line plots by state
Plot aggregated line plot by state
p_agg_tracks = ssvSignalLineplotAgg(bw_states_gr,
sample_ = "grp",
color_ = "grp")
gb = ggplot2::ggplot_build(p_agg_tracks)
yrng = range(gb$data[[1]]$y)
p_agg_tracks = p_agg_tracks +
scale_color_manual(values = state_colors) +
annotate("line", x = rep(-chmm_win/2, 2), y = yrng) +
annotate("line", x = rep(chmm_win/2, 2), y = yrng) +
labs(y = "FE", x = "bp", color = "state",
title = paste("Average FE by state,", target_data),
subtitle = paste("states sampled to",
max_per_state,
chmm_win,
"bp windows each\n",
flank_size,
"bp flanking each side")) +
theme(plot.title = element_text(hjust = 0))
p_agg_tracks
States heatmap
A heatmap using the same data from the aggregated line plot. Facetting is
done on state and regions are sorted in decreasing order of signal at center.
pdt = as.data.table(mcols(bw_states_gr))
pdt$grp_id = as.integer(pdt$grp_id)
# reassign grp_id to sort within each state set
dt_list = lapply(unique(pdt$grp), function(state){
dt = pdt[grp == state]
dtmax = dt[, .(ymax = y[which(x == x[order(abs(x))][1])]), by = grp_id]
dtmax = dtmax[order(ymax, decreasing = TRUE)]
dtmax[, grp_o := seq_len(.N)]
dtmax$ymax = NULL
dt = merge(dtmax, dt)
dt[, grp_id := grp_o]
dt$grp_o = NULL
dt
})
# reassemble
pdt = rbindlist(dt_list)
# heatmap facetted by state and sorted in decreasing order
p_state_hmap = ggplot(pdt) +
geom_raster(aes(x = x, y = grp_id, fill = y)) +
scale_y_reverse() +
facet_wrap("grp", nrow = 2) +
theme(axis.text.y = element_blank(),
axis.ticks.y = element_blank(),
axis.line.y = element_blank(),
axis.text.x = element_text(angle = 90, hjust = 1, vjust = .5),
strip.text = element_text(size= 8)) +
scale_fill_gradientn(colors = c("white", "orange", "red")) +
labs(y = "", fill = "FE",
x = "bp from local summit",
title = paste(max_per_state, "random regions per state"))
p_state_hmap
Figure assembly
Tie it all together into one figure with cowplot.
bar_height = .25
line_height = .3
ggdraw() +
draw_plot(p_state_raw_count + guides(fill = "none"),
x = 0,
y = 1 - bar_height,
width = .5,
height = bar_height) +
draw_plot(p_state_enrichment + guides(fill = "none"),
x = .5,
y = 1 - bar_height,
width = .5,
height = bar_height) +
draw_plot(p_agg_tracks,
x = 0,
y = 1 - bar_height - line_height,
width = 1,
height = line_height) +
draw_plot(p_state_hmap,
x = 0,
y = 0,
width = 1,
height = 1 - bar_height - line_height) +
draw_plot_label(LETTERS[1:4],
c(0, 0.48, 0, 0),
c(1, 1, 1 - bar_height, 1 - bar_height - line_height),
size = 15)
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knitr::opts_chunk$set( collapse = TRUE, dpi=60 )
Synopsis
seq - set - vis
r Githubpkg("jrboyd/seqsetvis") enables the visualization and analysis of
multiple genomic datasets. Although seqsetvis was designed for the comparison
of mulitple ChIP-seq datasets, this package is domain-agnostic and allows the
processing of multiple genomic coordinate files (bed-like files) and signal
files (bigwig files).
seqsetvis is ideal for the following tasks:
- Feature assessment and comparison.
- Differential enrichment or expression results assessment.
- Replicate comparison and reproducibility analysis.
- Chromatin state inspection.
- Clustering of histone modification and/or transcription factor enrichment at features such as promoters or enhancers.
seqsetvis accepts and outputs the Bioconductor standard GRanges object while leveraging the power and speed of the data.table package (an enhancement of data.frame) with the flexibility of ggplot2 to provide users with visualizations that are simultaneously publication ready while still being highly customizable. By default, data.table is used entirely internally so users unfamiliar with its use can safely ignore it. That said, fans of data.table can use seqsetvis in a data.table exclusive mode if they wish.
Features
- Mostly it "just works" with minimal assumed knowledge of R or other Bioconductor packages.
- All plots produced are
r CRANpkg("ggplot2")objects.- easily customized according to user preferences by appending ggplot2 functions.
- powerful plot arrangement with with
r CRANpkg("cowplot"),r CRANpkg("gridExtra"), etc.
- Although various plots are implemented, the underlying data are simple in
structure and easily accessible so you can do what you want with them.
- membership table for feature overlaps is just a logical matrix.
- signal data is stored in a tidy formatted
r CRANpkg("data.table"), ideal forr CRANpkg("ggplot2")visualization.
Although initially designed to focus on peak call sets, seqsetvis accepts any data in GRanges format and is therefore flexible enough to work dowstream of many genomics tools and packages. Indeed, set comparison visualizations are genomics agnostic and also accept generic data types defining sets.
Functions
ssv*- most primary user facing functions are prefixed by ssvssvOverlapIntervalSets- derives sets from a list of GRangesssvMakeMembTable- coerces various ways of defining sets to a membership tablessvFactorizeMembTable- converts a multi column membership table into a single factor.ssvFetch*- retrieves properly formatted data from .bigwig and .bam filesssvFeature*- visualizations of set counts and overlapsssvSignal*- visualizations of signal mapped to genomic coordinates in sets context
- utility functions
easyLoad_*- loads files into a list of GRanges for input intossv*functions- miscellaneous functions such as wrappers for data.table functions.
Installation and Loading
From Bioconductor
## try http:// if https:// URLs are not supported if (!requireNamespace("BiocManager", quietly=TRUE)) install.packages("BiocManager") BiocManager::install("seqsetvis")
Load the library
library(seqsetvis)
Load optional useful libraries
library(GenomicRanges) library(data.table) library(cowplot) theme_set(cowplot::theme_cowplot())
Defining sets
seqsetvis has several ways of accepting set information. Since seqsetvis was originally intended to focus on comparing sets of peak calls, a list of GRanges or a GRangesList is directly supported but there are more generic alternatives.
Overlapping a list of GRanges
Many functions in seqsetvis require some kind of sets definition
as input. Sets can be defined in one of several ways.
ssvOverlapIntervalSets() creates a single GRanges from a list of GRanges
with a logical membership table as metadata columns defining sets.
olaps = ssvOverlapIntervalSets(CTCF_in_10a_narrowPeak_grs) head(olaps)
ssvOverlapIntervalSets() also accepts a GRangesList if that's more natural.
olaps_fromGRangesList = ssvOverlapIntervalSets( GenomicRanges::GRangesList(CTCF_in_10a_narrowPeak_grs))
The membership data contained in the metadata columns of olaps is
the data structure seqsetvis set overlap functions use internally.
The S4 method ssvMakeMembTable() handles the necessary conversions and
can be extended
to accept a new class of data as long as it converts to an already accepted
data type and calls ssvMakeMembTable again.
For instance, ssvMakeMembTable(olaps) extracts the membership table from
the mcols of a single GRanges object.
head(ssvMakeMembTable(olaps))
Overlapping a list of more generic data
ssvMakeMembTable() can handle sets defined by a list of vectors.
These happen to be numeric but nearly anything that as.character() works on
should be acceptable.
my_set_list = list(1:3, 2:3, 3:6) ssvMakeMembTable(my_set_list)
Supplying a named list is recommended
names(my_set_list) = c("first", "second", "third") ssvMakeMembTable(my_set_list)
A list of character vectors works just as well.
my_set_list_char = lapply(my_set_list, function(x)letters[x]) ssvMakeMembTable(my_set_list_char)
Notice the rownames changed as the items changed.
Visualization of set overlaps, ssvFeature*
The ssvFeature* family of functions all accept a valid set definition and output a ggplot.
Barplot
Barplots are useful for for conveying the size of each set/group.
ssvFeatureBars(olaps)
Pie chart
Pie charts are an alternative to barplots but are generally more difficult to interpret.
ssvFeaturePie(olaps)
Venn diagram
Venn diagrams show the number of items falling into each overlap category. At release, seqsetvis is limited to displaying 3 sets via venn diagram. This will be increased in a future update.
ssvFeatureVenn(olaps)
Euler diagram
Often confused with Venn diagrams, Euler diagrams attempt to convey the size of each set and the degree to which the overlap with circles or ellipses of varying sizes. For data of any appreciable complexity, Euler diagrams are approximations by neccessity.
There is no hard limit to the number of sets seqsetvis can show in an Euler diagram but computation time increases rapidly along with the inaccuracy of approximation.
ssvFeatureEuler(olaps)
Binary Heatmap
An alternative to Venn/Euler diagrams, this "heatmap" contains one column per set and one row per item with item membership per set mapped to color. In this example black indicates TRUE for membership hence the top 2/3 is black for all three sets.
ssvFeatureBinaryHeatmap(olaps)
Visualization of genomic signal, ssvSignal*
The ssvSignal family of functions all take as input continuous signal data
mapped to the genome. This type of data is generally stored in bedGraph,
wiggle, or bigWig format. At this time, seqsetvis* directly supports the
bigwig format and creating pileups from indexed .bam files.
This is accomplished via the ssvFetchBigwig() and
ssvFetchBam() functions.
If you don't have .bigWig (or .bigwig or .bw) files you should be able to
easily generate some using r Biocpkg("rtracklayer") export.bw()
alongside import.bedGraph()
or import.wig() as appropriate.
Loading bigwig data
bigwig_files = c( system.file("extdata", "MCF10A_CTCF_FE_random100.bw", package = "seqsetvis"), system.file("extdata", "MCF10AT1_CTCF_FE_random100.bw", package = "seqsetvis"), system.file("extdata", "MCF10CA1_CTCF_FE_random100.bw", package = "seqsetvis") ) names(bigwig_files) = sub("_FE_random100.bw", "", basename(bigwig_files)) # names(bigwig_files) = letters[1:3] olap_gr = CTCF_in_10a_overlaps_gr target_size = quantile(width(olap_gr), .75) window_size = 50 target_size = round(target_size / window_size) * window_size olap_gr = resize(olap_gr, target_size, fix = "center") bw_gr = ssvFetchBigwig(bigwig_files, olap_gr, win_size = window_size) bw_gr
olap_gr = CTCF_in_10a_overlaps_gr bw_gr = CTCF_in_10a_profiles_gr
Overlap information must be transformed into a factor tied to id to be useful.
olap_groups = ssvFactorizeMembTable(mcols(olap_gr))
Line plots
Line plots are perhaps the most natural and common type of visualization for this type of continuous genomic data. Many genome browsers and other BioC packages support this type of visualization. These other approaches are very good at show lot's of different data of multiple types at single regions. In contrast, seqsetvis is focused on bringing in data from multiple regions simultaneously.
Individual line plots
Basic line plot of a subset of regions
# facet labels will display better if split into multiple lines bw_gr$facet_label = sub("_", "\n", bw_gr$sample) ssvSignalLineplot(bw_data = subset(bw_gr, id %in% 1:12), facet_ = "facet_label")
Facetting by region id is less overwhelming
ssvSignalLineplot(bw_data = subset(bw_gr, id %in% 1:4), facet_ = "id")
Aggregated line plots
Aggregate regions by mean
ssvSignalLineplotAgg(bw_data = bw_gr)
Apply spline of 10 to make a prettier plot
ssvSignalLineplotAgg(bw_data = bw_gr, spline_n = 10)
By adding some info to bw_gr, tweaking the group_ parameter, and adding a
facet call we can apply the aggregation function by peak call set and plot them
separately.
# append set info, modify aggregation group_ and add facet olap_2groups = ssvFactorizeMembTable(ssvMakeMembTable(olap_gr)[, 1:2]) grouped_gr = GRanges(merge(bw_gr, olap_2groups)) grouped_gr = subset(grouped_gr, sample %in% c("MCF10A_CTCF", "MCF10AT1_CTCF")) ssvSignalLineplotAgg(bw_data = grouped_gr, spline_n = 10, group_ = c("sample", "group")) + facet_wrap("group", ncol = 2) + labs(title = "Aggregated by peak call set", y = "FE", x = "bp from center")
Scatterplot
Scatterplots are a common way of viewing the relationship between two variables. seqsetvis allows users to easily apply a summary function (mean by default) to each region as measured in two samples and plot them on the x and y axis.
A basic scatterplot
ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF")
Using the result of olap_groups = ssvFactorizeMembTable(olap_gr) to add color
based on our peak calls.
ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF", color_table = olap_groups)
More than a few colors gets confusing quickly. Let's limit the color groups to the samples being plotted.
# by subsetting the matrix returned by ssvMakeMembTable() we have a lot of # control over the coloring. olap_2groups = ssvFactorizeMembTable(ssvMakeMembTable(olap_gr)[, 1:2]) ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF", color_table = olap_2groups)
Coloring by the third sample's peak call might be interesting.
olap_OutGroups = ssvFactorizeMembTable( ssvMakeMembTable(olap_gr)[, 3, drop = FALSE]) ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF", color_table = olap_OutGroups)
We can also get clever with ggplot2's facetting.
#tweaking group description will clean up plot labels a lot olap_groups$group = gsub("_CTCF", "", olap_groups$group) olap_groups$group = gsub(" & ", "\n", olap_groups$group) ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF", color_table = olap_groups) + facet_wrap("group") + guides(color = "none") + theme_linedraw()
Banded quantiles
These quantile bands are a more sophisticated way to aggregate the same data shown in the line plots.
ssvSignalBandedQuantiles(bw_gr, by_ = "sample", hsv_grayscale = TRUE, hsv_symmetric = TRUE, hsv_reverse = TRUE)
Heatmap
Heatmaps combined with clustering are a powerful way to find similarly marked regions of the genome. Calling ssvSignalHeatmap will perform k-means clustering automatically. k-means clusters sorted by similarity as determined by hierarchical clustering.
ssvSignalHeatmap(bw_gr, nclust = 3, facet_ = "facet_label")
Performing clustering manually allows for use of cluster info.
bw_clust = ssvSignalClustering(bw_gr, nclust = 3) bw_clust
Cluster selection:
subset(bw_clust, cluster_id == 3)
Plotting and pre-clustering (identical to just calling heatmap with same parameters)
ssvSignalHeatmap(bw_clust, facet_ = "facet_label")
Use case : CTCF in breast cancer
This vignette runs using a small subset of datasets that are publicly available via GEO. These data are fully described in GSE98551 and the associated paper: Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer
In brief, the three cell lines MCF10A, MCF10A-at1, and MCF10A-CA1a are used as
a model of breast cancer progression from normal-like MCF10A to
metastatic MCF10A-CA1a. The ChIP-seq target CTCF is both a transcriptional
regulator and an insulator frequently associated with the boundaries of genomic
structural domains.
Therefore, regions where CTCF changes are interesting for follow-up to see if
they are also associated wtih regulatory or structural changes and perhaps
important to breast cancer progression.
To download the full dataset see: narrowPeak from GEO and bigwig from GEO
We'll use r CRANpkg("cowplot") to arrange our plots in this section.
Setup
Set paths to bigwig files and narrowPeak files. There is some extra code here
to download the complete datasets using BiocFileCache IF the package
BiocFileCache is installed and cache_path already exists. If enabled, this
downloads ~3 GB of data.
pkgdata_path = system.file("extdata", package = "seqsetvis") cache_path = paste0(pkgdata_path, "/.cache") # the next line is enough to initialize the cache # BiocFileCache(cache = cache_path) use_full_data = dir.exists(cache_path) & require(BiocFileCache) use_full_data = FALSE if(use_full_data){ library(BiocFileCache) ssv_bfc = BiocFileCache(cache = cache_path) bw_files = vapply(seq_along(CTCF_in_10a_bigWig_urls), function(i){ rname = paste(names(CTCF_in_10a_bigWig_urls)[i], "bigwig", sep = ",") fpath = CTCF_in_10a_bigWig_urls[i] #bfcrpath calls bfcadd() if necessary and returns file path bfcrpath(ssv_bfc, rname = rname, fpath = fpath) }, "char") names(bw_files) = names(CTCF_in_10a_bigWig_urls) np_files = vapply(seq_along(CTCF_in_10a_narrowPeak_urls), function(i){ rname = paste(names(CTCF_in_10a_narrowPeak_urls)[i], "narrowPeak", sep = ",") fpath = CTCF_in_10a_narrowPeak_urls[i] #bfcrpath calls bfcadd() if necessary and returns file path bfcrpath(ssv_bfc, rname = rname, fpath = fpath) }, "a") names(np_files) = names(CTCF_in_10a_narrowPeak_urls) }else{ bw_files = vapply(c("MCF10A_CTCF", "MCF10AT1_CTCF", "MCF10CA1_CTCF"), function(x){ system.file("extdata", paste0(x, "_FE_random100.bw"), package = "seqsetvis") }, "char") # set filepaths np_files = c( system.file("extdata", "MCF10A_CTCF_random100.narrowPeak", package = "seqsetvis"), system.file("extdata", "MCF10AT1_CTCF_random100.narrowPeak", package = "seqsetvis"), system.file("extdata", "MCF10CA1_CTCF_random100.narrowPeak", package = "seqsetvis") ) names(np_files) = sub("_random100.narrowPeak", "", x = basename(np_files)) }
Loading narrowPeak files as GRanges
# load peak calls np_grs = easyLoad_narrowPeak(np_files)
Perform overlapping
olaps = ssvOverlapIntervalSets(np_grs)
Peak set desciption
Here we use a barplot (A) to convey the total number of peaks in each cell line. The Venn diagram (B) shows precisely how many peaks are in each overlap set. The Euler diagram (C) lacks the numerical precision (who could say how many peaks are only present in MCF10AT1?) but the basic trends are more obvious (MCF10A has the most peaks and the majority of peaks are common to all 3 cell lines).
p_bars = ssvFeatureBars(olaps, show_counts = FALSE) + theme(legend.position = "left") p_bars = p_bars + theme(axis.text.x = element_blank(), axis.ticks.x = element_blank(), legend.justification = "center") + labs(fill = "cell line") p_venn = ssvFeatureVenn(olaps, counts_txt_size = 4) + guides(fill = "none", color = "none") p_euler = ssvFeatureEuler(olaps) + guides(fill = "none", color = "none") cowplot::ggdraw() + cowplot::draw_plot(p_bars, x = 0, y = 0, width = .4, height = .7) + cowplot::draw_plot(p_venn, x = .4, y = .1, width = .3, height = .7) + cowplot::draw_plot(p_euler, x = 0.7, y = .1, width = 0.3, height = .7) + cowplot::draw_plot_label(c("CTCF binding in breast cancer cell lines", "A", "B", "C"), x = c(.04, .17, 0.4, .7), y = c(.92, .75, .75, .75), size = 10, hjust = 0)
Plot colors can be modified via the ggplot objects without recalling plot functions. (These colors aren't better per se.)
col_vals = c("MCF10A_CTCF" = 'red', "MCF10AT1_CTCF" = "blue", "MCF10CA1_CTCF" = "green") sf = scale_fill_manual(values = col_vals) sc = scale_color_manual(values = col_vals) cowplot::ggdraw() + cowplot::draw_plot(p_bars + sf, x = 0, y = 0, width = .4, height = .7) + cowplot::draw_plot(p_venn + sf + sc, x = .4, y = .1, width = .3, height = .7) + cowplot::draw_plot(p_euler + sf + sc, x = 0.7, y = .1, width = 0.3, height = .7) + cowplot::draw_plot_label(c("CTCF binding in breast cancer cell lines", "A", "B", "C"), x = c(.04, .17, 0.4, .7), y = c(.92, .75, .75, .75), size = 10, hjust = 0)
Annotation with ChIPpeakAnno
By using an annotation package like r Biocpkg("ChIPpeakAnno") we can
assign some biology to each peak.
library(ChIPpeakAnno) data(TSS.human.GRCh38) macs.anno <- annotatePeakInBatch(olaps, AnnotationData=TSS.human.GRCh38)
This gets us to easily selectable "gene lists" that everyone wants.
Apply a limit of 10 kb to reduce non-specific associations.
macs.anno = subset(macs.anno, distancetoFeature < 1000)
Gene ids associated with peaks present in MCF10A and MCF10AT1 but not MCF10CA1.
subset(macs.anno, MCF10AT1_CTCF & MCF10A_CTCF & !MCF10CA1_CTCF)$feature
Gene ids associated with the peaks present in MCF10A and neither MCF10AT1 or MCF10CA1.
subset(macs.anno, MCF10A_CTCF & !MCF10AT1_CTCF & !MCF10CA1_CTCF)$feature
With a meaningfully chosen set of substantial size we could then move on to gene ontology or a gene set enrichment type of analysis.
... if we're confident that this peak call is picking up real biological differences. Let's take a closer look at these peak calls in the next section.
Peak call validation
Digging into a new set of peak calls can be time-consuming and arbitrary. It's easy to spend a lot of time with a genome browser checking genes of interest or peaks at random and not come away with a clear idea how you well your peak call is performing.
seqsetvis is ideal for systematically assessing just what qualifies as a peak in each sample of an experiment.
Loading bigwig data
Before loading bigwigs, we need to decide on a uniform region size to retrieve. In this case we'll use the 75th quantile of merged peak widths and round up to the nearest multiple of window size. This is a bit arbitrary but a good fit in practice. We'll fully capture 75% of peak regions and not be influenced by outliers.
This is the same procedure ssvFetchBigwig() uses to automatically
set a fixed width.
window_size = 50 width_q75 = quantile(width(olaps), .75) width_q75 = ceiling(width_q75 / window_size) * window_size hist_res = hist(width(olaps)) lines(rep(width_q75, 2), c(0, max(hist_res$counts)), col = "red", lwd = 5) text(width_q75, max(hist_res$counts), "fixedSize", adj = c(-.1, 1), col = "red") ## apply width olaps_fixedSize = centerFixedSizeGRanges(olaps, width_q75)
Fetch profiles
Reading bigWig formatted files will not work at all on Windows OS. If you are able to read bigwigs on your system, there are few options.
Loading from the subsetted bigwigs inlcuded with seqsetvis.
This code chunk results in identical data to that stored in the
CTCF_in_10a_profiles_gr data object.
if(use_full_data){ bw_gr = ssvFetchBigwig(file_paths = bw_files, qgr = olaps_fixedSize, win_size = 50) }
Inspecting peaks
A) This scatterplot makes it clear that the peaks that looked unique to MCF10A have comparable fold-enrichment in MCF10AT1. This suggests that there may not be a meaningful biological difference between peaks that are present in MCF10A but absent in MCF10AT1.
B) Likewise for the comparison of MCF10A and MCF10CA1. In this case, there is a systematic reduction in fold-enrichment in CA1 compared to 10A. While it's possible there's some biology at play, it's far more likely that the ChIP procedure for CA1 simply wasn't as successful.
# shortening colnames will make group names less cumbersome in plot legend colnames(mcols(olaps_fixedSize)) = sub("_CTCF", "", colnames(mcols(olaps_fixedSize))) all_groups = levels(ssvFactorizeMembTable( ssvMakeMembTable(olaps_fixedSize))$group) all_colors = RColorBrewer::brewer.pal(length(all_groups), "Set1") all_colors[5:7] = safeBrew(3, "Dark2") names(all_colors) = all_groups olap_groups_12 = ssvFactorizeMembTable( ssvMakeMembTable(olaps_fixedSize)[, 1:2]) p_12 = ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10AT1_CTCF", color_table = olap_groups_12) + scale_color_manual(values = all_colors) olap_groups_13 = ssvFactorizeMembTable( ssvMakeMembTable(olaps_fixedSize)[, c(1,3)]) p_13 = ssvSignalScatterplot(bw_gr, x_name = "MCF10A_CTCF", y_name = "MCF10CA1_CTCF", color_table = olap_groups_13) + scale_color_manual(values = all_colors) if(use_full_data){ tp_12 = p_12 + scale_size_continuous(range = .1) + scale_alpha_continuous(range = .1) + geom_density2d(aes(color = group), h = 40, bins = 3) tp_13 = p_13 + scale_size_continuous(range = .1) + scale_alpha_continuous(range = .1) + geom_density2d(aes(color = group), h = 40, bins = 3) cowplot::plot_grid(tp_12 + labs(title = ""), tp_13 + labs(title = ""), label_y = .85, labels = "AUTO") }else{ cowplot::plot_grid(p_12 + labs(title = ""), p_13 + labs(title = ""), label_y = .85, labels = "AUTO") }
These observations are reinforced by the heatmap.
bw_gr$facet_label = sub("_", "\n", bw_gr$sample) clust_gr = ssvSignalClustering(bw_gr, nclust = 3, facet_ = "facet_label") ssvSignalHeatmap(clust_gr, facet_ = "facet_label") + labs(fill = "FE", y = "region", x = "bp from center")
The heatmap can be improved by using centerAtMax() to allow each region to
be shifted such that the maximum signal is at the center. A couple parameter
notes:
+ view_size controls how far each region can be shifted.
Shifting will cause overhangs which are trimmed by default so the resulting
regions are smaller. You may need to extened the origial query GRanges
set and repeat the fetch if the resulting regions are too small.
+ The by_ parameter is particularly tricky. Its default value of "id"
is probably what we want. This causes each region to be individually shifted
according to the maximum across samples. One alternative,
by_ = c("id", "sample") would have shifted each region in each sample.
Another alternative, by_ = "" or by_ = NULL would shift every region in
every sample identically.
center_gr = centerAtMax(clust_gr, view_size = 150, by_ = "id", check_by_dupes = FALSE) p_center_hmap = ssvSignalHeatmap(center_gr, facet_ = "facet_label") + labs(fill = "FE", y = "region", x = "bp from center") ## since center_gr still retains clustering information, clustering is not ## repeated by default, the following reclusters the data. clust_center_gr = ssvSignalClustering(center_gr, nclust = 3) p_center_hmap_reclust = ssvSignalHeatmap(clust_center_gr, facet_ = "facet_label") + labs(fill = "FE", y = "region", x = "bp from center") cowplot::plot_grid(p_center_hmap + labs(title = "original clustering"), p_center_hmap_reclust + labs(title = "reclustered"))
Cluster 1 and 2 seem to have the most robust enrichment. Let's extract them and annotate.
clust_df = as.data.frame(mcols(clust_gr)) clust_df = unique(clust_df[,c("id", "cluster_id")]) olap_clust_annot = olap_gr mcols(olap_clust_annot) = data.frame(id = seq_along(olap_clust_annot)) olap_clust_annot = GRanges(merge(olap_clust_annot, clust_df)) olap_clust_annot = subset(olap_clust_annot, cluster_id %in% 1:2) olap_clust_annot <- annotatePeakInBatch(olap_clust_annot, AnnotationData=TSS.human.GRCh38) olap_clust_annot$feature
Use case: ChromHMM inspection
In this demonstration we'll use the same CTCF dataset in breast cancer but bring in a chromHMM state file from an other dataset. This is meant to highlight the flexibility of seqsetvis.
Setup
If the previous CTCF in breast cancer Use Case made use of BiocFileCache, we'll continue caching external data sources. If not, we'll skip to a pre-cooked version of the same data.
The pre-cooked version is quite a narrow slice and there's a significant amount of data manipulation
target_data = "MCF10A_CTCF" chmm_win = 200 #window size is an important chromHMM parameter. # 200 is the default window size and matches the state segementation if(use_full_data){ # set all file paths chmm_bw_file = bfcrpath(ssv_bfc, rnames = paste(target_data, "bigwig", sep = ",")) chmm_np_file = bfcrpath(ssv_bfc, rnames = paste(target_data, "narrowPeak", sep = ",")) chmm_seg_file = bfcrpath(ssv_bfc, rnames = "MCF7,segmentation", fpath = chromHMM_demo_segmentation_url) query_chain = bfcquery(ssv_bfc, "hg19ToHg38,chain") if(nrow(query_chain) == 0){ chain_hg19ToHg38_gz = bfcrpath(ssv_bfc, rnames = "hg19ToHg38,gz", fpath = chromHMM_demo_chain_url) ch_raw = readLines(gzfile(chain_hg19ToHg38_gz)) ch_file = bfcnew(ssv_bfc, rname = "hg19ToHg38,chain") writeLines(ch_raw, con = ch_file) } chmm_chain_file = bfcrpath(ssv_bfc, rnames = "hg19ToHg38,chain") ch = rtracklayer::import.chain(chmm_chain_file) # load segmentation data chmm_gr = rtracklayer::import.bed(chmm_seg_file) #cleanup state names. chmm_gr$name = gsub("\\+", " and ", chmm_gr$name) chmm_gr$name = gsub("_", " ", chmm_gr$name) #setup color to state mapping colDF = unique(mcols(chmm_gr)[c("name", "itemRgb")]) state_colors = colDF$itemRgb names(state_colors) = colDF$name #liftover states from hg19 to hg38 ch = rtracklayer::import.chain(chmm_chain_file) chmm_gr_hg38 = rtracklayer::liftOver(chmm_gr, ch) chmm_gr_hg38 = unlist(chmm_gr_hg38) chmm_grs_list = as.list(GenomicRanges::split(chmm_gr_hg38, chmm_gr_hg38$name)) #transform narrowPeak ranges to summit positions chmm_np_grs = easyLoad_narrowPeak(chmm_np_file, file_names = target_data) chmm_summit_grs = lapply(chmm_np_grs, function(x){ start(x) = start(x) + x$relSummit end(x) = start(x) x }) qlist = append(chmm_summit_grs[1], chmm_grs_list) chmm_olaps = ssvOverlapIntervalSets(qlist, use_first = TRUE) #discard the columns for peak call and no_hit, not informative here. mcols(chmm_olaps)[[1]] = NULL chmm_olaps$no_hit = NULL #total width of genome assigned each state state_total_widths = sapply(chmm_grs_list, function(my_gr){ sum(as.numeric(width(my_gr))) }) #Expand state regions into 200 bp windows. state_wingrs = lapply(chmm_grs_list, function(my_gr){ st = my_gr$name[1] wgr = unlist(slidingWindows(my_gr, chmm_win, chmm_win)) wgr$state = st wgr }) state_wingrs = unlist(GRangesList(state_wingrs)) # fetch bw data for each state # it probably isn't useful to grab every single window for each state # so we can cap the number of each state carried forward max_per_state = 5000 # flank size zooms out a bit from each chromHMM window flank_size = 400 state_split = split(state_wingrs, state_wingrs$state) state_split = lapply(state_split, function(my_gr){ samp_gr = sample(my_gr, min(length(my_gr), max_per_state)) samp_gr = sort(samp_gr) names(samp_gr) = seq_along(samp_gr) samp_gr }) state_gr = unlist(GRangesList(state_split)) state_gr = resize(state_gr, width = chmm_win + 2 * flank_size, fix = "center") bw_states_gr = ssvFetchBigwig(file_paths = chmm_bw_file, qgr = state_gr, win_size = 50) bw_states_gr$grp = sub("\\..+", "", bw_states_gr$id) bw_states_gr$grp_id = sub(".+\\.", "", bw_states_gr$id) }else{ max_per_state = 20 flank_size = 400 state_colors = chromHMM_demo_state_colors bw_states_gr = chromHMM_demo_bw_states_gr chmm_olaps = chromHMM_demo_overlaps_gr state_total_widths = chromHMM_demo_state_total_widths }
Peak overlaps with states, counts
Create barplot of raw count of peaks overlapping states.
olaps_df = as.data.frame(mcols(chmm_olaps)) colnames(olaps_df) = gsub("\\.", " ", colnames(olaps_df)) p_state_raw_count = ssvFeatureBars(olaps_df, show_counts = FALSE) + labs(fill = "state", x = "") + scale_fill_manual(values = state_colors) + theme_cowplot() + guides(fill = "none") + theme(axis.text.x = element_text(angle = 90, size = 8, hjust = 1, vjust = .5)) p_state_raw_count
Peak overlaps with states, enrichment
Create barplot of enrichment of peaks overlapping states.
state_width_fraction = state_total_widths / sum(state_total_widths) state_peak_hits = colSums(olaps_df) state_peak_fraction = state_peak_hits / sum(state_peak_hits) enrichment = state_peak_fraction / state_width_fraction[names(state_peak_fraction)] enrich_df = data.frame(state = names(enrichment), enrichment = enrichment) p_state_enrichment = ggplot(enrich_df) + geom_bar(aes(x = state, fill = state, y = enrichment), stat = "identity") + labs(x = "") + theme_cowplot() + guides(fill = "none") + scale_fill_manual(values = state_colors) + theme(axis.text.x = element_text(angle = 90, size = 8, hjust = 1, vjust = .5)) p_state_enrichment
Aggregated line plots by state
Plot aggregated line plot by state
p_agg_tracks = ssvSignalLineplotAgg(bw_states_gr, sample_ = "grp", color_ = "grp") gb = ggplot2::ggplot_build(p_agg_tracks) yrng = range(gb$data[[1]]$y) p_agg_tracks = p_agg_tracks + scale_color_manual(values = state_colors) + annotate("line", x = rep(-chmm_win/2, 2), y = yrng) + annotate("line", x = rep(chmm_win/2, 2), y = yrng) + labs(y = "FE", x = "bp", color = "state", title = paste("Average FE by state,", target_data), subtitle = paste("states sampled to", max_per_state, chmm_win, "bp windows each\n", flank_size, "bp flanking each side")) + theme(plot.title = element_text(hjust = 0)) p_agg_tracks
States heatmap
A heatmap using the same data from the aggregated line plot. Facetting is done on state and regions are sorted in decreasing order of signal at center.
pdt = as.data.table(mcols(bw_states_gr)) pdt$grp_id = as.integer(pdt$grp_id) # reassign grp_id to sort within each state set dt_list = lapply(unique(pdt$grp), function(state){ dt = pdt[grp == state] dtmax = dt[, .(ymax = y[which(x == x[order(abs(x))][1])]), by = grp_id] dtmax = dtmax[order(ymax, decreasing = TRUE)] dtmax[, grp_o := seq_len(.N)] dtmax$ymax = NULL dt = merge(dtmax, dt) dt[, grp_id := grp_o] dt$grp_o = NULL dt }) # reassemble pdt = rbindlist(dt_list) # heatmap facetted by state and sorted in decreasing order p_state_hmap = ggplot(pdt) + geom_raster(aes(x = x, y = grp_id, fill = y)) + scale_y_reverse() + facet_wrap("grp", nrow = 2) + theme(axis.text.y = element_blank(), axis.ticks.y = element_blank(), axis.line.y = element_blank(), axis.text.x = element_text(angle = 90, hjust = 1, vjust = .5), strip.text = element_text(size= 8)) + scale_fill_gradientn(colors = c("white", "orange", "red")) + labs(y = "", fill = "FE", x = "bp from local summit", title = paste(max_per_state, "random regions per state")) p_state_hmap
Figure assembly
Tie it all together into one figure with cowplot.
bar_height = .25 line_height = .3 ggdraw() + draw_plot(p_state_raw_count + guides(fill = "none"), x = 0, y = 1 - bar_height, width = .5, height = bar_height) + draw_plot(p_state_enrichment + guides(fill = "none"), x = .5, y = 1 - bar_height, width = .5, height = bar_height) + draw_plot(p_agg_tracks, x = 0, y = 1 - bar_height - line_height, width = 1, height = line_height) + draw_plot(p_state_hmap, x = 0, y = 0, width = 1, height = 1 - bar_height - line_height) + draw_plot_label(LETTERS[1:4], c(0, 0.48, 0, 0), c(1, 1, 1 - bar_height, 1 - bar_height - line_height), size = 15)
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