regioneReloaded
25 April 2023
Contents
1 Introduction
In genetic and epigenetic research, high-throughput sequencing methods generate large amounts of data. The meaningful interpretation is one of the biggest challenges when analyzing multiple genomic datasets.
Sequencing results can be summarized as ‘region sets’: a list of genomic regions defined by specific coordinates in a reference genome. This is true for all frequently used epigenomic methods such as chromatin immuprecipation (ChIP)-seq, CUT&RUN, DNA-methylation profiling and chromosome conformation capture-based methods. Genome-sequencing studies provide information on mutations, copy number variations and inversions that can be expressed as region sets. Transcriptomic data can be converted in region sets for instance by extracting the genomic position of differentially expressed genes or their regulatory elements. All regions can be further associated with metadata further characterizing them. Any annotated feature of interest in the genome itself, such as repetitive elements, conserved regions, or regulatory elements, can be chosen as regions set for further analysis.
To interpret the meaning of overlaps between regions sets, in 2015 we published regioneR, an R package developed for the statistical evaluation of the association between sets of genomic regions. The main limitation of regioneR was that it can only perform pairwise association analysis. The resulting association value (“z-score”) of different pairwise analyses could not be directly compared since z-scores depend on the size and nature of the input region sets. When scaling up the number of pairwise comparisons of larger data sets, the required increase in calculation time becomes a serious limitation.
To address these issues, we now present regioneReloaded: an R package that is the logically designed evolution of regioneR allowing to calculate the statistical association between multiple regions sets at the same time. regionReloaded comprises a set of functions created to analyze clusters of associations and to facilitate the interpretation of results. Importantly, it includes strategies to improve p-value calculations and provides normalized z-scores allowing the direct comparison of multiple analysis. It also builds upon regioneR by adding new plotting functions for obtaining publication-ready graphs.
Taken together, regioneReloaded, aims to be a novel and valuable addition to the repertoire of available tools in the Bioconductor repository for the analysis of high-content genomic datasets.
1.1 regioneR and regioneReloaded
regioneR is an R package published in 2015 created to statistically test the positional association between genomic region sets. The core of regioneR is a permutation test framework specifically designed to work in a genomic environment. For a detailed insight into the permutation test strategy implemented in regioneR, including the different randomization and evaluation strategies, please see the regioneR vignette. The two main results that can be obtained with this statistical method can be summarized in two graphs. Figure 1A shows the association observed between the two region sets under study, highlighting the distance calculated in standard deviations from the random distribution. Figure 1B shows the local z-score: a narrow peak, as the one shown, indicates that the association is highly dependent on the exact location of the region. One the contrary, a flat profile would suggest a regional association.
(figure 1)
regioneR basic Graphs
1.2 Normalized Z-Score
regioneReloaded aims to integrate the framework previously developed for regioneR to calculate associations of different region sets simultaneously. However, the values of z-score obtained in different tests can not be directly compared. In particular, the value of the z-score is directly associated to the square root of the number of regions present in the first region set of the test (RS1).
To compare the z-scores obtained in multi pairwise tests between region sets, we introduce the concept of normalized z-score, which allows not only to directly compare different association events but also to work with subsets of data and speed up the calculations.
The normalized z-score is calculated as follows:
nZS = ZS / \(\sqrt{n}\)
We can empirically evaluate how the value of z-score between two well known strongly associated proteins, CTCF (ENCFF237OKO) and RAD21 (ENCFF072UEX) in HepG2 cell line from ENCODE portal, (Sloan et al. 2016, https://www.encodeproject.org/) , increases with the number of regions included in the test. On the other hand, the value of the normalized z-score is much more stable with different number of regions. Importantly, we see that the value of normalized z-score is stable approximately after including only 30% of the regions in the dataset. This demonstrates than sampling a portion of the dataset is a viable strategy to reduce calculation time without impacting the capacity to obtain meaningful normalized z-score values.
Normal z-score empirical calculations
Another crucial factor for the calculation of the normal z-score is the number of permutations performed in the test (ntimes). We suggest to use a minimum of 5000 permutations to achieve reproducible results.
Ntimes demonstration
2 Installation
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("regioneReloaded")
3 Quick start
library("regioneReloaded")
#> Loading required package: regioneR
#> Loading required package: GenomicRanges
#> Loading required package: stats4
#> Loading required package: BiocGenerics
#>
#> Attaching package: 'BiocGenerics'
#> The following objects are masked from 'package:stats':
#>
#> IQR, mad, sd, var, xtabs
#> The following objects are masked from 'package:base':
#>
#> Filter, Find, Map, Position, Reduce, anyDuplicated, aperm, append,
#> as.data.frame, basename, cbind, colnames, dirname, do.call,
#> duplicated, eval, evalq, get, grep, grepl, intersect, is.unsorted,
#> lapply, mapply, match, mget, order, paste, pmax, pmax.int, pmin,
#> pmin.int, rank, rbind, rownames, sapply, setdiff, sort, table,
#> tapply, union, unique, unsplit, which.max, which.min
#> Loading required package: S4Vectors
#>
#> Attaching package: 'S4Vectors'
#> The following object is masked from 'package:utils':
#>
#> findMatches
#> The following objects are masked from 'package:base':
#>
#> I, expand.grid, unname
#> Loading required package: IRanges
#> Loading required package: GenomeInfoDbThe permutation test can be performed on a list multiple region sets by the function crosswisePermTest(). This process is computing intensive and its calculation time depends on the number of cores called by the parameter mc.cores (see regioneR). The result of the permutation test obtained by running the code below is included as a pre-computed example dataset which can be loaded into the environment by running the command data("cw_Alien_RaR").
AlienGenome and AlienRSList are toy datasets described in the next section of the vignette.
#NOT RUN
set.seed(42)
cw_Alien_ReG<-crosswisePermTest(Alist = AlienRSList_narrow,
sampling = FALSE,
mc.cores= 25,
ranFUN = "resampleGenome",
evFUN = "numOverlaps",
genome = AlienGenome,
ntimes= 1000
)
#Once the multiple permutation tests are performed, we can generate a matrix of pairwise associations between the region sets with the function makeCrosswiseMatrix() and visualize it with plotCrosswiseMatrix().
data("cw_Alien")
cw_Alien_ReG<-makeCrosswiseMatrix(cw_Alien_ReG, pvcut = 1)
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.7497654 0.7857181 0.6514804 0.7363304 0.3459672 0.4561003 0.7516609
plotCrosswiseMatrix(cw_Alien_ReG)
4 Example dataset: the Alien Genome
Performing Permutation tests with region sets on an eukaryotic genome can take a long computation time. To demonstrate the different regionReloaded features on a small toy dataset with low computation times, we have created a fake genome called the ‘AlienGenome’ and an associated list of region sets. The Alien Genome consists of four chromosomes of lengths ranging from 2Mb to 0.1Mb.
Graphical descrition of the AlienGenome
AlienGenome <-
toGRanges(data.frame(
chr = c("AlChr1", "AlChr2", "AlChr3", "AlChr4"),
start = c(rep(1, 4)),
end = c(2e6, 1e6, 5e5, 1e5)
))
Four basic region sets were created on this genome. The first three (“regA”, “regB”, “regC”), were created using the function createRandomRegions() from the package regioneR. Each region set consists of 100 regions with an average width of 100 bp (and a standard deviation of 50 bp). Being randomly generated, the three region sets should show no significant associations with each other.
gnm <- AlienGenome
nreg=100
regA <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = gnm
)
regB <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50 ,
non.overlapping = TRUE,
genome = gnm
)
regC <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = gnm
)To generate “artificial” associations we can use the similarRegionSet() function, which produces sets of random regions with some degree of similarity to the original input region set. For each set of regions regA, regB and regC, we create a list of region sets that share a percentage (90% to 10%) of similarity with the original set.
vectorPerc <- seq(0.1, 0.9, 0.1)
RsetA <-
similarRegionSet(
GR = regA,
name = "regA",
genome = gnm,
vectorPerc = vectorPerc
)
RsetB <-
similarRegionSet(
GR = regB,
name = "regB",
genome = gnm,
vectorPerc = vectorPerc
)
RsetC <-
similarRegionSet(
GR = regC,
name = "regC",
genome = gnm,
vectorPerc = vectorPerc
)Next we create a region set of 100 regions that shares half the regions with regA and half with regB called regAB. From this region set we create similar region sets again using similarRegionSet().
vectorPerc2 <- seq(0.2, 0.8, 0.2)
regAB <- c(sample(regA, nreg / 2), sample(regB, nreg / 2))
RsetAB <-
similarRegionSet(
GR = regAB,
name = "regAB",
genome = gnm,
vectorPerc = vectorPerc2
)As a negative association control, we create random region sets that contain no regions of “regA”, “regB”, “regC”, “regAB or”regABC" using createRandomRegions() and substractRegions() functions from regioneR.
reg_no_A <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = subtractRegions(gnm, regA)
)
reg_no_B <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = subtractRegions(gnm, regB)
)
reg_no_C <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = subtractRegions(gnm, regC)
)
reg_no_AB <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = subtractRegions(gnm, c(regA, regB))
)
reg_no_ABC <-
createRandomRegions(
nregions = nreg,
length.mean = 100,
length.sd = 50,
non.overlapping = TRUE,
genome = subtractRegions(gnm, c(regA, regB, regC))
)To demonstrate the detection of “lateral” association (association with a region close to the one being tested), we create the “regD” region set. It is composed of regions in the vicinity of region set “regA” but does not overlap with the regions of “regA” itself.
dst <- sample(c(-300,300),length(regA),replace = TRUE)
regD <- regioneR::toGRanges(
data.frame(
chr=as.vector(GenomeInfoDb::seqnames(regA)),
start = start(regA) + dst,
end = end (regA) + dst
)
)
RsetD <-
similarRegionSet(
GR = regD,
name = "regD",
genome = gnm,
vectorPerc = vectorPerc2
)Finally, we store all the generated sets of regions in a list called AlienRSList which we can use as input for the crosswisePermTest() function to perform the multiple permutation test.
Rset_NO <- list(reg_no_A, reg_no_B, reg_no_C, reg_no_AB, reg_no_ABC)
names(Rset_NO) <- c("reg_no_A", "reg_no_B", "reg_no_C", "reg_no_AB", "reg_no_ABC")
AlienRSList_narrow <- c(RsetA, RsetB, RsetC, RsetD, RsetAB, Rset_NO)
summary(AlienRSList_narrow)
#> Length Class Mode
#> regA_01 100 GRanges S4
#> regA_02 100 GRanges S4
#> regA_03 100 GRanges S4
#> regA_04 100 GRanges S4
#> regA_05 100 GRanges S4
#> regA_06 100 GRanges S4
#> regA_07 100 GRanges S4
#> regA_08 100 GRanges S4
#> regA_09 100 GRanges S4
#> regA 100 GRanges S4
#> regB_01 100 GRanges S4
#> regB_02 100 GRanges S4
#> regB_03 100 GRanges S4
#> regB_04 100 GRanges S4
#> regB_05 100 GRanges S4
#> regB_06 100 GRanges S4
#> regB_07 100 GRanges S4
#> regB_08 100 GRanges S4
#> regB_09 100 GRanges S4
#> regB 100 GRanges S4
#> regC_01 100 GRanges S4
#> regC_02 100 GRanges S4
#> regC_03 100 GRanges S4
#> regC_04 100 GRanges S4
#> regC_05 100 GRanges S4
#> regC_06 100 GRanges S4
#> regC_07 100 GRanges S4
#> regC_08 100 GRanges S4
#> regC_09 100 GRanges S4
#> regC 100 GRanges S4
#> regD_02 100 GRanges S4
#> regD_04 100 GRanges S4
#> regD_06 100 GRanges S4
#> regD_08 100 GRanges S4
#> regD 100 GRanges S4
#> regAB_02 100 GRanges S4
#> regAB_04 100 GRanges S4
#> regAB_06 100 GRanges S4
#> regAB_08 100 GRanges S4
#> regAB 100 GRanges S4
#> reg_no_A 100 GRanges S4
#> reg_no_B 100 GRanges S4
#> reg_no_C 100 GRanges S4
#> reg_no_AB 100 GRanges S4
#> reg_no_ABC 100 GRanges S45 Multi Permutation Test with regioneReloaded
5.1 Crosswise Analysis and the genoMatriXeR object
crosswisePermTest() is the main statistical function of the package. “Crosswise” analysis (in contrast to “pairwise”) refers to the calculation of permutation tests between all possible combinations of elements in a first list of region sets (Alist) with those in a second list (Blist). The core statistical calculations of the permutation tests rely on permTest().
The result of crosswisePermTest() is stored in an S4 object of class genoMatriXeR that contains three slots: parameters, multiOverlaps and matrix.
- gMXR_obj
- @parameters
- Alist
- Blist
- sampling
- fraction
- min_sampling
- ranFUN
- evFUN
- universe
- adj_pv_method
- max_pv
- nc
- matOrder
- ntimes
- @multiOverlaps
- one field for each comparison
- @matrix
- GMat
- GMat_pv
- GMat_corX
- GMat_corY
- FitRow
- FitCol
- @parameters
5.1.1 Parameters
The slot “parameters” stores all the parameters that were used to obtain the genoMatriXeR object and can be retrieved with getParameters().
data("cw_Alien")
getParameters(cw_Alien_ReG)
#> parameter value
#> 1 Alist AlienRSList_narrow
#> 2 Blist AlienRSList_narrow
#> 3 sampling FALSE
#> 4 fraction 0.15
#> 5 min_sampling 0.15
#> 6 ranFUN resampleGenome
#> 7 evFUN numOverlaps
#> 8 ntimes 1000
#> 9 universe NULL
#> 10 adj_pv_method BH
#> 11 nc NULL
#> 12 matOrder NULL5.1.2 multiOverlaps
The “multiOverlaps” slot contains a list of data frames that contain the results of each permutation test performed. For each region set in “Alist”, a data frame is generated with 11 columns and a number of rows equal to the elements in “Blist”. The columns of the data frame contain the following information:
- order.id: order of comparison
- name: name of region sets in Blist
- n_regionA: number of permuted Alist regions
- n_regionB: number of regions in each regions set of Blist
- z_score: z-score calculated by permTest
- p_value: p.value calculated by permTest
- n_hits: number of hits computed using the evaluation function on a regions set of Alist versus each regions set of Blists
- mean_perm_test: mean of hits computed using the evaluation function on the permutated region set
- sd_perm_test: standard deviation of hits computed using the evaluation function on the permutated region set
- norm_zscore: normalized z-scores
- adj_p_value: adjusted p.value
5.1.3 Matrix
When the genoMatriXeR object is initially created by running crosswisePermTest(), the “matrix” slot will have a NULL value. See the section Matrix calculation and visualization on how to generate, access and visualize the association matrix.
5.2 Evaluation functions
regioneReloaded can use all the evaluation strategies present in regioneR, and it also allows the use of custom evaluation functions. The examples considered by this vignette will be based on the default evaluation function numOverlaps().
5.3 Randomization functions
regioneReloaded can use all the randomization strategies present in regioneR and it also allows the use of custom functions.
A notable difference with regioneR is that the randomization function resampleRegions() can work without the presence of the parameter “universe”. In this case a “universe” will be created by using all regions from the list of regions set “Alist” using the function createUniverse(). Three example pre-computed objects are included as sample data generated with three different randomization functions: “randomizeRegions”, “resampleRegions” and “resampleGenome”.
#NOT RUN
set.seed(42)
cw_Alien_RaR <- crosswisePermTest(
Alist = AlienRSList_narrow,
Blist = AlienRSList_narrow,
sampling = FALSE,
genome = AlienGenome,
per.chromosome=TRUE,
ranFUN = "randomizeRegions",
evFUN = "numOverlaps",
ntimes= 1000,
mc.cores = 20
)
set.seed(42)
cw_Alien_ReR <- crosswisePermTest(
Alist = AlienRSList_narrow,
Blist = AlienRSList_narrow,
sampling = FALSE,
genome = AlienGenome,
per.chromosome=TRUE,
ranFUN = "resampleRegions",
evFUN = "numOverlaps",
ntimes= 1000,
mc.cores = 20
)
set.seed(42)
cw_Alien_ReG <- crosswisePermTest(
Alist = AlienRSList_narrow,
Blist = AlienRSList_narrow,
sampling = FALSE,
genome = AlienGenome,
per.chromosome=TRUE,
ranFUN = "resampleGenome",
evFUN = "numOverlaps",
ntimes= 100,
mc.cores = 20
)
#
5.4 Matrix Calculation and visualization
Association matrix will be computed with the function makeCrosswiseMatrix(). This function returns a genoMatriXeR object with the matrix assigned to the “matrix” slot, the matrix will be able to be queried with the getMatrix() function. As a default value the matrix will be created using normalized z-score for a more in-depth description of the parameters that can be used to create the matrix we refer you to the makeCrosswiseMatrix() function manual. If no clustering method is chosen, automatically the most efficient clustering method will be chosen from those in hclust, as described in the chooseHclustMet() function manual.
cw_Alien_RaR <- makeCrosswiseMatrix(cw_Alien_RaR)
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.7592340 0.7805803 0.6610466 0.7222423 0.0402856 0.5192172 0.7231580
cw_Alien_ReG <- makeCrosswiseMatrix(cw_Alien_ReG)
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.7497654 0.7857181 0.6514804 0.7363304 0.3459672 0.4561003 0.7516609
cw_Alien_ReR <- makeCrosswiseMatrix(cw_Alien_ReR)
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.7515325 0.7716179 0.6542890 0.7136237 0.2223408 0.5618311 0.7245865To compare the output of the matrices created with different randomization functions, we need to use the fixed order of clusterization for all matrices.
modX <- getHClust(cw_Alien_ReG,hctype = "rows")
modY <- getHClust(cw_Alien_ReG,hctype = "cols")
X<-modX$labels[modX$order]
Y<-modY$labels[modX$order]
ord<-list(X=X,Y=Y)
p_ReG <- plotCrosswiseMatrix(cw_Alien_ReG, matrix_type = "association", ord_mat = ord)
p_ReR <- plotCrosswiseMatrix(cw_Alien_ReR, matrix_type = "association", ord_mat = ord)
plot(p_ReG)
plot(p_ReR)
getHClust() function returns an object of class hclust that you can use for further analysis.
plot(modX,cex = 0.8)
the matrix_type parameter plotCrosswiseMatrix() allows to choose between “association” matrix (where the z-score value obtained from the permutation test calculation will be shown), or “correlation” (where the Pearson’s R-value between each Regions Set will be used).
p_ReG_cor <- plotCrosswiseMatrix(cw_Alien_ReG, matrix_type = "correlation", ord_mat = ord)
# p_ReR_cor <- plotCrosswiseMatrix(cw_Alien_ReR, matrix_type = "correlation", ord_mat = ord)
plot(p_ReG_cor)
# plot(p_ReR_cor)5.5 Plot Single permutation test
regioneReloaded allows us to extract and visualize the results of a single Region Sets comparison, the visualization resumes the previous graphs in regioneR by exploiting the power of ggplot2.
plotSinglePT(cw_Alien_ReG, RS1 = "regA", RS2 = "regA_05")
p_sPT1 <- plotSinglePT(cw_Alien_ReG, RS1 = "regA", RS2 = "regC")
plot(p_sPT1)
5.6 Plot Dimensionality Reduction
Dimensionality reduction refers to a strategy capable of representing complex (high-density) data in a low-density space while retaining some meaningful properties of the original data. The plotCrosswiseDimRes function allows three of the most widely used algorithms (PCA, tRSNE, UMAP) to be applied to represent genoMatriXeR objects.
set.seed(42)
plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="PCA")
#> Warning: The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
The data is clustered using a clustering algorithm that can be selected from (hclust, kmeans or pam ), giving the possibility of importing an external clustering method.
set.seed(42)
p_cdr_hc <- plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="PCA", clust_met = "hclust")
# set.seed(42)
# p_cdr_pam <- plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="PCA", clust_met = "pam")
plot(p_cdr_hc)
#> Warning: The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
# plot(p_cdr_pam)
The parameters listRS and emphasize were added to emphasise the data found to be interesting.
lsRegSet<-list(regA="regA",regB="regB",regC="regC")
set.seed(42)
plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="PCA",listRS = lsRegSet)
#> Warning: The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
set.seed(42)
plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="PCA",listRS = lsRegSet,ellipse = TRUE, emphasize =TRUE)
#> Warning: The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
set.seed(67)
plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="tSNE",listRS = lsRegSet,ellipse = TRUE, emphasize =TRUE)
#> Warning: The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
set.seed(67)
plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="UMAP",listRS = lsRegSet,ellipse = TRUE, emphasize =TRUE)
#> Warning in MASS::cov.trob(data[, vars]): Probable convergence failure
#> Warning in MASS::cov.trob(data[, vars]): The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
The * return_table* parameter displays a table summarising the results of the clustering, incorporating the value ASW total mean of individual silhouette widths silohuette.
set.seed(67)
silTable <- plotCrosswiseDimRed(cw_Alien_ReG, nc = 6, type="UMAP",listRS = lsRegSet,return_table = TRUE)
#> Warning in MASS::cov.trob(data[, vars]): Probable convergence failure
#> Warning: The following aesthetics were dropped during statistical transformation: label
#> ℹ This can happen when ggplot fails to infer the correct grouping structure in
#> the data.
#> ℹ Did you forget to specify a `group` aesthetic or to convert a numerical
#> variable into a factor?
silTable
#> Name Cluster ASW
#> 1 regAB_08 clust_1 0.0871640
#> 2 regC_08 clust_1 0.0871640
#> 3 regC_07 clust_1 0.0871640
#> 4 regA_08 clust_1 0.0871640
#> 5 regC_09 clust_1 0.0871640
#> 6 regB_09 clust_1 0.0871640
#> 7 reg_no_B clust_1 0.0871640
#> 8 regA_09 clust_1 0.0871640
#> 9 reg_no_AB clust_1 0.0871640
#> 10 reg_no_C clust_1 0.0871640
#> 11 reg_no_ABC clust_1 0.0871640
#> 12 reg_no_A clust_1 0.0871640
#> 13 regAB_06 clust_2 0.3365061
#> 14 regAB_04 clust_2 0.3365061
#> 15 regAB clust_2 0.3365061
#> 16 regAB_02 clust_2 0.3365061
#> 17 regD_08 clust_3 0.3496858
#> 18 regD_06 clust_3 0.3496858
#> 19 regD_04 clust_3 0.3496858
#> 20 regD clust_3 0.3496858
#> 21 regD_02 clust_3 0.3496858
#> 22 regC_06 clust_4 0.4882054
#> 23 regC_05 clust_4 0.4882054
#> 24 regC_04 clust_4 0.4882054
#> 25 regC_03 clust_4 0.4882054
#> 26 regC_02 clust_4 0.4882054
#> 27 regC clust_4 0.4882054
#> 28 regC_01 clust_4 0.4882054
#> 29 regB_05 clust_5 0.2574193
#> 30 regB_04 clust_5 0.2574193
#> 31 regB_03 clust_5 0.2574193
#> 32 regB_02 clust_5 0.2574193
#> 33 regB clust_5 0.2574193
#> 34 regB_01 clust_5 0.2574193
#> 35 regB_08 clust_5 0.2574193
#> 36 regB_07 clust_5 0.2574193
#> 37 regB_06 clust_5 0.2574193
#> 38 regA_07 clust_6 0.3734175
#> 39 regA_06 clust_6 0.3734175
#> 40 regA_05 clust_6 0.3734175
#> 41 regA_04 clust_6 0.3734175
#> 42 regA_03 clust_6 0.3734175
#> 43 regA_02 clust_6 0.3734175
#> 44 regA clust_6 0.3734175
#> 45 regA_01 clust_6 0.37341756 Multi Local Zscore
The [multiLocalZscore] function allows the association of a single regions Set against a list of regions Sets to be analysed by applying the localZScore function in a neighbourhood defined by the window parameter a number of times defined by the step parameter.
#NOT RUN
mlz_Alien_ReG<-multiLocalZscore(A = AlienRSList_narrow$regA,
Blist = AlienRSList_narrow,
ranFUN = "resampleGenome",
evFUN = "numOverlaps",
window = 100,
step = 1,
max_pv = 1,
genome = AlienGenome)As with the crosswisePermTest() function, an object consisting of three slots paremeters multiLocalZscores and matrix will be created. These can be queried by the functions getParametrs(), getMultiEvaluation() and getMatrix() respectively. Again, the object thus created will have the matrix slot = NULL.
getParameters(mLZ_regA_ReG)
#> parameter value
#> 1 A AlienRSList_narrow$regA
#> 2 Blist AlienRSList_narrow
#> 3 sampling FALSE
#> 4 fraction 0.15
#> 5 min_sampling 0.15
#> 6 ranFUN resampleGenome
#> 7 evFUN numOverlaps
#> 8 universe NULL
#> 9 window 600
#> 10 step 10
#> 11 adj_pv_method BH
#> 12 max_pv 0.05
#> 13 Nregions 100
mlz_Me <- getMultiEvaluation(rR = mLZ_regA_ReG )
head(mlz_Me$resumeTable)
#> name p_value z_score mean_perm_test sd_perm_test n_overlaps
#> 1 regA_01 0.000999001 115.5593 0.576 0.7738364 90
#> 2 regA_02 0.000999001 104.9830 0.559 0.7662285 81
#> 3 regA_03 0.000999001 91.9693 0.568 0.7549476 70
#> 4 regA_04 0.000999001 79.3280 0.555 0.7493574 60
#> 5 regA_05 0.000999001 65.0379 0.598 0.7595874 50
#> 6 regA_06 0.000999001 52.3927 0.600 0.7710996 41
#> norm_zscore adj.p_value
#> 1 11.55593 0.003
#> 2 10.49830 0.003
#> 3 9.19693 0.003
#> 4 7.93280 0.003
#> 5 6.50379 0.003
#> 6 5.23927 0.003The matrix will be created by the function makeLZMatrix() in a similar manner as illustrated for the function makeCrosswiseMatrix(). Again, it is possible to pass a clustering method to the function, and if not, the best clustering function calculated using chooseHclustMet() will be chosen.
mLZ_regA_ReG <- makeLZMatrix(mLZ_regA_ReG)
#> [1] "method selected for hclustering: complete"
#> complete average single ward.D2 median centroid mcquitty
#> 0.9098230 0.8541174 0.7986368 0.8320493 0.8348816 0.8510903 0.8475492
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.8909663 0.8944326 0.7892161 0.8311822 0.8813696 0.8857083 0.8784679
plot(getHClust(mLZ_regA_ReG),cex = 0.8)
The plotLocalZScoreMatrix() function allows the visualisation of an object of class multilocalZScore. This type of visualization will also allow the association to be tested in the area adjacent to the tested Region Set.
plotLocalZScoreMatrix(mLZ_regA_ReG, maxVal = "max")
## Single Local ZScore
Similarly to as seen above for the plotSinglePT() function, the plotSingleLZ() function allows us to specifically display single associations drawn from a multiLocalZScore object.
plotSingleLZ(mLZ = mLZ_regA_ReG, RS =c("regA"), smoothing = TRUE)
It is also possible to simultaneously display multiple associations in the same graph by introducing a vector of region set names
plotSingleLZ(mLZ = mLZ_regA_ReG,RS =c("regA","regA_02","regA_06","regA_08","regD"),smoothing = TRUE)
7 Analyses of non-square matrices
regioneReloaded allows the association of Regions Sets of any length to be calculated. AlienRSList_broad is a list of Region Set created from AlienRSList_narrow, in which the extendRegions function has increased the width to an average of 5kb, to complete AlienRSList_broad a set of random region sets was added (RegR).
# average of the widths of AlienRSList_narrow
do.call("c",lapply(lapply(AlienRSList_narrow, width),mean))
#> regA_01 regA_02 regA_03 regA_04 regA_05 regA_06 regA_07
#> 104.05 104.05 104.05 104.05 104.05 104.05 104.05
#> regA_08 regA_09 regA regB_01 regB_02 regB_03 regB_04
#> 104.05 104.05 104.05 99.26 99.26 99.26 99.26
#> regB_05 regB_06 regB_07 regB_08 regB_09 regB regC_01
#> 99.26 99.26 99.26 99.26 99.26 99.26 101.46
#> regC_02 regC_03 regC_04 regC_05 regC_06 regC_07 regC_08
#> 101.46 101.46 101.46 101.46 101.46 101.46 101.46
#> regC_09 regC regD_02 regD_04 regD_06 regD_08 regD
#> 101.46 101.46 104.05 104.05 104.05 104.05 104.05
#> regAB_02 regAB_04 regAB_06 regAB_08 regAB reg_no_A reg_no_B
#> 98.48 98.48 98.48 98.48 98.48 104.79 101.69
#> reg_no_C reg_no_AB reg_no_ABC
#> 102.93 102.54 94.02
# average of the widths of AlienRSList_broad
do.call("c",lapply(lapply(AlienRSList_broad, width),mean))
#> regA_br_02 regA_br_04 regA_br_06 regA_br_08 regA_br regB_br_02 regB_br_04
#> 5104.05 5104.05 5104.05 5104.05 5104.05 5099.26 5099.26
#> regB_br_06 regB_br_08 regB_br regC_br_02 regC_br_04 regC_br_06 regC_br_08
#> 5099.26 5099.26 5099.26 5101.46 5101.46 5101.46 5101.46
#> regC_br regD_br_02 regD_br_04 regD_br_06 regD_br_08 regD_br regR_br_02
#> 5101.46 5104.05 5104.05 5104.05 5104.05 5104.05 5023.83
#> regR_br_04 regR_br_06 regR_br_08 regR_br
#> 5023.83 5023.83 5023.83 5023.83
##NOT RUN
cw_Alien_RaR_no_Square <- crosswisePermTest(
Alist = AlienRSList_narrow,
Blist = AlienRSList_broad,
sampling = FALSE,
genome = AlienGenome,
per.chromosome=TRUE,
ranFUN = "resampleGenome",
evFUN = "numOverlaps",
ntimes= 100,
mc.cores = 6
)
###the matrix calculated in this case is based on a double clustering of rows and columns
cw_Alien_ReG_no_Square <- makeCrosswiseMatrix(cw_Alien_ReG_no_Square)
#> Warning in makeCrosswiseMatrix(cw_Alien_ReG_no_Square): impossible to create
#> symmetrical matrix, number of columns is different from number of rows
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.7848737 0.7898638 0.7125290 0.7639944 0.7359119 0.7656280 0.7740310
#> [1] "method selected for hclustering: complete"
#> complete average single ward.D2 median centroid mcquitty
#> 0.7582517 0.7077825 0.4051347 0.6923967 0.7266403 0.7205035 0.7558722
plotCrosswiseMatrix(cw_Alien_ReG_no_Square)
In this case, the multiLocalZscore() function works in exactly the same way as in the previous cases, but in order to see the form of the association, it is recommended to enlarge window parameter.
#NOT RUN
mLZ_regA_ReG_br<-multiLocalZscore(A =
AlienRSList_narrow$regA,
Blist = AlienRSList_broad,
ranFUN = "resampleGenome",
genome = AlienGenome,
window = 5000,
step =100)
##mLZ_regA_ReG_br<-makeLZMatrix(mLZ_regA_ReG_br)
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.8736273 0.8783594 0.8460795 0.8706955 0.8744848 0.8776389 0.8773126
#> [1] "method selected for hclustering: average"
#> complete average single ward.D2 median centroid mcquitty
#> 0.9348281 0.9374008 0.9133837 0.9153168 0.8793742 0.9256832 0.8900115
plotLocalZScoreMatrix(mLZ_regA_ReG_br)
plotSingleLZ(mLZ = mLZ_regA_ReG_br,RS =c("regA_br","regA_br_02","regA_br_06","regA_br_08","regD_br"), smoothing = TRUE)
8 Session Info
sessionInfo()
#> R version 4.3.0 RC (2023-04-13 r84269)
#> Platform: x86_64-pc-linux-gnu (64-bit)
#> Running under: Ubuntu 22.04.2 LTS
#>
#> Matrix products: default
#> BLAS: /home/biocbuild/bbs-3.17-bioc/R/lib/libRblas.so
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_GB LC_COLLATE=C
#> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
#> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
#> [9] LC_ADDRESS=C LC_TELEPHONE=C
#> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
#>
#> time zone: America/New_York
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] regioneReloaded_1.2.0 regioneR_1.32.0 GenomicRanges_1.52.0
#> [4] GenomeInfoDb_1.36.0 IRanges_2.34.0 S4Vectors_0.38.0
#> [7] BiocGenerics_0.46.0 BiocStyle_2.28.0
#>
#> loaded via a namespace (and not attached):
#> [1] tidyselect_1.2.0 dplyr_1.1.2
#> [3] farver_2.1.1 Biostrings_2.68.0
#> [5] bitops_1.0-7 fastmap_1.1.1
#> [7] RCurl_1.98-1.12 GenomicAlignments_1.36.0
#> [9] XML_3.99-0.14 digest_0.6.31
#> [11] lifecycle_1.0.3 cluster_2.1.4
#> [13] magrittr_2.0.3 compiler_4.3.0
#> [15] rlang_1.1.0 sass_0.4.5
#> [17] tools_4.3.0 utf8_1.2.3
#> [19] yaml_2.3.7 rtracklayer_1.60.0
#> [21] knitr_1.42 askpass_1.1
#> [23] labeling_0.4.2 reticulate_1.28
#> [25] DelayedArray_0.26.0 plyr_1.8.8
#> [27] RColorBrewer_1.1-3 BiocParallel_1.34.0
#> [29] Rtsne_0.16 withr_2.5.0
#> [31] grid_4.3.0 fansi_1.0.4
#> [33] colorspace_2.1-0 ggplot2_3.4.2
#> [35] MASS_7.3-59 scales_1.2.1
#> [37] SummarizedExperiment_1.30.0 cli_3.6.1
#> [39] rmarkdown_2.21 crayon_1.5.2
#> [41] generics_0.1.3 umap_0.2.10.0
#> [43] RSpectra_0.16-1 reshape2_1.4.4
#> [45] rjson_0.2.21 cachem_1.0.7
#> [47] stringr_1.5.0 zlibbioc_1.46.0
#> [49] parallel_4.3.0 BiocManager_1.30.20
#> [51] XVector_0.40.0 restfulr_0.0.15
#> [53] matrixStats_0.63.0 vctrs_0.6.2
#> [55] Matrix_1.5-4 jsonlite_1.8.4
#> [57] bookdown_0.33 ggrepel_0.9.3
#> [59] magick_2.7.4 jquerylib_0.1.4
#> [61] glue_1.6.2 codetools_0.2-19
#> [63] stringi_1.7.12 gtable_0.3.3
#> [65] BiocIO_1.10.0 munsell_0.5.0
#> [67] tibble_3.2.1 pillar_1.9.0
#> [69] htmltools_0.5.5 openssl_2.0.6
#> [71] GenomeInfoDbData_1.2.10 BSgenome_1.68.0
#> [73] R6_2.5.1 evaluate_0.20
#> [75] lattice_0.21-8 Biobase_2.60.0
#> [77] highr_0.10 png_0.1-8
#> [79] Rsamtools_2.16.0 memoise_2.0.1
#> [81] bslib_0.4.2 Rcpp_1.0.10
#> [83] xfun_0.39 MatrixGenerics_1.12.0
#> [85] pkgconfig_2.0.3