Package Vignette for Genomic Interactions: ChIA-PET data
ChIA-PET
Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) is a recent method to study protein-mediated interactions at a genome-wide scale. Like most techniques for studying chromatin interaction it is based on chromosome conformation capture technology. Unlike 3C, 4C and 5C, however, it can detect interactions genome-wide, and includes a ChIP step to purify interactions involving a protein of interest.
The raw data from ChIA-PET is in the form of paired-end reads attached to one of two linker sequences. Reads with chimeric linkers are removed, and the data is aligned to the reference genome. The ChIA-PET tool can then be used to find pairs of regions (“anchors”) which have a significant number of reads mapping between them and therefore represent biologically meaningful chromatin interactions in the sample.
Imports
First we need to load the GenomicInteractions package, and the mm9 reference genome:
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We can then read in our data directly from the output of the ChIA-PET tool. At this stage we can also provide information about the cell type and a description tag for the experiment. The data is taken from Li et al., 2012, published in Cell. They have used antibodies against the initiation form of Pol II, which you would expect to find at active promoters, and we are looking at data from the K562 myelogenous leukemia cell line. The data should therefore give us an insight into the processes which regulate genes that are being actively transcribed.
chiapet.data = system.file("extdata/k562.rep1.cluster.pet3+.txt",
package="GenomicInteractions")
k562.rep1 = makeGenomicInteractionsFromFile(chiapet.data,
type="chiapet.tool",
experiment_name="k562",
description="k562 pol2 8wg16")This loads the data into a GenomicInteractions object,
which consists of two linked GenomicRanges objects
containing the anchors in each interaction, as well as the p-value, FDR
and the number of reads supporting each interaction.
GenomicInteractions Objects
The metadata we have added can easily be accesed, and edited:
## [1] "k562"As can the data from the ChIA-PET experiment:
## [1] 3 562 3 3 3 3## [1] 1.25703e-10 0.00000e+00 1.17148e-06 4.86859e-08 2.76777e-08 3.97019e-08
The two linked GRanges objects can be returned, but not
altered in-place:
## GRanges object with 64565 ranges and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr1 569922-571422 *
## [2] chr1 832761-905482 *
## [3] chr1 839092-842325 *
## [4] chr1 839393-841792 *
## [5] chr1 852731-855234 *
## ... ... ... ...
## [64561] chrX 154432946-154435728 *
## [64562] chrX 154436728-154439876 *
## [64563] chrX 154439789-154442306 *
## [64564] chrX 154459648-154462031 *
## [64565] chrX 154839050-154843949 *
## -------
## seqinfo: 25 sequences from an unspecified genome; no seqlengths## GRanges object with 64565 ranges and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chrM 8342-10675 *
## [2] chr1 838470-920603 *
## [3] chr1 935528-939051 *
## [4] chr1 955081-956755 *
## [5] chr1 933685-937006 *
## ... ... ... ...
## [64561] chrX 154442294-154446983 *
## [64562] chrX 154442540-154445105 *
## [64563] chrX 154448371-154451728 *
## [64564] chrX 154469339-154471852 *
## [64565] chrX 154843728-154848393 *
## -------
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsGenomicInteractions objects can easily handle
interactions detected between chromosomes, known as
trans-chromosomal interactions, since the anchors can be at any
point along the genome. is.trans returns a logical vector;
likewise is.cis is the opposite of this function.
sprintf("Percentage of trans-chromosomal interactions %.2f",
100*sum(is.trans(k562.rep1))/length(k562.rep1))## [1] "Percentage of trans-chromosomal interactions 1.00"The length of each interaction is not stored as metadata, but we can calculate the distance of each interaction using either the inner edge, outer edge or midpoints of the anchors. This is undefined for inter-chromosomal interactions, so NA is returned, so it is important to exclude these interactions from some analyses.
## [1] NA 10415 96581 115325 81363 79098GenomicRanges objects can be subsetted by either integer
or logical vectors like most R objects, and also BioConductor
Rle objects.
## GenomicInteractions object with 10 interactions and 3 metadata columns:
## seqnames1 ranges1 seqnames2 ranges2 | counts
## <Rle> <IRanges> <Rle> <IRanges> | <integer>
## [1] chr1 569922-571422 --- chrM 8342-10675 | 3
## [2] chr1 832761-905482 --- chr1 838470-920603 | 562
## [3] chr1 839092-842325 --- chr1 935528-939051 | 3
## [4] chr1 839393-841792 --- chr1 955081-956755 | 3
## [5] chr1 852731-855234 --- chr1 933685-937006 | 3
## [6] chr1 855856-858861 --- chr1 935669-937245 | 3
## [7] chr1 874165-879175 --- chr1 933340-938306 | 10
## [8] chr1 874190-877867 --- chr1 955674-959630 | 5
## [9] chr1 889676-896594 --- chr1 933897-938982 | 13
## [10] chr1 898753-907581 --- chr1 931133-939571 | 19
## p.value fdr
## <numeric> <numeric>
## [1] 1.62140e-12 1.25703e-10
## [2] 0.00000e+00 0.00000e+00
## [3] 4.21364e-08 1.17148e-06
## [4] 1.45938e-09 4.86859e-08
## [5] 7.85539e-10 2.76777e-08
## [6] 1.16802e-09 3.97019e-08
## [7] 1.23139e-25 3.58932e-23
## [8] 6.63691e-15 6.98795e-13
## [9] 4.91311e-36 2.33753e-33
## [10] 0.00000e+00 0.00000e+00
## -------
## regions: 129090 ranges and 0 metadata columns
## seqinfo: 25 sequences from an unspecified genome; no seqlengths## GenomicInteractions object with 100 interactions and 3 metadata columns:
## seqnames1 ranges1 seqnames2 ranges2 |
## <Rle> <IRanges> <Rle> <IRanges> |
## [1] chr17 81061878-81063593 --- chr17 81069140-81071677 |
## [2] chr12 92471123-92476371 --- chr12 92536058-92541905 |
## [3] chr12 94133179-94138581 --- chr12 94148748-94155117 |
## [4] chr1 16159386-16162714 --- chr1 16200021-16202543 |
## [5] chr8 30565559-30568584 --- chr8 30571442-30575013 |
## ... ... ... ... ... ... .
## [96] chr18 9142518-9147121 --- chr18 9149107-9151760 |
## [97] chr21 36305948-36310375 --- chr21 36321522-36324993 |
## [98] chr3 45731256-45733326 --- chr3 45742787-45744962 |
## [99] chr3 152540253-152542519 --- chr3 152551245-152555374 |
## [100] chr7 106682031-106689663 --- chr7 106807150-106811711 |
## counts p.value fdr
## <integer> <numeric> <numeric>
## [1] 3 4.73944e-15 5.10674e-13
## [2] 9 2.09928e-28 7.14475e-26
## [3] 7 4.61213e-25 1.29710e-22
## [4] 3 9.49998e-10 3.28555e-08
## [5] 3 3.11837e-15 3.46306e-13
## ... ... ... ...
## [96] 6 8.43027e-26 2.47951e-23
## [97] 4 2.82464e-17 4.17786e-15
## [98] 3 2.47586e-13 2.12956e-11
## [99] 4 1.50403e-16 2.04946e-14
## [100] 11 9.02628e-30 3.27619e-27
## -------
## regions: 129090 ranges and 0 metadata columns
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsThe length of each interaction is not stored as metadata, but we can
calculate the distance of each interaction using either the inner edge,
outer edge or midpoints of the anchors. Since this is undefinable for
trans-chromosomal interactions it is best to first subset only
cis interactions before calling
calculateDistances, otherwise NAs will be
present in the returned vector.
## [1] 10415 96581 115325 81363 79098 59153k562.short = k562.cis[calculateDistances(k562.cis) < 1e6] # subset shorter interactions
hist(calculateDistances(k562.short)) 
We can also subset based on the properties of the linked
GRanges objects.
Annotation
Genomic Interaction data is often used to look at the interactions between different elements in the genome, which are believed to have different functional roles. Interactions between promoters and their transcription termination sites, for example, are thought to be a by-product of the transcription process, whereas long-range interactions with enhancers play a role in gene regulation.
Since GenomicInteractions is based on
GenomicRanges, it is very easy to interrogate
GenomicInteractions objects using
GenomicRanges data. In the example, we want to annotate
interactions that overlap the promoters, transcription termination sites
or the body of any gene. Since this can be a time-consuming and
data-heavy process, this example runs the analysis for only chromosomes
17 & 18.
First we need the list of RefSeq transcripts:
library(GenomicFeatures)
hg19.refseq.db <- makeTxDbFromUCSC(genome="hg19", table="refGene")
refseq.genes = genes(hg19.refseq.db)
refseq.transcripts = transcriptsBy(hg19.refseq.db, by="gene")
non_pseudogene = names(refseq.transcripts) %in% unlist(refseq.genes$gene_id)
refseq.transcripts = refseq.transcripts[non_pseudogene] Rather than downloading the whole Refseq database, these are provided for chromosomes 17 & 18:
We can then use functions from GenomicRanges to call
promoters and terminators for these transcripts. We have taken promoter
regions to be within 2.5kb of an annotated TSS and terminators to be
within 1kb of the end of an annotated transcript. Since genes can have
multiple transcripts, they can also have multiple promoters/terminators,
so these are GRangesList objects, which makes handling
these objects slightly more complicated.
refseq.promoters = promoters(refseq.transcripts, upstream=2500, downstream=2500)
# unlist object so "strand" is one vector
refseq.transcripts.ul = unlist(refseq.transcripts)
# terminators can be called as promoters with the strand reversed
strand(refseq.transcripts.ul) = ifelse(strand(refseq.transcripts.ul) == "+", "-", "+")
refseq.terminators.ul = promoters(refseq.transcripts.ul, upstream=1000, downstream=1000)
# change back to original strand
strand(refseq.terminators.ul) = ifelse(strand(refseq.terminators.ul) == "+", "-", "+")
# `relist' maintains the original names and structure of the list
refseq.terminators = relist(refseq.terminators.ul, refseq.transcripts)These can be used to subset a GenomicInteractions object
directly from GRanges using the GenomicRanges
overlaps methods. findOverlaps called on a
GenomicInteractions object will return a list containing
Hits objects for both anchors.
We can finds any interactions involving a RefSeq promoter:
## GenomicInteractions object with 2907 interactions and 3 metadata columns:
## seqnames1 ranges1 seqnames2 ranges2 |
## <Rle> <IRanges> <Rle> <IRanges> |
## [1] chr17 616579-621961 --- chr17 620668-626263 |
## [2] chr17 632527-638035 --- chr17 636589-641349 |
## [3] chr17 634119-651606 --- chr17 642299-659172 |
## [4] chr17 654892-657597 --- chr17 683191-687275 |
## [5] chr17 656002-658841 --- chr17 679595-682692 |
## ... ... ... ... ... ... .
## [2903] chr18 77781151-77783476 --- chr18 77792968-77795855 |
## [2904] chr18 77784590-77787797 --- chr18 77792148-77795822 |
## [2905] chr18 77792093-77797983 --- chr18 77797455-77803127 |
## [2906] chr18 77793365-77797939 --- chr18 77864889-77868321 |
## [2907] chr18 77863992-77870413 --- chr18 77868294-77877151 |
## counts p.value fdr
## <integer> <numeric> <numeric>
## [1] 7 2.06358e-32 8.50563e-30
## [2] 9 2.39500e-36 1.15180e-33
## [3] 55 0.00000e+00 0.00000e+00
## [4] 6 4.86283e-24 1.27856e-21
## [5] 3 6.23098e-14 5.72161e-12
## ... ... ... ...
## [2903] 3 8.97548e-14 8.09366e-12
## [2904] 6 4.16341e-26 1.24576e-23
## [2905] 13 0.00000e+00 0.00000e+00
## [2906] 8 6.61618e-30 2.41717e-27
## [2907] 18 0.00000e+00 0.00000e+00
## -------
## regions: 129090 ranges and 0 metadata columns
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsHowever, one of the most powerful features in the
GenomicInteractions package is the ability to annotate each
anchor with a list of genomic regions and then summarise interactions
according to these features. This annotation is implemented as metadata
columns for the anchors in the GenomicInteractions object
and so is fast, and facilitates more complex analyses.
The order in which we annotate the anchors is important, since each
anchor can only have one node.class. The first listed take
precedence. Any regions not overlapping ranges in
annotation.features will be labelled as
distal.
annotation.features = list(promoter=refseq.promoters,
terminator=refseq.terminators,
gene.body=refseq.transcripts)
annotateInteractions(k562.rep1, annotation.features)## Annotating with promoter ...## Annotating with terminator ...## Annotating with gene.body ...## [1] "distal" "gene.body" "promoter" "terminator"We can now find interactions involving promoters using the annotated
node.class for each anchor:
p.one = anchorOne(k562.rep1)$node.class == "promoter"
p.two = anchorTwo(k562.rep1)$node.class == "promoter"
k562.rep1[p.one|p.two]## GenomicInteractions object with 2907 interactions and 3 metadata columns:
## seqnames1 ranges1 seqnames2 ranges2 |
## <Rle> <IRanges> <Rle> <IRanges> |
## [1] chr17 616579-621961 --- chr17 620668-626263 |
## [2] chr17 632527-638035 --- chr17 636589-641349 |
## [3] chr17 634119-651606 --- chr17 642299-659172 |
## [4] chr17 654892-657597 --- chr17 683191-687275 |
## [5] chr17 656002-658841 --- chr17 679595-682692 |
## ... ... ... ... ... ... .
## [2903] chr18 77781151-77783476 --- chr18 77792968-77795855 |
## [2904] chr18 77784590-77787797 --- chr18 77792148-77795822 |
## [2905] chr18 77792093-77797983 --- chr18 77797455-77803127 |
## [2906] chr18 77793365-77797939 --- chr18 77864889-77868321 |
## [2907] chr18 77863992-77870413 --- chr18 77868294-77877151 |
## counts p.value fdr
## <integer> <numeric> <numeric>
## [1] 7 2.06358e-32 8.50563e-30
## [2] 9 2.39500e-36 1.15180e-33
## [3] 55 0.00000e+00 0.00000e+00
## [4] 6 4.86283e-24 1.27856e-21
## [5] 3 6.23098e-14 5.72161e-12
## ... ... ... ...
## [2903] 3 8.97548e-14 8.09366e-12
## [2904] 6 4.16341e-26 1.24576e-23
## [2905] 13 0.00000e+00 0.00000e+00
## [2906] 8 6.61618e-30 2.41717e-27
## [2907] 18 0.00000e+00 0.00000e+00
## -------
## regions: 129090 ranges and 4 metadata columns
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsThis information can be used to categorise interactions into
promoter-distal, promoter-terminator etc. A table of interaction types
can be generated with categoriseInteractions:
## category count
## 1 distal-distal 396
## 2 distal-gene.body 76
## 3 distal-promoter 519
## 4 distal-terminator 101
## 5 gene.body-gene.body 795
## 6 gene.body-promoter 917
## 7 gene.body-terminator 164
## 8 promoter-promoter 1187
## 9 promoter-terminator 284
## 10 terminator-terminator 70Alternatively, we can subset the object based on interaction type:
## GenomicInteractions object with 164 interactions and 3 metadata columns:
## seqnames1 ranges1 seqnames2 ranges2 | counts
## <Rle> <IRanges> <Rle> <IRanges> | <integer>
## [1] chr17 1471460-1474306 --- chr17 1476212-1479585 | 4
## [2] chr17 1632603-1638741 --- chr17 1636657-1642967 | 12
## [3] chr17 3845975-3849573 --- chr17 3908008-3910817 | 5
## [4] chr17 4055645-4058706 --- chr17 4063341-4068158 | 4
## [5] chr17 4443889-4451615 --- chr17 4446814-4454998 | 11
## ... ... ... ... ... ... . ...
## [160] chr18 32873043-32876330 --- chr18 32911004-32914907 | 4
## [161] chr18 33566139-33569380 --- chr18 33571111-33574360 | 3
## [162] chr18 33689982-33692774 --- chr18 33695495-33699363 | 4
## [163] chr18 42638745-42641928 --- chr18 42647110-42649707 | 3
## [164] chr18 77914125-77916781 --- chr18 77919050-77922761 | 3
## p.value fdr
## <numeric> <numeric>
## [1] 1.14380e-18 1.92712e-16
## [2] 0.00000e+00 4.06377e-44
## [3] 1.46527e-19 2.69952e-17
## [4] 4.96495e-19 8.66918e-17
## [5] 7.22930e-42 4.17953e-39
## ... ... ...
## [160] 1.37802e-15 1.62547e-13
## [161] 5.37957e-15 5.74333e-13
## [162] 1.35703e-19 2.50960e-17
## [163] 1.03659e-15 1.24713e-13
## [164] 2.90970e-15 3.24878e-13
## -------
## regions: 129090 ranges and 4 metadata columns
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsThe 3 most common node.class values have short functions
defined for convenience (see ?is.pp for a complete
list):
k562.rep1[is.pp(k562.rep1)] # promoter-promoter interactions
k562.rep1[is.dd(k562.rep1)] # distal-distal interactions
k562.rep1[is.pt(k562.rep1)] # promoter-terminator interactionsSummary plots of interactions classes can easily be produced to get an overall feel for the data:
viewpoints will only take those interactions with a
certain node.class:
These are also combined in the function
plotSummaryStats.
Feature Summaries
The summariseByFeatures allows us to look in more detail
at interactions involving a specific set of loci. In this example we use
all RefSeq promoters, which we already have loaded in a
GRangesList object.
It is however possible to use any dataset which can be represented as
a named GRanges object, for example transcription-factor
ChIP data, predicted cis-regulatory sites or certain categories of
genes.
The categories are generated automatically from the annotated
node.class values in the object.
k562.rep1.promoter.annotation = summariseByFeatures(k562.rep1, refseq.promoters,
"promoter", distance.method="midpoint",
annotate.self=TRUE)
colnames(k562.rep1.promoter.annotation)## [1] "Promoter.id"
## [2] "numberOfPromoterInteractions"
## [3] "numberOfPromoterUniqueInteractions"
## [4] "numberOfPromoterInterChromosomalInteractions"
## [5] "numberOfPromoterUniqueInterChromosomalInteractions"
## [6] "numberOfPromoterDistalInteractions"
## [7] "numberOfPromoterGene.bodyInteractions"
## [8] "numberOfPromoterPromoterInteractions"
## [9] "numberOfPromoterTerminatorInteractions"
## [10] "numberOfUniquePromoterDistalInteractions"
## [11] "numberOfUniquePromoterGene.bodyInteractions"
## [12] "numberOfUniquePromoterPromoterInteractions"
## [13] "numberOfUniquePromoterTerminatorInteractions"
## [14] "PromoterDistanceMedian"
## [15] "PromoterDistanceMean"
## [16] "PromoterDistanceMinimum"
## [17] "PromoterDistanceMaximum"
## [18] "PromoterDistanceWeightedMedian"
## [19] "numberOfSelfPromoterGene.bodyInteractions"
## [20] "numberOfSelfPromoterPromoterInteractions"
## [21] "numberOfSelfPromoterTerminatorInteractions"
## [22] "numberOfSelfUniquePromoterGene.bodyInteractions"
## [23] "numberOfSelfUniquePromoterPromoterInteractions"
## [24] "numberOfSelfUniquePromoterTerminatorInteractions"This allows us to very quickly generate summaries of the data and
provides a quick method to isolate genes of interest. In this case we
produce lists of RefSeq IDs, which can easily be converted to EntrezIDs
or gene symbols through existing BioConductor packages (in this case
org.Hs.eg.db provides bimaps between common human genome
annotations).
Which promoters have the strongest Promoter-Promoter interactions based on PET-counts?
i = order(k562.rep1.promoter.annotation$numberOfPromoterPromoterInteractions,
decreasing=TRUE)[1:10]
k562.rep1.promoter.annotation[i,"Promoter.id"]## [1] "100506779" "9256" "406934" "54894" "100616220" "6827"
## [7] "56155" "5889" "5034" "396"Which promoters are contacting the largest number of distal elements?
i = order(k562.rep1.promoter.annotation$numberOfUniquePromoterDistalInteractions,
decreasing=TRUE)[1:10]
k562.rep1.promoter.annotation[i,"Promoter.id"]## [1] "10140" "400604" "7050" "100130581" "100616277" "26118"
## [7] "100874261" "101927666" "140735" "5366"What percentage of promoters are in contact with transcription termination sites?
total = sum(k562.rep1.promoter.annotation$numberOfPromoterTerminatorInteractions > 0)
sprintf("%.2f%% of promoters have P-T interactions", 100*total/nrow(k562.rep1.promoter.annotation))## [1] "16.43% of promoters have P-T interactions"References
Li, Guoliang, et al. “Software ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing.” Genome Biol 11 (2010): R22.
Li, Guoliang, et al. “Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation.” Cell 148.1 (2012): 84-98