1 Abstract

ChIPseeker is an R package for annotating ChIP-seq data analysis. It supports annotating ChIP peaks and provides functions to visualize ChIP peaks coverage over chromosomes and profiles of peaks binding to TSS regions. Comparison of ChIP peak profiles and annotation are also supported. Moreover, it supports evaluating significant overlap among ChIP-seq datasets. Currently, ChIPseeker contains 17,000 bed file information from GEO database. These datasets can be downloaded and compare with user’s own data to explore significant overlap datasets for inferring co-regulation or transcription factor complex for further investigation.

2 Citation

If you use ChIPseeker1 in published research, please cite:

G Yu, LG Wang, QY He. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 2015, 31(14):2382-2383. doi:[10.1093/bioinformatics/btv145](

3 Introduction

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) has become standard technologies for genome wide identification of DNA-binding protein target sites. After read mappings and peak callings, the peak should be annotated to answer the biological questions. Annotation also create the possibility of integrating expression profile data to predict gene expression regulation. ChIPseeker1 was developed for annotating nearest genes and genomic features to peaks.

ChIP peak data set comparison is also very important. We can use it as an index to estimate how well biological replications are. Even more important is applying to infer cooperative regulation. If two ChIP seq data, obtained by two different binding proteins, overlap significantly, these two proteins may form a complex or have interaction in regulation chromosome remodelling or gene expression. ChIPseeker1 support statistical testing of significant overlap among ChIP seq data sets, and incorporate open access database GEO for users to compare their own dataset to those deposited in database. Protein interaction hypothesis can be generated by mining data deposited in database. Converting genome coordinations from one genome version to another is also supported, making this comparison available for different genome version and different species.

Several visualization functions are implemented to visualize the coverage of the ChIP seq data, peak annotation, average profile and heatmap of peaks binding to TSS region.

Functional enrichment analysis of the peaks can be performed by my Bioconductor packages DOSE2, ReactomePA3, clusterProfiler4.

## loading packages
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene

4 ChIP profiling

The datasets CBX6 and CBX7 in this vignettes were downloaded from GEO (GSE40740)5 while ARmo_0M, ARmo_1nM and ARmo_100nM were downloaded from GEO (GSE48308)6 . ChIPseeker provides readPeakFile to load the peak and store in GRanges object.

files <- getSampleFiles()
## $ARmo_0M
## [1] "/tmp/Rtmp0BMK7h/Rinst4b7a39cfad38/ChIPseeker/extdata/GEO_sample_data/GSM1174480_ARmo_0M_peaks.bed.gz"
## $ARmo_1nM
## [1] "/tmp/Rtmp0BMK7h/Rinst4b7a39cfad38/ChIPseeker/extdata/GEO_sample_data/GSM1174481_ARmo_1nM_peaks.bed.gz"
## $ARmo_100nM
## [1] "/tmp/Rtmp0BMK7h/Rinst4b7a39cfad38/ChIPseeker/extdata/GEO_sample_data/GSM1174482_ARmo_100nM_peaks.bed.gz"
## $CBX6_BF
## [1] "/tmp/Rtmp0BMK7h/Rinst4b7a39cfad38/ChIPseeker/extdata/GEO_sample_data/GSM1295076_CBX6_BF_ChipSeq_mergedReps_peaks.bed.gz"
## $CBX7_BF
## [1] "/tmp/Rtmp0BMK7h/Rinst4b7a39cfad38/ChIPseeker/extdata/GEO_sample_data/GSM1295077_CBX7_BF_ChipSeq_mergedReps_peaks.bed.gz"
peak <- readPeakFile(files[[4]])
## GRanges object with 1331 ranges and 2 metadata columns:
##          seqnames                 ranges strand |             V4        V5
##             <Rle>              <IRanges>  <Rle> |       <factor> <numeric>
##      [1]     chr1     [ 815093,  817883]      * |    MACS_peak_1    295.76
##      [2]     chr1     [1243288, 1244338]      * |    MACS_peak_2     63.19
##      [3]     chr1     [2979977, 2981228]      * |    MACS_peak_3    100.16
##      [4]     chr1     [3566182, 3567876]      * |    MACS_peak_4    558.89
##      [5]     chr1     [3816546, 3818111]      * |    MACS_peak_5     57.57
##      ...      ...                    ...    ... .            ...       ...
##   [1327]     chrX [135244783, 135245821]      * | MACS_peak_1327     55.54
##   [1328]     chrX [139171964, 139173506]      * | MACS_peak_1328    270.19
##   [1329]     chrX [139583954, 139586126]      * | MACS_peak_1329    918.73
##   [1330]     chrX [139592002, 139593238]      * | MACS_peak_1330    210.88
##   [1331]     chrY [ 13845134,  13845777]      * | MACS_peak_1331     58.39
##   -------
##   seqinfo: 24 sequences from an unspecified genome; no seqlengths

4.1 ChIP peaks coverage plot

After peak calling, we would like to know the peak locations over the whole genome, covplot function calculates the coverage of peak regions over chromosomes and generate a figure to visualize. GRangesList is also supported and can be used to compare coverage of multiple bed files.

covplot(peak, weightCol="V5")

covplot(peak, weightCol="V5", chrs=c("chr17", "chr18"), xlim=c(4.5e7, 5e7))

4.2 Profile of ChIP peaks binding to TSS regions

First of all, for calculating the profile of ChIP peaks binding to TSS regions, we should prepare the TSS regions, which are defined as the flanking sequence of the TSS sites. Then align the peaks that are mapping to these regions, and generate the tagMatrix.

## promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
## tagMatrix <- getTagMatrix(peak, windows=promoter)
## to speed up the compilation of this vignettes, we use a precalculated tagMatrix
tagMatrix <- tagMatrixList[[4]]

In the above code, you should notice that tagMatrix is not restricted to TSS regions. The regions can be other types that defined by the user.

4.2.1 Heatmap of ChIP binding to TSS regions

tagHeatmap(tagMatrix, xlim=c(-3000, 3000), color="red")
Heatmap of ChIP peaks binding to TSS regions

Heatmap of ChIP peaks binding to TSS regions

ChIPseeker provide a one step function to generate this figure from bed file. The following function will generate the same figure as above.

peakHeatmap(files[[4]], TxDb=txdb, upstream=3000, downstream=3000, color="red")

4.2.2 Average Profile of ChIP peaks binding to TSS region

plotAvgProf(tagMatrix, xlim=c(-3000, 3000),
            xlab="Genomic Region (5'->3')", ylab = "Read Count Frequency")
Average Profile of ChIP peaks binding to TSS region

Average Profile of ChIP peaks binding to TSS region

The function plotAvgProf2 provide a one step from bed file to average profile plot. The following command will generate the same figure as shown above.

plotAvgProf2(files[[4]], TxDb=txdb, upstream=3000, downstream=3000,
             xlab="Genomic Region (5'->3')", ylab = "Read Count Frequency")

Confidence interval estimated by bootstrap method is also supported for characterizing ChIP binding profiles.

plotAvgProf(tagMatrix, xlim=c(-3000, 3000), conf = 0.95, resample = 1000)

4.3 Profile of ChIP peaks binding to start site of Exon/Intron

Referring to the issue #16, we developed getBioRegion function to support centering all peaks to the start region of Exon/Intron. Users can also create heatmap or average profile of ChIP peaks binding to these regions.

5 Peak Annotation

peakAnno <- annotatePeak(files[[4]], tssRegion=c(-3000, 3000),
                         TxDb=txdb, annoDb="")
## >> loading peak file...               2017-04-24 07:29:30 PM 
## >> preparing features information...      2017-04-24 07:29:30 PM 
## >> identifying nearest features...        2017-04-24 07:29:31 PM 
## >> calculating distance from peak to TSS...   2017-04-24 07:29:32 PM 
## >> assigning genomic annotation...        2017-04-24 07:29:32 PM 
## >> adding gene annotation...          2017-04-24 07:29:47 PM 
## >> assigning chromosome lengths           2017-04-24 07:29:47 PM 
## >> done...                    2017-04-24 07:29:47 PM

Peak Annotation is performed by annotatePeak. User can define TSS (transcription start site) region, by default TSS is defined from -3kb to +3kb. The output of annotatePeak is csAnno instance. ChIPseeker provides as.GRanges to convert csAnno to GRanges instance, and to convert csAnno to data.frame which can be exported to file by write.table.

TxDb object contained transcript-related features of a particular genome. Bioconductor provides several package that containing TxDb object of model organisms with multiple commonly used genome version, for instance TxDb.Hsapiens.UCSC.hg38.knownGene, TxDb.Hsapiens.UCSC.hg19.knownGene for human genome hg38 and hg19, TxDb.Mmusculus.UCSC.mm10.knownGene and TxDb.Mmusculus.UCSC.mm9.knownGene for mouse genome mm10 and mm9, etc. User can also prepare their own TxDb object by retrieving information from UCSC Genome Bioinformatics and BioMart data resources by R function makeTxDbFromBiomart and makeTxDbFromUCSC. TxDb object should be passed for peak annotation.

All the peak information contained in peakfile will be retained in the output of annotatePeak. The position and strand information of nearest genes are reported. The distance from peak to the TSS of its nearest gene is also reported. The genomic region of the peak is reported in annotation column. Since some annotation may overlap, ChIPseeker adopted the following priority in genomic annotation.

Downstream is defined as the downstream of gene end.

ChIPseeker also provides parameter genomicAnnotationPriority for user to prioritize this hierachy.

annotatePeak report detail information when the annotation is Exon or Intron, for instance “Exon (uc002sbe.3/9736, exon 69 of 80)”, means that the peak is overlap with an Exon of transcript uc002sbe.3, and the corresponding Entrez gene ID is 9736 (Transcripts that belong to the same gene ID may differ in splice events), and this overlaped exon is the 69th exon of the 80 exons that this transcript uc002sbe.3 prossess.

Parameter annoDb is optional, if provided, extra columns including SYMBOL, GENENAME, ENSEMBL/ENTREZID will be added. The geneId column in annotation output will be consistent with the geneID in TxDb. If it is ENTREZID, ENSEMBL will be added if annoDb is provided, while if it is ENSEMBL ID, ENTREZID will be added.

5.1 Visualize Genomic Annotation

To annotate the location of a given peak in terms of genomic features, annotatePeak assigns peaks to genomic annotation in “annotation” column of the output, which includes whether a peak is in the TSS, Exon, 5’ UTR, 3’ UTR, Intronic or Intergenic. Many researchers are very interesting in these annotations. TSS region can be defined by user and annotatePeak output in details of which exon/intron of which genes as illustrated in previous section.

Pie and Bar plot are supported to visualize the genomic annotation.

Genomic Annotation by pieplot

Genomic Annotation by pieplot

Genomic Annotation by barplot

Genomic Annotation by barplot

Since some annotation overlap, user may interested to view the full annotation with their overlap, which can be partially resolved by vennpie function.

Genomic Annotation by vennpie

Genomic Annotation by vennpie

We extend UpSetR to view full annotation overlap. User can user upsetplot function.


We can combine vennpie with upsetplot by setting vennpie = TRUE.

upsetplot(peakAnno, vennpie=TRUE)

5.2 Visualize distribution of TF-binding loci relative to TSS

The distance from the peak (binding site) to the TSS of the nearest gene is calculated by annotatePeak and reported in the output. We provide plotDistToTSS to calculate the percentage of binding sites upstream and downstream from the TSS of the nearest genes, and visualize the distribution.

              title="Distribution of transcription factor-binding loci\nrelative to TSS")
Distribution of Binding Sites

Distribution of Binding Sites

6 Functional enrichment analysis

Once we have obtained the annotated nearest genes, we can perform functional enrichment analysis to identify predominant biological themes among these genes by incorporating biological knowledge provided by biological ontologies. For instance, Gene Ontology (GO)7 annotates genes to biological processes, molecular functions, and cellular components in a directed acyclic graph structure, Kyoto Encyclopedia of Genes and Genomes (KEGG)8 annotates genes to pathways, Disease Ontology (DO)9 annotates genes with human disease association, and Reactome10 annotates gene to pathways and reactions.

ChIPseeker also provides a function, seq2gene, for linking genomc regions to genes in a many-to-many mapping. It consider host gene (exon/intron), promoter region and flanking gene from intergenic region that may under control via cis-regulation. This function is designed to link both coding and non-coding genomic regions to coding genes and facilitate functional analysis.

Enrichment analysis is a widely used approach to identify biological themes. I have developed several Bioconductor packages for investigating whether the number of selected genes associated with a particular biological term is larger than expected, including DOSE2 for Disease Ontology, ReactomePA for reactome pathway, clusterProfiler4 for Gene Ontology and KEGG enrichment analysis.


pathway1 <- enrichPathway($geneId)
head(pathway1, 2)
##              ID                                              Description
## 186712   186712                      Regulation of beta-cell development
## 3769402 3769402 Deactivation of the beta-catenin transactivating complex
##         GeneRatio  BgRatio       pvalue     p.adjust       qvalue
## 186712     12/431 33/10281 4.030830e-09 3.313342e-06 3.313342e-06
## 3769402     9/431 43/10281 5.820255e-05 2.392125e-02 2.392125e-02
##                                                                    geneID
## 186712  2494/5080/3651/3175/6928/390874/3642/4821/4825/2255/222546/389692
## 3769402                   607/55553/6662/6657/51176/83595/64321/7088/6658
##         Count
## 186712     12
## 3769402     9
gene <- seq2gene(peak, tssRegion = c(-1000, 1000), flankDistance = 3000, TxDb=txdb)
pathway2 <- enrichPathway(gene)
head(pathway2, 2)
##            ID                            Description GeneRatio  BgRatio
## 186712 186712    Regulation of beta-cell development    10/349 33/10281
## 383280 383280 Nuclear Receptor transcription pathway     9/349 51/10281
##              pvalue     p.adjust       qvalue
## 186712 8.241737e-08 6.379104e-05 6.359151e-05
## 383280 4.654074e-05 1.331545e-02 1.327380e-02
##                                                       geneID Count
## 186712 2494/3651/4821/4825/2255/222546/389692/5080/6928/3642    10
## 383280          2494/5241/2100/4306/7025/7101/2516/2649/9971     9

More information can be found in the vignettes of Bioconductor packages DOSE2, ReactomePA, clusterProfiler4, which also provide several methods to visualize enrichment results. The clusterProfiler4 is designed for comparing and visualizing functional profiles among gene clusters, and can directly applied to compare biological themes at GO, DO, KEGG, Reactome perspective.

7 ChIP peak data set comparison

7.1 Profile of several ChIP peak data binding to TSS region

Function plotAvgProf and tagHeatmap can accept a list of tagMatrix and visualize profile or heatmap among several ChIP experiments, while plotAvgProf2 and peakHeatmap can accept a list of bed files and perform the same task in one step.

7.1.1 Average profiles

## promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
## tagMatrixList <- lapply(files, getTagMatrix, windows=promoter)
## to speed up the compilation of this vigenette, we load a precaculated tagMatrixList
plotAvgProf(tagMatrixList, xlim=c(-3000, 3000))
Average Profiles of ChIP peaks among different experiments

Average Profiles of ChIP peaks among different experiments

plotAvgProf(tagMatrixList, xlim=c(-3000, 3000), conf=0.95,resample=500, facet="row")