Astrid Louise Deschenes, Elsa Bernatchez, Charles Joly Beauparlant, Fabien Claude Lamaze, Rawane Samb, Pascal Belleau and Arnaud Droit.
This package and the underlying similaRpeak code are distributed under the Artistic license 2.0. You are free to use and redistribute this software.
The similaRpeak package calculates metrics to estimate the level of similarity between two ChIP-Seq profiles.
The metrics are:
RATIO_AREA: The ratio between the profile areas.
The first profile is always divided by the second profile. NA is returned
if minimal area threshold is not respected for at least one of the profiles.
DIFF_POS_MAX: The difference between the maximal peaks positions. The
difference is always the first profile value minus the second profile value.
NA is returned if minimal peak value is not respected. A profile can have
more than one position with the maximum value. In that case, the median
position is used. A threshold argument can be set to consider all positions
within a certain range of the maximum value. A threshold argument can also be
set to ensure that the distance between two maximum values is not too wide.
When this distance is not respected, it is assumed that more than one peak
is present in the profile and NA is returned.
RATIO_MAX_MAX: The ratio between the peaks values
in each profile. The first profile is always divided by the second profile.
NA if minimal peak value threshold is not respected for at least one of the
profiles.
RATIO_INTERSECT: The ratio between the intersection area and the total
area of the two profiles. NA if minimal area threshold is not respected for
the intersection area.
RATIO_NORMALIZED_INTERSECT: The ratio between the intersection area and
the total area of two normalized profiles. The profiles are normalized by
dividing them by their average value. NA if minimal area threshold is not
respected for the intersection area.
SPEARMAN_CORRELATION: The Spearman's rho statistic between profiles.
suppressMessages(library(similaRpeak))
ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. For a specific region, the read count of aligned sequences at each position of the region is used to generate the ChIP-Seq profile for the region.
To estimate the level of similarity between two ChIP-Seq profiles for a
specific region, two vector containing the profiles values, as reported in
reads per million (RPM) for each position of the selected region, are needed.
Both vector should have the same length and should not contain any negative
value.
Aligned sequences are usually stored in BAM files. As example, a slimmed BAM
file (align1.bam) is selected as well as a specific region
(chr1:172081530-172083529). Using BAM file and
region information, represented by as a GRanges object, the coverage for the
specified region is extracted using specialized Bioconductor packages.
suppressMessages(library(GenomicAlignments))
suppressMessages(library(rtracklayer))
suppressMessages(library(Rsamtools))
bamFile01 <- system.file("extdata/align1.bam", package = "similaRpeak")
region <- GRanges(Rle(c("chr1")), IRanges(start = 172081530, end = 172083529),
strand= Rle(strand(c("*"))))
param <- ScanBamParam(which = region)
alignments01 <- readGAlignments(bamFile01, param = param)
coveragesRegion01 <- coverage(alignments01)[region]
coveragesRegion01
## RleList of length 1
## $chr1
## integer-Rle of length 2000 with 92 runs
## Lengths: 297 30 2 4 14 18 19 36 ... 21 15 35 36 65 36 307
## Values : 0 1 2 3 2 1 0 1 ... 2 1 0 1 0 2 0
The coverages01 can
easily be transformed to a vector of numerical value to obtain the raw
ChIP-Seq profile for the selected region.
coveragesRegion01 <- as.numeric(coveragesRegion01$chr1)
length(coveragesRegion01)
## [1] 2000
summary(coveragesRegion01)
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 0.000 0.000 0.000 0.907 1.000 6.000
To convert the raw coverage to reads per million (RPM), the total number of
reads present in the BAM file is needed to assign a weight at the coverage
function.
param <- ScanBamParam(flag = scanBamFlag(isUnmappedQuery=FALSE))
count01 <- countBam(bamFile01, param = param)$records
coveragesRPMRegion01 <- coverage(alignments01, weight=1000000/count01)[region]
coveragesRPMRegion01 <- as.numeric(coveragesRPMRegion01$chr1)
length(coveragesRPMRegion01)
## [1] 2000
summary(coveragesRPMRegion01)
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 0.0 0.0 0.0 195.7 215.7 1294.0
The read per millions values are quite low for the coveragesRPMRegion01
because the original BAM file has been reduced in size to simplify the example.
Other examples are available on the worflows section of the Bioconductor website.
Finally, the metagene package , available on Bioconductor, can also be used to generate ChIP-Seq profiles. An example is available on metagene wiki.
Mathematically, a metric is considered as a function that quantifies the similarity between two objects.The function must return zero when the two objects are perfectly similar (identity of indiscernibles) and a non-negative value when are dissimilar.
The metrics present in the similaRpeak package do not strictly respect this standard but can all be translated to pseudometrics. A pseudometric is a function d which satisfies the axioms for a metric, except that instead of the identity of indiscernibles axiom, the metric must only return zero when it compare an object to itself.
By using the absolute value of the DIFF_POS_MAX metric, the definition of a pseudometric is formally respected. However, the respective position of the maximum peak of profiles is lost.
\[ |DIFF\_POS\_MAX| \]
By using the absolute value of the logarithm of the RATIO_AREA, RATIO_MAX_MAX, RATIO_INTERSECT and RATIO_NORMALIZED_INTERSECT metrics, the definition of a pseudometric is formally respected.
\[ |\log(RATIO)| \]
To ease comparison, the same ChIP-Seq profiles are used in each metric description section. Those are ChIP-Seq profiles of two histone post-transcriptional modifications linked to highly active enhancers H3K27ac (DCC accession: ENCFF000ASG) and H3K4me1 (DCC accession: ENCFF000ARY) from the Encyclopedia of DNA Elements (ENCODE) data (Dunham I et al. 2012).
Here is how to load the demoProfiles data used in following sections. The
ChIP-Seq profiles of the enhancers H3K27ac and H3K4me1 for 4 specifics regions
are in reads per million (RPM).
data(demoProfiles)
str(demoProfiles)
## List of 4
## $ chr2.70360770.70361098 :List of 2
## ..$ H3K27ac: num [1:329] 13 12 12 11 11 11 9 9 7 7 ...
## ..$ H3K4me1: num [1:329] 6 7 7 7 7 8 8 8 8 9 ...
## $ chr3.73159773.73160145 :List of 2
## ..$ H3K27ac: num [1:373] 1 0 0 0 0 0 0 0 0 0 ...
## ..$ H3K4me1: num [1:373] 0 0 0 0 0 0 0 0 0 0 ...
## $ chr8.43092918.43093442 :List of 2
## ..$ H3K27ac: num [1:525] 82 82 81 81 80 80 79 78 78 76 ...
## ..$ H3K4me1: num [1:525] 1404 1398 1392 1383 1367 ...
## $ chr19.27739373.27739767:List of 2
## ..$ H3K27ac: num [1:395] 4 3 3 3 2 2 0 0 0 0 ...
## ..$ H3K4me1: num [1:395] 17 15 13 13 11 6 3 0 0 0 ...
The RATIO_AREA metric is the ratio between the profile areas. The first
profile (profile1 parameter) is always divided by the second profile
(profile2 parameter). NA is returned if minimal area threshold
(ratioAreaThreshold parameter) is not respected for at least one of the
profiles.
The RATIO_AREA metric can be useful to detect regions with similar coverage even if the profiles are different.
metrics <- similarity(demoProfiles$chr2.70360770.70361098$H3K27ac,
demoProfiles$chr2.70360770.70361098$H3K4me1)
metrics$metric$RATIO_AREA
## [1] 1.025942
metrics <- similarity(demoProfiles$chr8.43092918.43093442$H3K27ac,
demoProfiles$chr8.43092918.43093442$H3K4me1)
metrics$metric$RATIO_AREA
## [1] 0.05928535
metrics <- similarity(demoProfiles$chr3.73159773.73160145$H3K27ac,
demoProfiles$chr3.73159773.73160145$H3K4me1)
metrics$metric$RATIO_AREA
## [1] 2.228563
metrics <- similarity(demoProfiles$chr19.27739373.27739767$H3K27ac,
demoProfiles$chr19.27739373.27739767$H3K4me1)
metrics$metric$RATIO_AREA
## [1] 0.1245884
The DIFF_POS_MAX metric is the difference between the maximal peaks
positions. The difference is always the first profile value
(profile1 parameter) minus the second profile value
(profile2 parameter). NA is returned if minimal peak value is not
respected. A profile can have more than one position with the maximum value.
In that case, the median position is used. A threshold (diffPosMaxTolerance
parameter) can be set to consider all positions within a certain range of the
maximum value. A threshold (diffPosMaxThresholdMaxDiff parameter) can also
be set to ensure that the distance between two maximum values is not too wide.
When this distance is not respected, it is assumed that more than one peak is
present in the profile and NA is returned. Finally, a threshold
(diffPosMaxThresholdMinValue parameter) can be set to ensure that the
maximum value is egal or superior to a minimal value. When this minimum value
is not respected, it is assumed that no peak is present in the profile and
NA is returned.
The DIFF_POS_MAX metric can be useful to detect regions with shifted peaks.
metrics <- similarity(demoProfiles$chr2.70360770.70361098$H3K27ac,
demoProfiles$chr2.70360770.70361098$H3K4me1)
metrics$metric$DIFF_POS_MAX
## [1] -20
metrics <- similarity(demoProfiles$chr8.43092918.43093442$H3K27ac,
demoProfiles$chr8.43092918.43093442$H3K4me1)
metrics$metric$DIFF_POS_MAX
## [1] -0.5
metrics <- similarity(demoProfiles$chr3.73159773.73160145$H3K27ac,
demoProfiles$chr3.73159773.73160145$H3K4me1)
metrics$metric$DIFF_POS_MAX
## [1] 2.5
metrics <- similarity(demoProfiles$chr19.27739373.27739767$H3K27ac,
demoProfiles$chr19.27739373.27739767$H3K4me1)
metrics$metric$DIFF_POS_MAX
## [1] 2.5
The RATIO_MAX_MAX metric is the ratio between the peaks values
in each profile. The first profile (profile1 parameter) is always divided by
the second profile (profile2 parameter). NA if minimal peak value threshold
(ratioMaxMaxThreshold parameter) is not respected for at least one of the
profiles.
The RATIO_MAX_MAX metric can be useful to detect regions with peaks with similar (or dissimilar) amplitude.
metrics <- similarity(demoProfiles$chr2.70360770.70361098$H3K27ac,
demoProfiles$chr2.70360770.70361098$H3K4me1)
metrics$metric$RATIO_MAX_MAX
## [1] 0.952381
metrics <- similarity(demoProfiles$chr8.43092918.43093442$H3K27ac,
demoProfiles$chr8.43092918.43093442$H3K4me1)
metrics$metric$RATIO_MAX_MAX
## [1] 0.05840456
metrics <- similarity(demoProfiles$chr3.73159773.73160145$H3K27ac,
demoProfiles$chr3.73159773.73160145$H3K4me1)
metrics$metric$RATIO_MAX_MAX
## [1] 2.5
metrics <- similarity(demoProfiles$chr19.27739373.27739767$H3K27ac,
demoProfiles$chr19.27739373.27739767$H3K4me1)
metrics$metric$RATIO_MAX_MAX
## [1] 0.1210191
The RATIO_INTERSECT metric is the ratio between the intersection area and
the total area of the two profiles. NA if minimal area threshold
(ratioIntersectThreshold parameter) is not respected for the intersection
area.
The RATIO_INTERSECT metric can be useful to detect regions with possibily similar profiles.
metrics <- similarity(demoProfiles$chr2.70360770.70361098$H3K27ac,
demoProfiles$chr2.70360770.70361098$H3K4me1)
metrics$metric$RATIO_INTERSECT
## [1] 0.6307208
metrics <- similarity(demoProfiles$chr8.43092918.43093442$H3K27ac,
demoProfiles$chr8.43092918.43093442$H3K4me1)
metrics$metric$RATIO_INTERSECT
## [1] 0.05926862
metrics <- similarity(demoProfiles$chr3.73159773.73160145$H3K27ac,
demoProfiles$chr3.73159773.73160145$H3K4me1)
metrics$metric$RATIO_INTERSECT
## [1] 0.4276368
metrics <- similarity(demoProfiles$chr19.27739373.27739767$H3K27ac,
demoProfiles$chr19.27739373.27739767$H3K4me1)
metrics$metric$RATIO_INTERSECT
## [1] 0.1231793
The RATIO_NORMALIZED_INTERSECT metric is the ratio between the
intersection area and the total area of the two normalized profiles. The
profiles are normalized by divinding them by their average value (total area
of the profile divided by profile lenght). NA if minimal area threshold
(ratioNormalizedIntersectThreshold parameter) is not respected for
the intersection area.
The RATIO_NORMALIZED_INTERSECT metric can be useful to detect regions with possibily similar profiles even if their have different amplitude.
metrics <- similarity(demoProfiles$chr2.70360770.70361098$H3K27ac,
demoProfiles$chr2.70360770.70361098$H3K4me1)
metrics$metric$RATIO_NORMALIZED_INTERSECT
## [1] 0.6316846
metrics <- similarity(demoProfiles$chr8.43092918.43093442$H3K27ac,
demoProfiles$chr8.43092918.43093442$H3K4me1)
metrics$metric$RATIO_NORMALIZED_INTERSECT
## [1] 0.8914731
metrics <- similarity(demoProfiles$chr3.73159773.73160145$H3K27ac,
demoProfiles$chr3.73159773.73160145$H3K4me1)
metrics$metric$RATIO_NORMALIZED_INTERSECT
## [1] 0.7832524
metrics <- similarity(demoProfiles$chr19.27739373.27739767$H3K27ac,
demoProfiles$chr19.27739373.27739767$H3K4me1)
metrics$metric$RATIO_NORMALIZED_INTERSECT
## [1] 0.8439258
The SPEARMAN_CORRELATION metric is the Spearman's rho statistic calculated
usign the two profiles.NA if minimal standard deviation
(spearmanCorrSDThreashold parameter) is not respected for at least one of the
profiles.
The SPEARMAN_CORRELATION assesses how well the relationship between the two ChIP-Seq profiles can be described using a monotonic function. As the RATIO_NORMALIZED_INTERSECT metric, the SPEARMAN_CORRELATION metric can be useful to detect regions with possibily similar profiles even if their have different amplitude.
metrics <- similarity(demoProfiles$chr2.70360770.70361098$H3K27ac,
demoProfiles$chr2.70360770.70361098$H3K4me1)
metrics$metric$SPEARMAN_CORRELATION
## [1] 0.05945987
metrics <- similarity(demoProfiles$chr8.43092918.43093442$H3K27ac,
demoProfiles$chr8.43092918.43093442$H3K4me1)
metrics$metric$SPEARMAN_CORRELATION
## [1] 0.819497
metrics <- similarity(demoProfiles$chr3.73159773.73160145$H3K27ac,
demoProfiles$chr3.73159773.73160145$H3K4me1)
metrics$metric$SPEARMAN_CORRELATION
## [1] 0.9515006
metrics <- similarity(demoProfiles$chr19.27739373.27739767$H3K27ac,
demoProfiles$chr19.27739373.27739767$H3K4me1)
metrics$metric$SPEARMAN_CORRELATION
## [1] 0.6164638
Highly active enhancer regions are thought to be important for the cell fate (Andersson et al. 2014, FANTOM5 consortium, Hnisz et al. 2013). Highly active enhancers regions have been selected in GM12878 cells. Similarity of ChIP-seq profiles has been tested using two histone post-transcriptional modifications linked to highly active enhancers H3K27ac (DCC accession: ENCFF000ASG) and H3K4me1 (DCC accession: ENCFF000ARY) from the Encyclopedia of DNA Elements (ENCODE) data (Dunham I et al. 2012). Accordingly with the literature, similarity between the profiles of these two histone marks has been identified.
First, the similaRpeak package must be loaded.
suppressMessages(library(similaRpeak))
A region, chr7:61968807-61969730, shows interesting profiles for both histones. Let's load the data for this region.
data(chr7Profiles)
str(chr7Profiles)
## List of 1
## $ chr7.61968807.61969730:List of 2
## ..$ H3K27ac: num [1:924] 31 31 31 31 30 29 27 24 24 26 ...
## ..$ H3K4me1: num [1:924] 319 317 310 300 284 274 259 257 258 253 ...
H3K27ac and H3K4me1 profiles have those shapes:
The metrics are calculated using the similarity function which takes as
arguments the two ChIP-Seq profiles vectors and the threshold values.
metrics <- similarity(chr7Profiles$chr7.61968807.61969730$H3K27ac,
chr7Profiles$chr7.61968807.61969730$H3K4me1,
ratioAreaThreshold=5,
ratioMaxMaxThreshold=2,
ratioIntersectThreshold=5,
ratioNormalizedIntersectThreshold=2,
diffPosMaxThresholdMinValue=10,
diffPosMaxThresholdMaxDiff=100,
diffPosMaxTolerance=0.01)
The similarity function returns a list which contains the general
information about both ChIP-Seq profiles and a list of all calculated metrics.
metrics
## $nbrPosition
## [1] 924
##
## $areaProfile1
## [1] 22140
##
## $areaProfile2
## [1] 182991
##
## $maxProfile1
## [1] 77
##
## $maxProfile2
## [1] 646
##
## $maxPositionProfile1
## [1] 557
##
## $maxPositionProfile2
## [1] 657
##
## $metrics
## $metrics$RATIO_AREA
## [1] 0.1209896
##
## $metrics$DIFF_POS_MAX
## [1] -49.5
##
## $metrics$RATIO_MAX_MAX
## [1] 0.119195
##
## $metrics$RATIO_INTERSECT
## [1] 0.1208916
##
## $metrics$RATIO_NORMALIZED_INTERSECT
## [1] 0.8478406
##
## $metrics$SPEARMAN_CORRELATION
## [1] 0.9689683
Each specific information can be directly accessed. Some examples:
metrics$areaProfile1
## [1] 22140
metrics$areaProfile2
## [1] 182991
metrics$metrics$RATIO_INTERSECT
## [1] 0.1208916
The RATIO_INTERSECT value of 0.12 and the RATIO_MAX_MAX value of 0.12 are quite low. Both values can be explained by the large difference in coverage between profiles. Those values could be interpreted as two profiles with low level of similarity. However, the RATIO_NORMALIZED_INTERSECT of 0.85 is much closer to 1. It could be a sign that the profiles, once normalized, are quite similar. This hypothesis can be validated by looking at a graph of the normalized profiles :
It is possible to create only one selected metric by using the MetricFactory object (with the possibility of specifying the thresholds) and by passing the name of the metric to create (RATIO_AREA, DIFF_POS_MAX, RATIO_MAX_MAX, RATIO_INTERSECT or RATIO_NORMALIZED_INTERSECT):
factory = MetricFactory$new(diffPosMaxTolerance=0.04)
The factory has to be iniatized only once and can be used as many times as necessary. It can calculate the same metrics but with different profiles or different metrics with same profiles as long as the thresholds values remain the same:
ratio_max_max <- factory$createMetric(metricType="RATIO_MAX_MAX",
profile1=demoProfiles$chr2.70360770.70361098$H3K27ac,
profile2=demoProfiles$chr2.70360770.70361098$H3K4me1)
ratio_max_max
## $RATIO_MAX_MAX
## [1] 0.952381
ratio_normalized_intersect <- factory$createMetric(
metricType="RATIO_NORMALIZED_INTERSECT",
profile1=demoProfiles$chr2.70360770.70361098$H3K27ac,
profile2=demoProfiles$chr2.70360770.70361098$H3K4me1)
ratio_normalized_intersect
## $RATIO_NORMALIZED_INTERSECT
## [1] 0.6316846
Andersson R, Gebhard C, Miguel-Escalada I, Hoof I, Bornholdt J, et al. (2014) An atlas of active enhancers across human cell types and tissues. Nature, 507(7493), 455-461.
Dunham I, Kundaje A, Aldred SF, et al. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012 Sep 6;489(7414):57-74.
Forrest AR, Kawaji H, Rehli M, Baillie JK, de Hoon MJ, et al. (2014) A promoter-level mammalian expression atlas. Nature, 507(7493):462-470.
Hnisz D, Abraham BJ, Lee TI, Lau A, Saint-André V, et al. (2013) Super-enhancers in the control of cell identity and disease. Cell, 155(4), 934-947.