BiocStyle::markdown()
enrichR()
calls ChIP-seq enrichment over control for coordinate-sorted and
indexed bamfilese <- enrichR(treatment = "ChIP.bam", control = "Control.bam", genome = "hg19")
diffR()
calls differential enrichment between two conditions, i.e. two
ChIP-seq tracksde <- diffR(treatment = "ChIP1.bam", control = "ChIP2.bam", genome = "hg19")
regimeR()
calls k
enrichment regimes in a ChIP-seq experiment over
controlre <- regimeR(treatment = "ChIP.bam", control = "Control.bam", genome = "hg19", models = k)
exportR()
writes above results to bed, bedGraph or bigWig#export enriched regions with FDR<=10% for downstream analysis exportR(obj = e, filename = "enriched.bed", type = "bed", fdr = 0.1) #or #write normalized differential enrichment to bigWig for genome browser display exportR(obj = de, filename = "diffEnrichment.bw", type = "bigWig")
citation("normr")
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) provides genome-wide localization data for DNA-associated proteins. To infer the regions bound by such proteins the read densities obtained by such a ChIP-seq experiment are compared to the corresponding read profile obtained by a control experiment. A meaningful comparison requires normalization to mitigate the effects of technical biases, e.g. different sequencing depth. But more importantly the effect of the enrichment of certain regions on the overall read statistics. Normalization requires knowledge of the regions that remained unchanged, such that normalization and difference calling are inseparable.
This package, normR (normR obeys regime mixture Rules),
follows this logic and performs normalization and difference calling
simultaneously to identify genomic regions enriched by the ChIP-procedure
(enrichR()
). In addition, normR enables the comparison between ChIP-seq data
obtained from different conditions allowing for unraveling genomic regions that
change their association with the ChIP-target (diffR()
). Lastly, normR is
capable to differentiate multiple regimes of enrichment, i.e. broad domains
and sharp peaks (regimeR()
). In brief, all these routines encompass three
steps:
This vignette explains a common workflow of normR analysis on NGS data for calling enrichment, identification of differential ChIP-seq enrichment and stratification of enrichment regimes. Herein, we provide examples for the export of results to common data formats like bigWig and bed. We show how analysis statistics and diagnostic plots are helpful for studying results. In a latter section, we cover more advanced topics including the alteration of read counting strategies, the application of normR on user-defined regions (e.g. promoters) and the integration of Copy Number Variation information in differential ChIP-seq enrichment calls.
enrichR()
: Calling Enrichment with an Input ControlIn this first section, we would like to call regions significantly enriched for reads in the ChIP-seq experiment given a Control alignment. Here, we analyze ChIP-seq data for both H3K4me3 (pointy enrichment) and H3K36me3 (broad enrichment) given an Input-seq control alignment originating from a human immortalized myelogenous leukemia line (K562). Using normR, we show that our representative region on human chromsome 1 (chr1:22500000-25000000) is enriched for H3K4me3 mostly at promoters and precludes H3K36me3 enrichment which is overrepresented in gene bodies.
IGV browser shot of Input (grey), H3K4me3 (green) and H3K36me3 (purple)
alignment data on chr1 22.5Mb to 25Mb with genes (black) drawn.
As part of the normR package, we provide 3 alignment files in bam format
(Input, H3K4me3 and H3K36me3 ChIP-seq) containing reads for human chr1 22.5Mb
to 25Mb. Note, bam files used in normR need to be sorted by read coordinates
(samtools sort x.bam x_sorted
) and indexed (samtools index x_sorted.bam
).
Our example files already fullfil this criteria.
Firstly, we retrieve filepaths for toy data:
#Loading required package library("normr") inputBamfile <- system.file("extdata", "K562_Input.bam", package="normr") k4me3Bamfile <- system.file("extdata", "K562_H3K4me3.bam", package="normr") k36me3Bamfile <- system.file("extdata", "K562_H3K36me3.bam", package="normr")
Secondly and lastly, we need to specify the genome of the alignment. The
genome
argument can be a character specifying a UCSC genome identifier,
e.g. "hg19", or we can provide a 2-dimensional data.frame
with columns
seqnames and length. We will follow the later option: You can generate a genome
data.frame
yourself or can use GenomeInfoDb
for retrieving the data.frame
from UCSC for given genome identifier:
#Fetch chromosome information genome <- GenomeInfoDb::getChromInfoFromUCSC("hg19", assembled.molecules.only = TRUE) #Delete unnecessary columns genome <- genome[,1:2] genome #Toy data has only "chr1" genome <- genome[genome[,1] == "chr1",] genome
Now, we are all set to do a enrichment call with default parameters:
#Enrichment Calling for H3K4me3 and H3K36me3 k4me3Fit <- enrichR(treatment = k4me3Bamfile, control = inputBamfile, genome = genome, verbose = FALSE) k36me3Fit <- enrichR(treatment = k36me3Bamfile, control = inputBamfile, genome = genome, verbose = FALSE)
That was easy and fast! You must know that all results are stored as
NormRFit-class
objects. They provide convenient access to count data and
fitting results. For example, let's have a look at some simple fitting
statistics for H3K4me3:
k4me3Fit
The "Type" of the NormRFit
object is defined by the function generating it,
i.e. enrichR()
, diffR()
or regimeR()
. The "Number of Regions" is the
number of 250bp bins along the specified genome (default binsize). The "Number
of Components" is 2 (background and enriched) in the case of enrichR()
. The
parameter $\theta^$ ("Theta (naive bg)") describes a naive background
parametrization if the effect of enrichment is not taken into account. The
actual $\theta_B$ is with ~0.09 much smaller than $\theta^*$ which allows for
more sensitive enrichment calling. Furthermore, by looking at the "Mixture
Proportions" we find H3K4me3 is enriched in ~5% of the windows. For H3K36me3,
we find ~23% of the regions enriched.
k36me3Fit
We can use some methods provided by the NormRFit-class
to get a grasp on the
quality of our normR call, e.g. print a more concise summary that gives the
number of significant regions under different False Discovery Rates ($FDR$).
summary(k4me3Fit)
Note, summary()
prints an additional section containing information on the
statistical testing. The "T-Filter threshold" filters out regions that are not
considered for multiple testing correction due to low power. The sum of counts
in treatment and control is less than this quantity (Dialsingh et al.,
Bioinformatics, 2015, 1–7). The "Number of Regions filtered out" is very large
in our example because the toy alignment files are filtered for reads within
chr1 22.5Mb to 25Mb. However, the specified genome covers chr1:0-249250621
which results in alot of zero counts. This does not influence the fit but for
testing the regions are filtered. Based on computed q-vlaues, H3K4me3 shows
587 (24+433+67+63) regions significantly enriched for $FDR \le 0.05$. The
summary for H3K36me3 enrichment calls confirms 2,378 (0+1951+212+215) regions
significantly enriched for $FDR \le 0.05$:
summary(k36me3Fit)
Based on the fitted background, normR can calculate a standardized, regularized enrichment for further processing:
#background normalized enrichment k4me3Enr <- getEnrichment(k4me3Fit) #restrict to regions with non-zero counts idx <- which(rowSums(do.call(cbind, getCounts(k4me3Fit))) != 0) summary(k4me3Enr[idx])
If we compare H3K4me3 and H3K36me3 enrichment in non-zero regions, we can see some mutual exclusivity of both marks represented by off-diagonal elements):
x <- k4me3Enr[idx] y <- getEnrichment(k36me3Fit)[idx] d.x <- density(x); d.y <- density(y) limits <- range(x,y) layout( matrix( c(0,2,2,1,3,3,1,3,3),ncol=3) ) plot(d.x$x, d.x$y, xlim=limits, type='l', main="H3K36me3 normalized Enrichment", xlab="", ylab="Density") abline(v=0, lty=3, lwd=2, col=4) plot(d.y$y, d.y$x, ylim=limits, xlim=rev(range(d.y$y)), type='l', main="H3K4me3 normalized Enrichment", xlab="Density", ylab="") abline(h=0, lty=3, lwd=2, col=4) color <- rep("grey10", length(idx)) plot(x, y, xlim=limits, ylim=limits, pch=20, xlab="", ylab="", col=adjustcolor(color, alpha.f=.2)) abline(0, 1, lty=2, lwd=3, col=2) abline(v=0, lty=3, lwd=2, col=4) abline(h=0, lty=3, lwd=2, col=4)
To analyze mutually exclusivity of H3K4me3 and H3K36me3, we would like to
recover the regions signficantly enriched in k4me3Fit
and k36me3Fit
and
color these regions in the scatter plot above.
#integer vector with <NA> set to non-significant regions k4me3Classes <- getClasses(k4me3Fit, fdr = 0.05) k36me3Classes <- getClasses(k36me3Fit, fdr = 0.05) #Color scatter plot based on enrichment color[!is.na(k4me3Classes[idx])] <- "#2C9500" color[!is.na(k36me3Classes[idx])] <- "#990099" color[!is.na(k4me3Classes+k36me3Classes)[idx]] <- "#971621" plot(x, y, xlim=limits, ylim=limits, pch=20, col=adjustcolor(color, alpha.f=.5), xlab="H3K4me3 normalized Enrichment", ylab="H3K36me3 normalized Enrichment") legend("topright", pch=20, col=unique(color), cex=.6, bg="white", legend=c("Background", "H3K36me3 enriched", "H3K4me3 enriched", "H3K4me3/K36me3 enriched") )
Processing of regions within R can be facilitated by getting significantly
enriched ($FDR \le 0.05%$) regions as a GRanges
object:
k4me3Ranges <- getRanges(k4me3Fit)[!is.na(k4me3Classes)] #Alternatively you can extract ranges without storing the class vector k4me3Ranges <- getRanges(k4me3Fit, fdr = 0.05) #as expected we get 587 regions length(k4me3Ranges)
As a representative analysis, we would like check for overrepresentation of enriched regions with genes and promoters by using Fisher's exact test. Let's start with H3K4me3:
#example gene annotation for representative region (chr1:22500000-25000000) genes <- read.delim(file = system.file("extdata", "genes.bed",package="normr"), header = FALSE, stringsAsFactors = FALSE) library("GenomicRanges") genes <- GRanges(seqnames = genes[, 1], ranges = IRanges(start = genes[, 2], end = genes[, 3]), strand = genes[, 6], ENSTID = genes[, 4]) genes <- unique(genes) #Fisher-test provides significance of overlap #(total specifies number of bins in representative region) overlapOdds <- function(query, subject, total = 10000) { subject <- reduce(subject, ignore.strand = TRUE) ov1 <- countOverlaps(query, subject) m <- matrix(c(sum(ov1 != 0), sum(ov1 == 0), ceiling(sum(width(subject))/width(query)[1]-sum(ov1 != 0)), 0), ncol = 2) m[2,2] <- total - sum(m) fisher.test(m, alternative="greater") } #Overlap of H3K4me3 with genes overlapOdds(k4me3Ranges, genes) #Overlap of H3K4me3 with promoters promoters <- promoters(genes, upstream = 2000, downstream = 2000) overlapOdds(k4me3Ranges, promoters)
It is known that promoters are marked by H3K4me3 if their gene's expression is initiated. Our analysis above shows that H3K4me3-enriched regions are indeed significantly overrepresented within genes (Fisher's signed-exact test; P-value<0.001; odds ratio = 1.72) and, more pronounced, in promoter regions (odds ratio = 7.74).
By comparing H3K36me3 and H3K4me3 ranges, we can identify significant overlap of H3K36me3 and H3K4me3 (odds ratio = 1.53) that is most pronounced in promoter regions (odds ratio = 9.68) than compared to gene bodies (odds ratio = 2.85).
#Overlap of H3K36me3 with H3K4me3 k36me3Ranges <- getRanges(k36me3Fit, fdr = 0.05) overlapOdds(k36me3Ranges, k4me3Ranges) #Overlap of H3K36me3 with H3K4me3 at promoter regions overlapOdds(k36me3Ranges[countOverlaps(k36me3Ranges, promoters) != 0], k4me3Ranges[countOverlaps(k4me3Ranges, promoters) != 0]) #Overlap of H3K36me3 with H3K4me3 in genes overlapOdds(k36me3Ranges[countOverlaps(k36me3Ranges, genes) != 0], k4me3Ranges[countOverlaps(k4me3Ranges, genes) != 0])
H3K36me3 is associated to transcriptional elongation in the gene body. The presence of H3K36me3 within the gene body marks transcribed genes. Indeed, H3K36me3 enrichment is significantly overrepresented mostly at genes (odds ratio = 5.52) and, to a lower extend, at promoters (odds ratio = 2.68).
#Overlap of H3K36me3 in genes overlapOdds(k36me3Ranges, genes) #Overlap of H3K36me3 with promoters overlapOdds(k36me3Ranges, promoters(genes, 1500, 1500))
While there exist a plethora of analysis options of normR results within R,
exportR()
provides functionality to write results to a file. To export
coordinates of enriched regions, the widely used
BED format is applicable.
It is human-readable and can be imported in common genome browsers, e.g.
UCSC genome browser or
IGV. To export the background-normalized enrichment, the
binary bigWig format is
used. Check ?exportR
for more options.
#export coordinates of significantly (FDR <= 0.05) enriched regions exportR(k4me3Fit, filename = "k4me3Fit.bed", type = "bed", fdr = 0.05) exportR(k36me3Fit, filename = "k36me3Fit.bed", type = "bed", fdr = 0.05) #export background-normalized enrichment exportR(k4me3Fit, filename = "k4me3Fit.bw", type = "bigWig") exportR(k36me3Fit, filename = "k36me3Fit.bw", type = "bigWig")
IGV browser shot of Input (grey), H3K4me3 (green) and H3K36me3 (purple)
alignment data (bars), normalized enrichment, i.e. "bigWig" files, (lines)
and enriched regions, i.e. "bed" files (boxes below respective tracks).
diffR()
: Calling Differential Enrichment without a Control ExperimentNormalization and difference calling are inseparable in calling ChIP-seq
enrichment. Following this notion, a direct comparison of two ChIP-seq tracks
can be performed with diffR()
. In many studies, researchers are interested in
conditional changes in ChIP-seq enrichment. Below, we exemplify this analysis
by joint analysis of H3K4me3 and H3K36me3 ChIP-seq data. Because we already
counted k4me3Bamfile
and k36me3Bamfile
already in k4me3Fit
and
k36me3Fit
, respectively, we can use these counts directly. Note that, in this
case, the genome
has be set to a GenomicRanges
object specifying the
genomic regions. We can extract this from either one of the NormRFit
objects.
#We could use read counts from above NormRFit objects k4k36Dif <- diffR(treatment = getCounts(k4me3Fit)$treatment, control = getCounts(k36me3Fit)$treatment, genome = getRanges(k4me3Fit), verbose = FALSE) #<or> (unnecessarily) count again #k4k36Dif <- diffR(treatment = k4me3Bamfile, control = k36me3Bamfile, # genome = genome, verbose = FALSE) #summary statistics summary(k4k36Dif)
The "Type" of the object has changed to 'diffR' because it was generated by
this function. The "Number of Regions" did not change because we use the same
binning strategy. However, the "Number of Components" is now 3 representing (i)
H3K36me3 enrichment without H3K4me3-enrichment, (ii) H3K36me3 and H3K4me3
(non)-enriched and (iii) H3K4me3 enrichment without H3K36me3-enrichment. The
"Backgroundcomponent B" is 2 in this case: diffR()
identifies significant
enrichment and depletion by a two-sided test on the background. Looking at the
"Mixture Proportions", regions are classified to (i) in ~49.8%, (ii) in
~29.5% and (iii) in ~20.7% of the regions. 2,629 regions are significantly
different from background with $FDR \le 0.05$. These regions are either
H3K4me3-positive or H3K36me3 positive. We can export these regions and the
normalized enrichment with exportR
:
exportR(k4k36Dif, filename = "k4k36Dif.bed", type = "bed", fdr = 0.05) exportR(k4k36Dif, filename = "k4k36Dif.bw", type = "bigWig")
IGV browser shot of Input (grey), H3K4me3 (green) and H3K36me3 (purple)
alignment data. Background normalized difference is plotted as a heatmap,
i.e. "bigWig" file, and differential regions are plotted as boxes, i.e.
"bed" file (blue: treatment (H3K4me3) enriched, red: control (H3K36me3)
enriched).
regimeR()
: Identify Enrichment Regimes in ChIP-seq ExperimentsThe two sections above aimed at discerning enrichment from background. The
extendable normR approach also allows for identification of different
enrichment regimes with regimeR()
by increasing the number of model
components.
Let's start with 3 components (Background + 2 Enrichment Regimes) for H3K4me3. By using two enrichment regimes, we may uncover effects of sample heterogeneity affecting transcriptional initiation of certian genes.
k4me3Regimes <- regimeR(treatment = getCounts(k4me3Fit)$treatment, control = getCounts(k4me3Fit)$control, genome = getRanges(k4me3Fit), models = 3, verbose = FALSE) summary(k4me3Regimes)
~10.5% of the regions show enrichment which gets segmented into ~7.7% low and ~2.8% high enrichment. 692 regions are significant ($FDR \le 0.05$).
Now, we would like to use two enrichment regimes for H3K36me3. In this way, we might be able to classify genes of low and high transcriptional rates:
k36me3Regimes <- regimeR(treatment = getCounts(k36me3Fit)$treatment, control = getCounts(k36me3Fit)$control, genome = getRanges(k36me3Fit), models = 3, verbose = FALSE) summary(k36me3Regimes)
We can now export the called regimes as bed files for browser display. Each track has two enrichment regimes which are shaded for their degree of significance. The export is done analogously to the cases described above:
exportR(k4me3Regimes, filename = "k4me3Regimes.bed", type = "bed", fdr=0.05) exportR(k36me3Regimes, filename = "k36me3Regimes.bed", type = "bed", fdr=0.05)
IGV browser shot of Input (grey), H3K4me3 (green) and H3K36me3 (purple)
alignment data. Regime calls are plotted as boxes below respective tracks
(Yellow = low enrichment, Pink = high enrichment)
In addition to the information covered above, it is recommend to have a look at
help pages (?
) of normR functions. Here, we would like to discuss three
important points:
NormRCountConfig-class
It is very important how we count reads contained in the bamfile. The
NormRCountConfig-class
provides methods to define the counting strategy on
single-end and paired-end alignment data:
#Single End: # Count in 500bp bins. # Consider only reads with Mapping Quality >= 20. # Filter reads for marked duplicates (e.g. with picard mark-duplicates) # Shift the counting position for a read 100 bp downstream. countConfigSE <- countConfigSingleEnd(binsize = 500, mapq = 20, filteredFlag = 1024, shift = 100) #Paired End: # Count in 500bp bins. # Consider only reads with Mapping Quality >= 30. # Count the midpoint of the aligned fragment instead of 5' ends. # Consider only reads corresponding to fragments with size from 100 to 300bp countConfigPE <- countConfigPairedEnd(binsize = 500, mapq = 30, midpoint=TRUE, tlenFilter = c(100, 300)) #Plug in the counting configuration into normR, e.g. in enrichR() fit <- enrichR(treatment = k4me3Bamfile, control = inputBamfile, genome = genome, countConfig = countConfigPE)
You could do a fit on a set of pre-defined regions like promoters or known
transcription factor binding sites. You need to count beforehand with
bamsignals
. Note, for the fit to work correctly these regions should be of
same size.
promoters <- promoters(genes, 1500, 1500) #regions have identical size? all(width(promoters) == 3000)
#Fit only on promoters promotersFit <- enrichR(treatment = k4me3Bamfile, control = inputBamfile, genome = promoters, verbose = FALSE) summary(promotersFit)
Copy Number Variations (CNVs) are an important feature of cancerous cells like
tumour samples. The difference calling on two ChIP-seq experiments with
diffR()
is sensitive to CNVs if the underlying sequence is amplified in the
genome. However, you can harness diffR()
's functionality to call differences
in two Input tracks to detect CNVs in a treatment respective to a control. To
allow for coarse-grained detection of difference in Input, a sufficiently large
binsize has to be used, e.g. 22kb.
cnvs <- diffR(treatment = treatmentInputBamfile, control = controlInputBamfile, genome = genome, countConfig = countConfigSingleEnd(binsize = 2.5e4)) #export the CNV calls exportR(cnvs, "CNVs.bed") #Filter previous ChIP-seq difference calls for CNVs ov <- countOverlaps(getRanges(diffFit, fdr = .05), getRanges(cnvs, fdr = .05)) idx <- which(ov == 0) cnvCleanedGR <- getRanges(diffFit, fdr = .05)[idx]
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