---
title: "Detection of consensus regions inside a group of experiments using genomic positions and genomic ranges"
author: Astrid Deschenes, Fabien Claude Lamaze, Pascal Belleau and Arnaud Droit
date: '`r format(Sys.time(), "%d %B, %Y")`'
output:
BiocStyle::html_document:
toc: true
bibliography: consensusSeekeR.bibtex
vignette: >
%\VignetteIndexEntry{Detection of consensus regions inside a group of experiences using genomic positions and genomic ranges}
%\VignetteEngine{knitr::rmarkdown}
%\VignettePackage{consensusSeekeR}
%\VignetteEncoding{UTF-8}
---
```{r style, echo = FALSE, results = 'asis', message=FALSE}
BiocStyle::markdown()
library(knitr)
```
<br />
# Licensing and citing
This package and the underlying `r Rpackage("consensusSeekeR")` code are
distributed under the Artistic license 2.0. You are free to use and
redistribute this software.
If you use this package for a publication, we would ask you to cite
the following:
>Samb R, Khadraoui K, Belleau P, et al. (2015) Using informative Multinomial-Dirichlet prior in a t-mixture with reversible jump estimation of nucleosome positions for genome-wide profiling. Statistical Applications in Genetics and Molecular Biology. Published online before print December 10, 2015. <a href="http://dx.doi.org/10.1515/sagmb-2014-0098">doi:10.1515/sagmb-2014-0098</a>
# Introduction
Genome data, such as genes, nucleosomes or
single-nucleotide polymorphisms (SNPs) are linked to the genome by
occupying either a range of
positions or a single position on the sequence. Genomic related data
integration is made possible by treating the data as
ranges on the genome [@Lawrence2013].
Bioconductor has developed an infrastructure, including packages such as
`r Biocpkg("GenomicRanges")`, `r Biocpkg("IRanges")` and
`r Biocpkg("GenomicFeatures")`, which facilitate
the integrative statistical analysis of range-based genomic data.
Ranges format is a convenient way for the analysis of diffent experimental
genomic data. As
an example, the peak calling step, in the analysis of ChIP-seq data, commonly
generates NarrowPeak outputs. The NarrowPeak format, wich is used by the
ENCODE project [@Dunham2012a],
includes a peak position located inside
a genomic range.
In genomic analysis where feature identification generate a position value
surrounded by a genomic range, such as ChIP-Seq peaks and nucleosome postions,
the replication of an experiment may result in slight differences between
predicted values. Conciliation of the results can be difficult, especially
when many replicates are done. One current approach used to identify
consensus regions in a group of
results consist of extracting the overlapping regions of the genomic
ranges. This approach,
when used on a large number of experiments, can miss, as a side effect,
regions when one of the experiment has missing or slightly shift
features. On the
other hand, the use of the union of the regions can result in wide
consensus ranges.
As an example, shown using Integrative genomics viewer [@Interests2011], of
two ChIP-Seq peaks from [ENCODE](https://www.encodeproject.org)
for the FOSL2 transcription
factor (DCC accession: ENCFF002CFN). The data have been analyzed using
MACS2 [@Zhang2008]
with the default parameters and the q-value set to 0.05. The ChIP-Seq peak
is a genomic feature
that can be defined by a position value (the peak position) and a genome
range (the enriched region). This example shows that the peak position is not
necessarily at the center of the enriched region.
The `r Rpackage("consensusSeekeR")` package implements a novative way to
identify consensus which use the features positions, instead of the most
commonly used genomic ranges.
# The consensusSeekeR package
The `r Rpackage("consensusSeekeR")` package implements a novative way to
identify consensus
ranges in a group of experiments which generated position values
surrounded by genomic ranges. The `r Rpackage("consensusSeekeR")` package is
characterized by its use of the position values, instead of the genomic
ranges, to identify the consensus regions. The positions values have the
double advantages of being, most of the time,
the most important information from features and allowing creation of
consensius regions of smaller ranges.
Using iterative steps on ordered features position values from all
experiments, a window of fixed size (specified by user) with the
current feature position
as starting point is set. All
features which reside inside the window are gathered to calculate a
median feature position which is then used to recreate a new window. This
time, the
new window has twice the size fixed by user and its center is the median
feature position. An
update of the features located inside the window is done and the
median feature position is recalculated. This step is repeated up to the
moment that
the set of features remains identical between two iterations.
The final set of features positions is used to fix
the central position of the consensus region. This final
region must respect the minimum number of experiments with at least one
feature inside it to be retained as a final consensus region. The minimum
number of experiments is set by the user. At last, the consensus region can be
extended or/and shrinked to fit the regions associated to the position values
present inside. If new features positions are added during the consensus
region resizing, the iterative steps are not reprocessed. It is possible
that the extension step adds new features in the extended consensus region.
However, those new features ranges won't be taken into account during the
extension step.
# Loading consensusSeekeR package
As with any R package, the `r Rpackage("consensusSeekeR")` package should
first be loaded with the following command:
```{r loadingPackage, warning=FALSE, message=FALSE}
library(consensusSeekeR)
```
# Inputs
## Positions and Ranges
The main function of the `r Rpackage("consensusSeekeR")` is
`findConsensusPeakRegions`. The mains inputs of the `findConsensusPeakRegions`
function are:
1. a `GRanges` of the feature `positions` of all experiments with a
metadata field called `name`.
2. a `GRanges` of the feature `ranges` for all experiments with a metadata
field called `name`.
Beware that the `GRanges` of the feature `ranges` is only mandatory if the
`expandToFitPeakRegion` parameter and/or the `shrinkToFitPeakRegion` parameter
are set to `TRUE`.
Both inputs must satify these conditions:
* All rows of each `GRanges` must be named after the experiment source. All
entries from the same experiment must be assigned the same name.
* Each feature must have one entry in both `GRanges`. The metadata
field `name` is used to associate the feature position to its range.
This is an example showing how a metadata field `name` can easily
be created and row names can be assigned:
```{r input, echo=FALSE}
### Load dataset
data(A549_FOSL2_01_NarrowPeaks_partial)
### Remove dataset metadata field "name"
A549_FOSL2_01_NarrowPeaks_partial$name <- NULL
A549_FOSL2_01_NarrowPeaks_partial$score <- NULL
A549_FOSL2_01_NarrowPeaks_partial$qValue <- NULL
A549_FOSL2_01_NarrowPeaks_partial$pValue <- NULL
A549_FOSL2_01_NarrowPeaks_partial$signalValue <- NULL
A549_FOSL2_01_NarrowPeaks_partial$peak <- NULL
```
```{r setName, collapse=TRUE}
### Initial dataset without metadata field
head(A549_FOSL2_01_NarrowPeaks_partial, n = 3)
### Adding a new metadata field "name" unique to each entry
A549_FOSL2_01_NarrowPeaks_partial$name <- paste0("FOSL2_01_entry_",
1:length(A549_FOSL2_01_NarrowPeaks_partial))
### Assign the same row name to each entry
names(A549_FOSL2_01_NarrowPeaks_partial) <- rep("FOSL2_01",
length(A549_FOSL2_01_NarrowPeaks_partial))
### Final dataset with metadata field 'name' and row names
head(A549_FOSL2_01_NarrowPeaks_partial, n = 3)
```
```{r inputClean, echo=FALSE, message=FALSE}
### Remove dataset
rm(A549_FOSL2_01_NarrowPeaks_partial)
```
## Chromosomes information
The chromosomes information is mandatory. It ensures that the consensus regions
do not exceed the length of the chromosomes.
The chromosomes information is contained inside a `Seqinfo` object. The
information from some UCSC genomes can be fetched automatically using
the `r Biocpkg("GenomeInfoDb")` package.
```{r knownGenome, collapse=TRUE}
### Import library
library(GenomeInfoDb)
### Get the information for Human genome version 19
hg19Info <- Seqinfo(genome="hg19")
### Subset the object to keep only the analyzed chromosomes
hg19Subset <- hg19Info[c("chr1", "chr10", "chrX")]
```
A `Seqinfo` object can also be created using the chromosomes information
specific to the analyzed genome.
```{r newGenome, collapse=FALSE}
### Create an Seqinfo Object
chrInfo <- Seqinfo(seqnames=c("chr1", "chr2", "chr3"),
seqlengths=c(1000, 2000, 1500), isCircular=c(FALSE, FALSE, FALSE),
genome="BioconductorAlien")
```
```{r deleteChr, echo=FALSE, message=FALSE}
rm(chrInfo)
rm(hg19Subset)
rm(hg19Info)
```
# Read NarrowPeak files
The NarrowPeak format is often used to provide called peaks of signal
enrichment based on pooled, normalized data. The `r Biocpkg("rtracklayer")`
package has functions which faciliates the loading of NarrowPeak files.
Since the main function of the `r Rpackage("consensusSeekeR")` package needs 2
`GRanges` objects, some manipulations are needed to create one `GRanges` for
the regions and one `GRanges` for the peaks.
```{r rtracklayer, collapse=TRUE}
### Load the needed packages
library(rtracklayer)
library(GenomicRanges)
### Demo file contained within the consensusSeekeR package
file_narrowPeak <- system.file("extdata",
"A549_FOSL2_ENCSR000BQO_MZW_part_chr_1_and_12.narrowPeak", package = "consensusSeekeR")
### Information about the extra columns present in the file need
### to be specified
extraCols <- c(signalValue = "numeric", pValue = "numeric", qValue = "numeric", peak = "integer")
### Create genomic ranges for the regions
regions <- import(file_narrowPeak, format = "BED", extraCols = extraCols)
### Create genomic ranges for the peaks
peaks <- regions
ranges(peaks) <- IRanges(start = (start(regions) + regions$peak), width = rep(1, length(regions$peak)))
### First rows of each GRanges object
head(regions, n = 2)
head(peaks, n = 2)
```
```{r secretRM, echo=FALSE, collapse=TRUE}
rm(peaks)
rm(regions)
```
A simpler way is to use the `readNarrowPeakFile` function of the
`r Rpackage("consensusSeekeR")` package which generates both
the peaks and the narrowPeak `GRanges`.
```{r readNarrowPeak_1, collapse=TRUE}
library(consensusSeekeR)
### Demo file contained within the consensusSeekeR package
file_narrowPeak <- system.file("extdata",
"A549_FOSL2_ENCSR000BQO_MZW_part_chr_1_and_12.narrowPeak", package = "consensusSeekeR")
### Create genomic ranges for both the regions and the peaks
result <- readNarrowPeakFile(file_narrowPeak, extractRegions = TRUE, extractPeaks = TRUE)
### First rows of each GRanges object
head(result$narrowPeak, n = 2)
head(result$peak, n = 2)
```
```{r secretRM_02, echo=FALSE, collapse=TRUE}
rm(result)
```
# Case study: nucleosome positioning
Global gene expression patterns are established and maintained by the
concerted actions of Transcription Factors (TFs) and the proteins that
constitute chromatin. The key structural element of chromatin is the
nucleosome, which consists of an octameric histone core wrapped by 147 bps
of DNA and connected to its neighbor by approximately 10-80 pbs of linker
DNA [@Kornberg1999]. Nucleosome occupancy and positioning have been proved to be
dynamic. It also has a major impact on expression, regulation, and evolution
of eukaryotic genes [@Jiang2015].
## Comparing nucleosome positioning results from different software
With the development of Next-generation sequencing, nucleosome
positioning using MNase-Seq data or MNase- or sonicated- ChIP-Seq data
combined with either single-end or paired-end sequencing have evolved as
popular techniques. Software such as PING [@Woo2013] and NOrMAL
[@Polishko2012], generates output which contains the positions of the
predicted nucleosomes, which simply are one base pair positions on
the reference genome. This position represents the center of the
predicted nucleosome. A range of $\pm$ 73 bps is usually superposed to the
predicted nucleosome to repesent the nucleosome occupancy.
First, the `r Rpackage("consensusSeekeR")` package must be loaded.
```{r libraryLoadNucl, warning=FALSE, message=FALSE}
library(consensusSeekeR)
```
The datasets, which are included in the `r Rpackage("consensusSeekeR")`
package, have to be loaded. Those include results obtained using syntethic
reads distributed following a normal distribution with a variance of
20 from three different
nucleosome positioning software: PING [@Woo2013], NOrMAL [@Polishko2012] and
NucPosSimulator [@Schopflin2013]. The genomic ranges have been obtained by
adding $\pm$ 73 bps to the detected positions.
```{r loadingDatasetsNucl}
### Loading dataset from NOrMAL
data(NOrMAL_nucleosome_positions) ; data(NOrMAL_nucleosome_ranges)
### Loading dataset from PING
data(PING_nucleosome_positions) ; data(PING_nucleosome_ranges)
### Loading dataset from NucPosSimulator
data(NucPosSimulator_nucleosome_positions) ; data(NucPosSimulator_nucleosome_ranges)
```
```{r prepareData, echo=FALSE}
rownames(NOrMAL_nucleosome_positions) <- NULL
rownames(NOrMAL_nucleosome_ranges) <- NULL
```
For the positions and ranges dataset from the same software, the `name`
field is paired to ensure that each position can be associated to its range.
The metadata field `name` must be unique to each feature for all datasets.
```{r datasetNuc, collapse=TRUE}
### Each entry in the positions dataset has an equivalent metadata
### "name" entry in the ranges dataset
head(NOrMAL_nucleosome_positions, n = 2)
head(NOrMAL_nucleosome_ranges, n = 2)
```
To be able to identify all entries from the same software, each row of the
dataset has to be assigned a name. All positions and ranges
from the same source must be assigned identical row names. In this
exemple, datasets are going to be identified by the name of their source
software.
```{r nuclAssignment, collapse=TRUE}
### Assigning software name "NOrMAL"
names(NOrMAL_nucleosome_positions) <- rep("NOrMAL", length(NOrMAL_nucleosome_positions))
names(NOrMAL_nucleosome_ranges) <- rep("NOrMAL", length(NOrMAL_nucleosome_ranges))
### Assigning experiment name "PING"
names(PING_nucleosome_positions) <- rep("PING", length(PING_nucleosome_positions))
names(PING_nucleosome_ranges) <- rep("PING", length(PING_nucleosome_ranges))
### Assigning experiment name "NucPosSimulator"
names(NucPosSimulator_nucleosome_positions) <- rep("NucPosSimulator",
length(NucPosSimulator_nucleosome_positions))
names(NucPosSimulator_nucleosome_ranges) <- rep("NucPosSimulator",
length(NucPosSimulator_nucleosome_ranges))
### Row names are unique to each software
head(NOrMAL_nucleosome_positions, n = 2)
head(PING_nucleosome_positions, n = 2)
head(NucPosSimulator_nucleosome_positions, n = 2)
```
The consensus regions for chromosome 1 only are calculated
with a defaut region size of 50 bases pairs (2 * `extendingSize`)
The regions are extended to include all nucleosome regions
(`expandToFitPeakRegion` = `TRUE` and `shrinkToFitPeakRegion` = `TRUE`).
To be retained as a consensus region, nucleosomes from at least 2
software must be present in the region (`minNbrExp` = `2`).
.
```{r nuclConsensus, collapse=FALSE}
### Only choromsome 1 is going to be analyzed
chrList <- Seqinfo("chr1", 135534747, NA)
### Find consensus regions with both replicates inside it
results <- findConsensusPeakRegions(
narrowPeaks = c(NOrMAL_nucleosome_ranges, PING_nucleosome_ranges,
NucPosSimulator_nucleosome_ranges),
peaks = c(NOrMAL_nucleosome_positions, PING_nucleosome_positions,
NucPosSimulator_nucleosome_positions),
chrInfo = chrList,
extendingSize = 25,
expandToFitPeakRegion = TRUE,
shrinkToFitPeakRegion = TRUE,
minNbrExp = 2,
nbrThreads = 1)
```
The output of `findConsensusPeakRegions` function is a list containing an
object `call` and an object `consensusRanges`. The object `call` contains the
matched call while the object `consensusRanges` is a `GRanges` containing the
consensus regions.
```{r nuclResults, collapse=TRUE}
### Print the call
results$call
### Print the 3 first consensus regions
head(results$consensusRanges, n = 3)
```
A total of `r length(results$consensusRanges)` consensus regions have been
found. An exemple of the consensus regions (in dark blue) is shown here using
Integrative genomics viewer [@Interests2011]:
```{r removeDatasetsNucl, echo=FALSE}
### Remove dataset from NOrMAL
rm(NOrMAL_nucleosome_positions)
rm(NOrMAL_nucleosome_ranges)
### Remove dataset from PING
rm(PING_nucleosome_positions)
rm(PING_nucleosome_ranges)
### Remove dataset from NucPosSimulator
rm(NucPosSimulator_nucleosome_positions)
rm(NucPosSimulator_nucleosome_ranges)
```
# Case study: ChIP-Seq data
Next-generation DNA sequencing coupled with chromatin immunoprecipitation
(ChIP-seq) has changed the ability to interrogate the genomic landscape
of histone
modifications, transcriptional cofactors and
transcription-factors binding in living cells [@Mundade2014].
Consortium,
such as [ENCODE](https://www.encodeproject.org) have developed and are
constantly updating a set of standards and guidelines for ChIP-Seq
experiments [@Landt2012].
ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel
DNA sequencing. The
obtained sequence reads are first mapped to the reference genome of the
organism used in the experiments. Binding sites are then detected using
software specialized in transcript factor binding sites identification, such as
MACS2 [@Zhang2008] and PeakRanger [@Feng2011]. Peaks
are defined as a single base pair position while statistically
enriched regions are defined as genomic ranges.
## ChIP-Seq replicates from one experiment
The Encyclopedia of DNA Elements (ENCODE) Consortium is an international
collaboration of research groups funded by the National Human Genome Research
Institute. The [ENCODE website](https://www.encodeproject.org) is a portal
giving access to the data generated by the ENCODE Consortium. The amount of
data gathered is extensive. Moreover, for some experiments, more than
one ChIP-Seq replicate is often available.
The software used to identify transcript factor binding sites generally
generates a peak position and an enriched region for each binding site.
However, it is quite unlikely that the exact peak position is exactly the same
across replicates. Even more, there is not yet a consensus on how to analyze
multiple-replicate ChIP-seq samples [@Yang2014].
The `r Rpackage("consensusSeekeR")` package can be used to identify consensus
regions for two or more replicates ChIP-Seq samples. The consensus regions
are being found by using the peak positions.
The transcription factor binding for the CTCF transcription factor have been
analyzed and 2 replicates are available in BAM files format on
[ENCODE website](https://www.encodeproject.org) (DCCs:
ENCFF000MYJ and ENCFF000MYN). The NarrowPeaks were generated using
MACS2 [@Zhang2008]
with the default parameters and the q-value set to 0.05.
To simplify this demo, only part of genome hg19, chr1:246000000-249250621 and
chr10:10000000-12500000, have been retained in
the datasets.
First, the `r Rpackage("consensusSeekeR")` package must be loaded.
```{r libraryLoad, warning=FALSE, message=FALSE}
library(consensusSeekeR)
```
The datasets, which are included in the `r Rpackage("consensusSeekeR")`
package, have to be loaded.
```{r loadingDatasets}
### Loading datasets
data(A549_CTCF_MYN_NarrowPeaks_partial) ; data(A549_CTCF_MYN_Peaks_partial)
data(A549_CTCF_MYJ_NarrowPeaks_partial) ; data(A549_CTCF_MYJ_Peaks_partial)
```
To be able to identify data from the same source, each row of the dataset
has to be assigned a source name. Beware that `NarrowPeak` and `Peak`
datasets from the same source must be assigned identical names. In this
exemple, datasets are replicates of the same experiment. So, the names
"rep01" and "rep02" are going to be assigned to each dataset.
```{r repAssignment}
### Assigning experiment name "rep01" to the first replicate
names(A549_CTCF_MYJ_NarrowPeaks_partial) <- rep("rep01", length(A549_CTCF_MYJ_NarrowPeaks_partial))
names(A549_CTCF_MYJ_Peaks_partial) <- rep("rep01", length(A549_CTCF_MYJ_Peaks_partial))
### Assigning experiment name "rep02" to the second replicate
names(A549_CTCF_MYN_NarrowPeaks_partial) <- rep("rep02", length(A549_CTCF_MYN_NarrowPeaks_partial))
names(A549_CTCF_MYN_Peaks_partial) <- rep("rep02", length(A549_CTCF_MYN_Peaks_partial))
```
The consensus regions for chromosome 10 only are calculated
with a defaut region size of 200 bases pairs (2 * `extendingSize`)
The regions are extended to include all peaks regions
(`expandToFitPeakRegion` = `TRUE` and `shrinkToFitPeakRegion` = `TRUE`).
A peak from both replicates must be present withinin a region for it to be retained as a
consensus region.
```{r replicateConsensus, collapse=FALSE}
### Only choromsome 10 is going to be analyzed
chrList <- Seqinfo("chr10", 135534747, NA)
### Find consensus regions with both replicates inside it
results <- findConsensusPeakRegions(
narrowPeaks = c(A549_CTCF_MYJ_NarrowPeaks_partial, A549_CTCF_MYN_NarrowPeaks_partial),
peaks = c(A549_CTCF_MYJ_Peaks_partial, A549_CTCF_MYN_Peaks_partial),
chrInfo = chrList,
extendingSize = 100,
expandToFitPeakRegion = TRUE,
shrinkToFitPeakRegion = TRUE,
minNbrExp = 2,
nbrThreads = 1)
```
The output of `findConsensusPeakRegions` function is a list containing an
object `call` and an object `conesensusRanges`. The object `call` contains the
matched call while the object `conesensusRanges` is a `GRanges` containing the
consensus regions.
```{r replicateResults, collapse=TRUE}
### Print the call
results$call
### Print the 3 first consensus regions
head(results$consensusRanges, n = 3)
```
A total of `r length(results$consensusRanges)` consensus regions have been
found. An example of the consensus regions (in green) is shown here using
Integrative genomics viewer [@Interests2011]:
```{r rmCurrentDatasets, echo=FALSE, message=FALSE}
### Remove datasets
rm(A549_CTCF_MYN_NarrowPeaks_partial)
rm(A549_CTCF_MYN_Peaks_partial)
rm(A549_CTCF_MYJ_NarrowPeaks_partial)
rm(A549_CTCF_MYJ_Peaks_partial)
```
## ChIP-Seq data from multiple experiments
The `r Rpackage("consensusSeekeR")` package can also be used to identify
consensus regions for two or more ChIP-Seq samples from multiple experiments.
The peak positions are the feature used to identify the consensus regions.
The transcription factor binding for the NR3C1 transcription factor have been
analyzed in more than one experiment. For each experiment, replicates have
been analyzed together using the irreproducible discovery rate method [@Li2011].
Results are available in bed narrowPeak format on
[ENCODE website](https://www.encodeproject.org) (DCCs:
ENCFF002CFQ, ENCFF002CFR and ENCFF002CFS) [@Dunham2012a].
To simplify this demo, only part of genome hg19, chr2:40000000-50000000 and
chr3:10000000-13000000, have been retained in
the datasets.
First, the `r Rpackage("consensusSeekeR")` package must be loaded.
```{r libLoad, warning=FALSE, message=FALSE}
library(consensusSeekeR)
```
The datasets, which are included in the `r Rpackage("consensusSeekeR")`
package, have to be loaded.
```{r loadingDatasetsExp}
### Loading datasets
data(A549_NR3C1_CFQ_NarrowPeaks_partial) ; data(A549_NR3C1_CFQ_Peaks_partial)
data(A549_NR3C1_CFR_NarrowPeaks_partial) ; data(A549_NR3C1_CFR_Peaks_partial)
data(A549_NR3C1_CFS_NarrowPeaks_partial) ; data(A549_NR3C1_CFS_Peaks_partial)
```
To be able to identify data from the same source, each row of the dataset
has to be assigned an experiment name. Beware that NarrowPeak and Peak
datasets from the same source must be assigned identical names. In this
exemple, datasets are coming from different experiments for the same
transcription factor. So, the short name of each experiment
"ENCFF002CFQ", "ENCFF002CFR" and "ENCFF002CFS" is going to be
assigned to each dataset.
```{r expAssignment}
### Assign experiment name "ENCFF002CFQ" to the first experiment
names(A549_NR3C1_CFQ_NarrowPeaks_partial) <- rep("ENCFF002CFQ", length(A549_NR3C1_CFQ_NarrowPeaks_partial))
names(A549_NR3C1_CFQ_Peaks_partial) <- rep("ENCFF002CFQ", length(A549_NR3C1_CFQ_Peaks_partial))
### Assign experiment name "ENCFF002CFQ" to the second experiment
names(A549_NR3C1_CFR_NarrowPeaks_partial) <- rep("ENCFF002CFR", length(A549_NR3C1_CFR_NarrowPeaks_partial))
names(A549_NR3C1_CFR_Peaks_partial) <- rep("ENCFF002CFR", length(A549_NR3C1_CFR_Peaks_partial))
### Assign experiment name "ENCFF002CFQ" to the third experiment
names(A549_NR3C1_CFS_NarrowPeaks_partial) <- rep("ENCFF002CFS", length(A549_NR3C1_CFS_NarrowPeaks_partial))
names(A549_NR3C1_CFS_Peaks_partial) <- rep("ENCFF002CFS", length(A549_NR3C1_CFS_Peaks_partial))
```
In ENCODE bed narrowPeak format, entries don't have a specific metadata field
called `name`. So, to be able to use the `findConsensusPeakRegions` function,
specific names must manually be added to each entry.
```{r rowNames}
### Assign specific name to each entry of to first experiment
### NarrowPeak name must fit Peaks name for same experiment
A549_NR3C1_CFQ_NarrowPeaks_partial$name <- paste0("NR3C1_CFQ_region_",
1:length(A549_NR3C1_CFQ_NarrowPeaks_partial))
A549_NR3C1_CFQ_Peaks_partial$name <- paste0("NR3C1_CFQ_region_",
1:length(A549_NR3C1_CFQ_NarrowPeaks_partial))
### Assign specific name to each entry of to second experiment
### NarrowPeak name must fit Peaks name for same experiment
A549_NR3C1_CFR_NarrowPeaks_partial$name <- paste0("NR3C1_CFR_region_",
1:length(A549_NR3C1_CFR_NarrowPeaks_partial))
A549_NR3C1_CFR_Peaks_partial$name <- paste0("NR3C1_CFR_region_",
1:length(A549_NR3C1_CFR_Peaks_partial))
### Assign specific name to each entry of to third experiment
### NarrowPeak name must fit Peaks name for same experiment
A549_NR3C1_CFS_NarrowPeaks_partial$name <- paste0("NR3C1_CFS_region_",
1:length(A549_NR3C1_CFS_NarrowPeaks_partial))
A549_NR3C1_CFS_Peaks_partial$name <- paste0("NR3C1_CFS_region_",
1:length(A549_NR3C1_CFS_Peaks_partial))
```
The consensus regions for chromosome 2 only are calculated
with a defaut region size of 400 bases pairs (2 * `extendingSize`)
The regions are not extended to include all peaks regions but are shrinked
when exceeding peaks regions
(`expandToFitPeakRegion` = `FALSE` and `shrinkToFitPeakRegion` = `TRUE`).
A peak from 2 out of 3 experiments must be present in a region for it to be retained as
a consensus region.
```{r experimentConsensus, collapse=FALSE}
### Only choromsome 2 is going to be analyzed
chrList <- Seqinfo("chr2", 243199373, NA)
### Find consensus regions with both replicates inside it
results <- findConsensusPeakRegions(
narrowPeaks = c(A549_NR3C1_CFQ_NarrowPeaks_partial, A549_NR3C1_CFR_NarrowPeaks_partial,
A549_NR3C1_CFS_NarrowPeaks_partial),
peaks = c(A549_NR3C1_CFQ_Peaks_partial, A549_NR3C1_CFR_Peaks_partial,
A549_NR3C1_CFS_Peaks_partial),
chrInfo = chrList,
extendingSize = 200,
expandToFitPeakRegion = FALSE,
shrinkToFitPeakRegion = TRUE,
minNbrExp = 2,
nbrThreads = 1)
```
The output of `findConsensusPeakRegions` function is a list containing an
object `call` and an object `consensusRanges`. The object `call` contains the
matched call while the object `consensusRanges` is a `GRanges` containing the
consensus regions.
```{r experimentResults, collapse=TRUE}
### Print the call
results$call
### Print the first 3 consensus regions
head(results$consensusRanges, n = 3)
```
A total of `r length(results$consensusRanges)` consensus regions have been
found. A example of the consensus regions (in green) is shown here using
Integrative genomics viewer [@Interests2011]:
```{r rmDatasetsExp, echo=FALSE, message=FALSE}
### Remove datasets
rm(A549_NR3C1_CFQ_NarrowPeaks_partial)
rm(A549_NR3C1_CFQ_Peaks_partial)
rm(A549_NR3C1_CFR_NarrowPeaks_partial)
rm(A549_NR3C1_CFR_Peaks_partial)
rm(A549_NR3C1_CFS_NarrowPeaks_partial)
rm(A549_NR3C1_CFS_Peaks_partial)
```
# Parameters
## Effect of the shrinkToFitPeakRegion parameter
The `shrinkToFitPeakRegion` allows the resizing of the consensus region to fit
the minimum regions of the included features when those values are
included inside the initial consensus region. When the
`extendingSize` parameter is large, the effect can be quite visible on the
final consensus regions. For exemple, the following figure shows the same
region, from the NR3C1 example with `extendingSize` of 200, when the
`shrinkToFitPeakRegion` is set to `TRUE` (green color) and to `FALSE`
(orange color) using Integrative
genomics viewer [@Interests2011]:
## Effect of the expandToFitPeakRegion parameter
The `expandToFitPeakRegion` allows the resizing of the consensus region to fit
the maximum of the included features when those values are
outside the initial consensus region. When the
`extendingSize` parameter is small, the effect can be quite visible on the
final consensus regions. For exemple, the following figure shows the same
region, from the CTCF example with `extendingSize` of 100, when the
`shrinkToFitPeakRegion` is set
to `FALSE` (orange color) and to `TRUE` (green color).
## Effect of the extendingSize parameter
The value of the `extendingSize` parameter can affect the final number of
consensus regions. While small `extendingSize` value can miss some regions,
large `extendingSize` value can gather consensus regions. Testing a range
of `extendingSize` parameters can be an option worth considering.
As an example, the number of consensus regions obtained with different values
of `extendingSize` is calculated.
``` {r sizeEffect, collapse=TRUE, eval=FALSE}
### Set different values for the extendingSize parameter
size <- c(1, 10, 50, 100, 300, 500, 750, 1000)
### Only chrompsome 10 is going to be analyzed
chrList <- Seqinfo("chr10", 135534747, NA)
### Find consensus regions using all the size values
resultsBySize <- lapply(size, FUN = function(size) findConsensusPeakRegions(
narrowPeaks = c(A549_CTCF_MYJ_NarrowPeaks_partial, A549_CTCF_MYN_NarrowPeaks_partial),
peaks = c(A549_CTCF_MYJ_Peaks_partial, A549_CTCF_MYN_Peaks_partial),
chrInfo = chrList,
extendingSize = size,
expandToFitPeakRegion = TRUE,
shrinkToFitPeakRegion = TRUE,
minNbrExp = 2,
nbrThreads = 1))
### Extract the number of consensus regions obtained for each extendingSize
nbrRegions <- mapply(resultsBySize, FUN = function(x) return(length(x$consensusRanges)))
```
A graph can be used to visualize the variation of the number of consensus
regions in function of the `extendingSize` parameter.
```{r graphSizeEffect, warning=FALSE, eval=FALSE}
library(ggplot2)
data <- data.frame(extendingSize = size, nbrRegions = nbrRegions)
ggplot(data, aes(extendingSize, nbrRegions)) + scale_x_log10("Extending size") +
stat_smooth(se = FALSE, method = "loess", size=1.4) +
ylab("Number of consensus regions") +
ggtitle(paste0("Number of consensus regions in function of the extendingSize"))
```
# Parallelizing consensusSeekeR
Due to the size of the analyzed genomes, the `findConsensusPeakRegions`
function can take a while to process. However, a job can be separated by
chromosome and run in parallel. This takes advantage of multiple
processors and reduce the total execution time. The number of threads
used can be set with
the `nbrThreads` parameter in the `findConsensusPeakRegions` function.
```{r parallelExample, eval=FALSE, collapse=TRUE}
### Load data
data(A549_FOSL2_01_NarrowPeaks_partial) ; data(A549_FOSL2_01_Peaks_partial)
data(A549_FOXA1_01_NarrowPeaks_partial) ; data(A549_FOXA1_01_Peaks_partial)
### Assigning name "FOSL2"
names(A549_FOSL2_01_NarrowPeaks_partial) <- rep("FOSL2", length(A549_FOSL2_01_NarrowPeaks_partial))
names(A549_FOSL2_01_Peaks_partial) <- rep("FOSL2", length(A549_FOSL2_01_Peaks_partial))
### Assigning name "FOXA1"
names(A549_FOXA1_01_NarrowPeaks_partial) <- rep("FOXA1", length(A549_FOXA1_01_NarrowPeaks_partial))
names(A549_FOXA1_01_Peaks_partial) <- rep("FOXA1", length(A549_FOXA1_01_Peaks_partial))
### Two chromosomes to analyse
chrList <- Seqinfo(paste0("chr", c(1,10)), c(249250621, 135534747), NA)
### Find consensus regions using 2 threads
results <- findConsensusPeakRegions(
narrowPeaks = c(A549_FOSL2_01_NarrowPeaks_partial, A549_FOXA1_01_Peaks_partial),
peaks = c(A549_FOSL2_01_Peaks_partial, A549_FOXA1_01_NarrowPeaks_partial),
chrInfo = chrList, extendingSize = 200, minNbrExp = 2,
expandToFitPeakRegion = FALSE, shrinkToFitPeakRegion = FALSE,
nbrThreads = 4)
```
# Acknowledgment
We thank Imène Boudaoud for her advice on the vignette content.
# Session info
Here is the output of `sessionInfo()` on the system on which this document was
compiled:
```{r sessionInfo, echo=FALSE}
sessionInfo()
```
# References