1 Overview

Here, we perform a window-based DB analysis to identify regions of differential H3K9ac enrichment between pro-B and mature B cells (Revilla-I-Domingo et al. 2012). H3K9ac is associated with active promoters and tends to exhibit relatively narrow regions of enrichment relative to other marks such as H3K27me3. The experimental design contains two biological replicates for each of the two cell types.

2 Aligning reads in the H3K9ac libraries

The first task is to download the relevant ChIP-seq libraries from the NCBI Gene Expression Omnibus (GEO) (Edgar, Domrachev, and Lash 2002). These files are obtained from the data series GSE38046, using the Sequence Read Accession (SRA) numbers listed below. Multiple technical replicates exist for some of the biological replicates, and are indicated as those files with the same grouping.

sra.numbers <- c("SRR499718", "SRR499719", "SRR499720", "SRR499721",
    "SRR499734", "SRR499735", "SRR499736", "SRR499737", "SRR499738")
grouping <- c("proB-8113", "proB-8113", "proB-8108", "proB-8108",
    "matureB-8059", "matureB-8059", "matureB-8059", "matureB-8059", "matureB-8086")
all.sra <- paste0(sra.numbers, ".lite.sra")
data.frame(SRA=all.sra, Condition=grouping)
##                  SRA    Condition
## 1 SRR499718.lite.sra    proB-8113
## 2 SRR499719.lite.sra    proB-8113
## 3 SRR499720.lite.sra    proB-8108
## 4 SRR499721.lite.sra    proB-8108
## 5 SRR499734.lite.sra matureB-8059
## 6 SRR499735.lite.sra matureB-8059
## 7 SRR499736.lite.sra matureB-8059
## 8 SRR499737.lite.sra matureB-8059
## 9 SRR499738.lite.sra matureB-8086

These files are downloaded in the SRA format, and need to be unpacked to the FASTQ format prior to alignment. This can be done using the fastq-dump utility from the SRA Toolkit.

for (sra in all.sra) {
    code <- system(paste("fastq-dump", sra))
all.fastq <- paste0(sra.numbers, ".fastq")

Reads from technical replicates are pooled together into a single FASTQ file prior to further processing. This reflects the fact that they originate from a single library of DNA fragments. <- split(all.fastq, grouping)
for (group in names( {
    code <- system(paste(c("cat",[[group]], ">",
        paste0(group, ".fastq")), collapse=" "))
group.fastq <- paste0(names(, ".fastq")

Reads in each library are aligned to the mm10 build of the mouse genome, using the align function in the Rsubread package (Liao, Smyth, and Shi 2013). This assumes that an index has already been constructed with the prefix index/mm10. The function uses a seed-and-vote paradigm to quickly and accurately map reads to the genome by focusing on locations that receive a number of votes above some consensus threshold. Here, a threshold of 2 votes is used instead of the default of 3, to accommodate the short length of the reads (32–36 bp). The type parameter is also set to optimize for genomic alignment, rather than alignment to the transcriptome.

bam.files <- paste0(names(, ".bam")
align(index="index/mm10", readfile1=group.fastq, TH1=2, type=1,
    input_format="FASTQ", output_file=bam.files)

In each of the resulting BAM files, alignments are re-sorted by their mapping locations. This is required for input into csaw, but is also useful for other programs like genome browsers that depend on sorting and indexing for rapid retrieval of reads.

for (bam in bam.files) {
    out <- suppressWarnings(sortBam(bam, "h3k9ac_temp"))
    file.rename(out, bam)

Potential PCR duplicates are marked using the MarkDuplicates tool from the Picard software suite. These are identified as alignments at the same genomic location, such that they may have originated from PCR-amplified copies of the same DNA fragment.

temp.bam <- "h3k9ac_temp.bam"
temp.file <- "h3k9ac_metric.txt"
temp.dir <- "h3k9ac_working"
for (bam in bam.files) {
    code <- system(sprintf("MarkDuplicates I=%s O=%s M=%s \\
        TMP_DIR=%s AS=true REMOVE_DUPLICATES=false \\
        VALIDATION_STRINGENCY=SILENT", bam, temp.bam,   
        temp.file, temp.dir))
    file.rename(temp.bam, bam)

The behaviour of the alignment pipeline for this data set is easily summarized with some statistics. Ideally, the proportion of mapped reads should be high (70-80% or higher), while the proportion of marked reads should be low (below 20%). Note that only reads with unique mapping locations are reported by Rsubread as being successfully mapped.

diagnostics <- list()
for (bam in bam.files) {
    total <- countBam(bam)$records
    mapped <- countBam(bam, param=ScanBamParam(
    marked <- countBam(bam, param=ScanBamParam(
        flag=scanBamFlag(isUnmapped=FALSE, isDuplicate=TRUE)))$records
    diagnostics[[bam]] <- c(Total=total, Mapped=mapped, Marked=marked)
diag.stats <- data.frame(, diagnostics))
diag.stats$Prop.mapped <- diag.stats$Mapped/diag.stats$Total*100
diag.stats$Prop.marked <- diag.stats$Marked/diag.stats$Mapped*100
##                     Total  Mapped  Marked Prop.mapped Prop.marked
## matureB-8059.bam 16675372 7752077 1054591    46.48818   13.603980
## matureB-8086.bam  6347683 4899961  195100    77.19291    3.981664
## proB-8108.bam    10413135 8213980  297796    78.88095    3.625478
## proB-8113.bam    10724526 9145743  489177    85.27876    5.348685

Finally, the libraries are indexed for rapid retrieval by genomic location. This generates a number of index files at the same location as the BAM files.


3 Obtaining the ENCODE blacklist for mm10

A number of genomic regions contain high artifactual signal in ChIP-seq experiments. These often correspond to genomic features like telomeres or microsatellite repeats. For example, multiple tandem repeats in the real genome are reported as a single unit in the genome build. Alignment of all (non-specifically immunoprecipitated) reads from the former will result in artificially high coverage of the latter. Moreover, differences in repeat copy numbers between conditions can lead to detection of spurious DB.

As such, these problematic regions must be removed prior to further analysis. This is done with an annotated blacklist for the mm10 build of the mouse genome. Genomic intervals in the blacklist are loaded to memory using the import method from the rtracklayer package. All reads mapped to those intervals will be ignored during processing in csaw. The blacklist itself was constructed by identifying consistently problematic regions in the ENCODE and modENCODE data sets (ENCODE Project Consortium 2012).

blacklist <- import("mm10.blacklist.bed.gz")
## GRanges object with 164 ranges and 0 metadata columns:
##         seqnames              ranges strand
##            <Rle>           <IRanges>  <Rle>
##     [1]    chr10     3110061-3110270      *
##     [2]    chr10   22142531-22142880      *
##     [3]    chr10   22142831-22143070      *
##     [4]    chr10   58223871-58224100      *
##     [5]    chr10   58225261-58225500      *
##     ...      ...                 ...    ...
##   [160]     chr9     3038051-3038300      *
##   [161]     chr9   24541941-24542200      *
##   [162]     chr9   35305121-35305620      *
##   [163]     chr9 110281191-110281400      *
##   [164]     chr9 123872951-123873160      *
##   -------
##   seqinfo: 19 sequences from an unspecified genome; no seqlengths

Any user-defined set of regions can be used as a blacklist in this analysis. For example, one could use predicted repeat regions from the UCSC genome annotation (Rosenbloom et al. 2015). This tends to remove a greater number of problematic regions (especially microsatellites) compared to the ENCODE blacklist. However, the size of the UCSC list means that genuine DB sites may also be removed. Thus, the ENCODE blacklist is preferred for most applications. Alternatively, if negative control libraries are available, they can be used to empirically identify problematic regions with the GreyListChIP package. These regions should be ignored as they have high coverage in the controls and are unlikely to be genuine binding sites.

3.1 Setting up the analysis parameters

Here, the settings for the DB analysis are specified. Recall that the paths to the BAM files are stored in the bam.files vector after alignment. The cell type for each file is conveniently extracted from the file name.

celltype <- sub("-.*", "", bam.files)
data.frame(BAM=bam.files, CellType=celltype)
##                BAM CellType
## 1 matureB-8059.bam  matureB
## 2 matureB-8086.bam  matureB
## 3    proB-8108.bam     proB
## 4    proB-8113.bam     proB

In the csaw package, the readParam object determines which reads are extracted from the BAM files. The idea is to set this up once and to re-use it in all relevant functions. For this analysis, reads are ignored if they map to blacklist regions or do not map to the standard set of mouse nuclear chromosomes.

standard.chr <- paste0("chr", c(1:19, "X", "Y"))
param <- readParam(minq=50, discard=blacklist, restrict=standard.chr)

Reads are also ignored if they have a mapping quality (MAPQ) score below 50. This avoids spurious results due to weak or non-unique alignments. While a MAPQ threshold of 50 is conservative, a stringent threshold is necessary here due to the short length of the reads. Note that the range of MAPQ scores will vary between aligners, so some inspection of the BAM files is necessary to choose an appropriate value.

4 Computing the average fragment length

Strand bimodality is often observed in ChIP-seq experiments involving narrow binding events like H3K9ac marking. This refers to the presence of distinct subpeaks on each strand and is quantified with cross-correlation plots (Kharchenko, Tolstorukov, and Park 2008). A strong peak in the cross-correlations should be observed if immunoprecipitation was successful. The delay distance at the peak corresponds to the distance between forward-/reverse-strand subpeaks. This is identified from Figure 1 and is used as the average fragment length for this analysis.

x <- correlateReads(bam.files, param=reform(param, dedup=TRUE))
frag.len <- maximizeCcf(x)
## [1] 148
plot(1:length(x)-1, x, xlab="Delay (bp)", ylab="CCF", type="l")
abline(v=frag.len, col="red")
text(x=frag.len, y=min(x), paste(frag.len, "bp"), pos=4, col="red")
Cross-correlation function (CCF) against delay distance for the H3K9ac data set. The delay with the maximum correlation is shown as the red line.

Figure 1: Cross-correlation function (CCF) against delay distance for the H3K9ac data set
The delay with the maximum correlation is shown as the red line.

Only unmarked reads (i.e., not potential PCR duplicates) are used to calculate the cross-correlations. This reduces noise from variable PCR amplification and decreases the size of the “phantom” peak at the read length (Landt et al. 2012). However, removal of marked reads is risky as it caps the signal in high-coverage regions of the genome. This can result in loss of power to detect DB, or introduction of spurious DB when the same cap is applied to libraries of different sizes. Thus, the marking status of each read will be ignored in the rest of the analysis, i.e., no duplicates will be removed in downstream steps.

5 Counting reads into windows

csaw uses a sliding window strategy to quantify binding intensity across the genome. Each read is directionally extended to the average fragment length, to represent the DNA fragment from which that read was sequenced. The number of extended reads overlapping a window is counted. The window is then moved to its next position on the genome, and counting is repeated. (Each read is usually counted into multiple windows, which will introduce correlations between adjacent windows but will not otherwise affect the analysis.) This is done for all libraries such that a count is obtained for each window in each library. The windowCounts function produces a RangedSummarizedExperiment object containing these counts in matrix form, where each row corresponds to a window and each column represents a library. <- windowCounts(bam.files, param=param, width=150, ext=frag.len)
## class: RangedSummarizedExperiment 
## dim: 1578941 4 
## metadata(6): spacing width ... param final.ext
## assays(1): counts
## rownames: NULL
## rowData names(0):
## colnames: NULL
## colData names(4): bam.files totals ext rlen

To analyze H3K9ac data, a window size of 150 bp is used here. This corresponds roughly to the length of the DNA in a nucleosome (Humburg et al. 2011), which is the smallest relevant unit for studying histone mark enrichment. The spacing between windows is set to the default of 50 bp, i.e., the start positions for adjacent windows are 50 bp apart. Smaller spacings can be used to improve spatial resolution, but will increase memory usage and runtime by increasing the number of windows required to cover the genome. This is unnecessary as increased resolution confers little practical benefit for this data set – counts for very closely spaced windows will be practically identical. Finally, windows with very low counts (by default, less than a sum of 10 across all libraries) are removed to reduce memory usage. This represents a preliminary filter to remove uninteresting windows corresponding to likely background regions.

5.1 Filtering windows by abundance

As previously mentioned, low-abundance windows contain no binding sites and need to be filtered out. This improves power by removing irrelevant tests prior to the multiple testing correction; avoids problems with discreteness in downstream statistical methods; and reduces computational work for further analyses. Here, filtering is performed using the average abundance of each window (McCarthy, Chen, and Smyth 2012), which is defined as the average log-count per million for that window. This performs well as an independent filter statistic for NB-distributed count data (Lun and Smyth 2014).

The filter threshold is defined based on the assumption that most regions in the genome are not marked by H3K9ac. Reads are counted into large bins and the median coverage across those bins is used as an estimate of the background abundance. This estimate is then compared to the average abundances of the windows, after rescaling to account for differences in the window and bin sizes. A window is only retained if its coverage is 3-fold higher than that of the background regions, i.e., the abundance of the window is greater than the background abundance estimate by log2(3) or more. This removes a large number of windows that are weakly or not marked and are likely to be irrelevant.

bins <- windowCounts(bam.files, bin=TRUE, width=2000, param=param)
filter.stat <- filterWindows(, bins, type="global")
min.fc <- 3
keep <- filter.stat$filter > log2(min.fc)
##    Mode   FALSE    TRUE 
## logical  907923  671018

The effect of the fold-change threshold is examined visually in Figure 2. The chosen threshold is greater than the abundances of most bins in the genome – presumably, those that contain background regions. This suggests that the filter will remove most windows lying within background regions.

hist(filter.stat$back.abundances, main="", breaks=50,
    xlab="Background abundance (log2-CPM)")
threshold <- filter.stat$abundances[1] - filter.stat$filter[1] + log2(min.fc)
abline(v=threshold, col="red")
Histogram of average abundances across all 2 kbp genomic bins. The filter threshold is shown as the red line.

Figure 2: Histogram of average abundances across all 2 kbp genomic bins
The filter threshold is shown as the red line.

The actual filtering itself is done by simply subsetting the RangedSummarizedExperiment object. <-[keep,]

6 Normalizing for library-specific trended biases

Normalization is required to eliminate confounding library-specific biases prior to any comparisons between libraries. Here, a trended bias is present between libraries in Figure 3. This refers to a systematic fold-difference in window coverage between libraries that changes according to the average abundance of the window.

win.ab <- scaledAverage(
adjc <- calculateCPM(, use.offsets=FALSE)
logfc <- adjc[,1] - adjc[,4]
smoothScatter(win.ab, logfc, ylim=c(-6, 6), xlim=c(0, 5),
    xlab="Average abundance", ylab="Log-fold change")

lfit <- smooth.spline(logfc~win.ab, df=5)
o <- order(win.ab)
lines(win.ab[o], fitted(lfit)[o], col="red", lty=2)
Abundance-dependent trend in the log-fold change between two H3K9ac libraries (mature B over pro-B), across all windows retained after filtering. A smoothed spline fitted to the log-fold change against the average abundance is also shown in red.

Figure 3: Abundance-dependent trend in the log-fold change between two H3K9ac libraries (mature B over pro-B), across all windows retained after filtering
A smoothed spline fitted to the log-fold change against the average abundance is also shown in red.

Trended biases cannot be removed by scaling methods like TMM normalization (Robinson and Oshlack 2010), as the amount of scaling required varies with the abundance of the window. Rather, non-linear normalization methods must be used. csaw implements a version of the fast loess method (Ballman et al. 2004) that has been modified to handle count data (Lun and Smyth 2015). This produces a matrix of offsets that can be used during GLM fitting. <- normOffsets(, type="loess")
offsets <- assay(, "offset")
##            [,1]       [,2]      [,3]      [,4]
## [1,] -0.5877976 -0.4025337 0.3956788 0.5946524
## [2,] -0.5666204 -0.3795870 0.3771290 0.5690783
## [3,] -0.6261282 -0.4722153 0.4397219 0.6586216
## [4,] -0.6529113 -0.5451086 0.4786559 0.7193639
## [5,] -0.6713865 -0.5836750 0.5012739 0.7537877
## [6,] -0.7027471 -0.6462644 0.5385283 0.8104832

The effect of non-linear normalization is visualized with another mean-difference plot. Once the offsets are applied to adjust the log-fold changes, the trend is eliminated from the plot (Figure 4). The cloud of points is also centred at a log-fold change of zero. This indicates that normalization was successful in removing the differences between libraries.

norm.adjc <- calculateCPM(, use.offsets=TRUE)
norm.fc <- norm.adjc[,1]-norm.adjc[,4]
smoothScatter(win.ab, norm.fc, ylim=c(-6, 6), xlim=c(0, 5),
    xlab="Average abundance", ylab="Log-fold change")

lfit <- smooth.spline(norm.fc~win.ab, df=5)
lines(win.ab[o], fitted(lfit)[o], col="red", lty=2)
Effect of non-linear normalization on the trended bias between two H3K9ac libraries. Normalized log-fold changes are shown for all windows retained after filtering. A smoothed spline fitted to the log-fold change against the average abundance is also shown in red.

Figure 4: Effect of non-linear normalization on the trended bias between two H3K9ac libraries
Normalized log-fold changes are shown for all windows retained after filtering. A smoothed spline fitted to the log-fold change against the average abundance is also shown in red.

The implicit assumption of non-linear methods is that most windows at each abundance are not DB. Any systematic difference between libraries is attributed to bias and is removed. The assumption of a non-DB majority is reasonable for this data set, given that the cell types being compared are quite closely related. However, it is not appropriate in cases where large-scale DB is expected, as removal of the difference would result in loss of genuine DB. An alternative normalization strategy for these situations will be described later in the CBP analysis.

7 Statistical modelling of biological variability

7.1 Introduction

Counts are modelled using NB GLMs in the edgeR package (McCarthy, Chen, and Smyth 2012; Robinson, McCarthy, and Smyth 2010). The NB distribution is useful as it can handle low, discrete counts for each window. The NB dispersion parameter allows modelling of biological variability between replicate libraries. GLMs can also accommodate complex experimental designs, though a simple design is sufficient for this study.

celltype <- factor(celltype)
design <- model.matrix(~0+celltype)
colnames(design) <- levels(celltype)
##   matureB proB
## 1       1    0
## 2       1    0
## 3       0    1
## 4       0    1
## attr(,"assign")
## [1] 1 1
## attr(,"contrasts")
## attr(,"contrasts")$celltype
## [1] "contr.treatment"

As a general rule, the experimental design should contain at least two replicates in each of the biological conditions. This ensures that the results for each condition are replicable and are not the result of technical artifacts such as PCR duplicates. Obviously, more replicates will provide more power to detect DB accurately and reliability, albeit at the cost of time and experimental resources.

7.2 Estimating the NB dispersion

The RangedSummarizedExperiment object is coerced into a DGEList object (plus offsets) for use in edgeR. Estimation of the NB dispersion is performed using the estimateDisp function. Specifically, a NB dispersion trend is fitted to all windows against the average abundance. This means that empirical mean-dispersion trends can be flexibly modelled.

y <- asDGEList(
y <- estimateDisp(y, design)
##    Min. 1st Qu.  Median    Mean 3rd Qu.    Max. 
## 0.03156 0.04223 0.04303 0.04201 0.04339 0.04401

The NB dispersion trend is visualized in Figure 5 as the biological coefficient of variation (BCV), i.e., the square root of the NB dispersion. Note that only the trended dispersion will be used in the downstream steps – the common and tagwise values are only shown for diagnostic purposes. Specifically, the common BCV provides an overall measure of the variability in the data set, averaged across all windows. Data sets with common BCVs ranging from 10 to 20% are considered to have low variability, i.e., counts are highly reproducible. The tagwise BCVs should also be dispersed above and below the fitted trend, indicating that the fit was successful.