Epiregulon tutorial with MultiAssayExperiment
Introduction
Gene regulatory networks model the underlying gene regulation hierarchies that drive gene expression and cell states. The main functions of this package are to construct gene regulatory networks and infer transcription factor (TF) activity at the single cell level by integrating scATAC-seq and scRNA-seq data and incorporating of public bulk TF ChIP-seq data.
There are three related packages: epiregulon,
epiregulon.extra
and epiregulon.archr, the two of which are available
through Bioconductor and the last of which is only available through github. The
basic epiregulon package takes in gene expression and
chromatin accessibility matrices in the form of
SingleCellExperiment objects, constructs gene regulatory
networks (“regulons”) and outputs the activity of transcription factors
at the single cell level. The epiregulon.extra package
provides a suite of tools for enrichment analysis of target genes,
visualization of target genes and transcription factor activity, and
network analysis which can be run on the epiregulon output.
If the users would like to start from ArchR projects
instead of SingleCellExperiment objects, they may choose to
use epiregulon.archr package, which allows for seamless
integration with the ArchR
package, and continue with the rest of the workflow offered in
epiregulon.extra.
For full documentation of the epiregulon package, please
refer to the epiregulon
book.
Installation
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("epiregulon")Alternatively, you could install from github
Load package
Data preparation
Prior to using epiregulon, single cell preprocessing
needs to performed by user’s favorite methods. The following components
are required:
1. Peak matrix from scATAC-seq containing the
chromatin accessibility information
2. Gene expression matrix from
either paired or unpaired scRNA-seq. RNA-seq integration needs to be
performed for unpaired dataset.
3. Dimensionality reduction matrix
from either single modality dataset or joint scRNA-seq and scATAC-seq
This tutorial demonstrates the basic functions of
epiregulon, using the reprogram-seq dataset which can be
downloaded from the scMultiome
package. In this example, prostate cancer cells (LNCaP) were infected in
separate wells with viruses encoding 4 transcription factors (NKX2-1,
GATA6, FOXA1 and FOXA2) and a positive control (mNeonGreen) before
pooling. The identity of the infected transcription factors was tracked
through cell hashing (available in the field
hash_assignment of the colData) and serves as
the ground truth.
# load the MAE object
library(scMultiome)
mae <- scMultiome::reprogramSeq()
# extract peak matrix
PeakMatrix <- mae[["PeakMatrix"]]
# extract expression matrix
GeneExpressionMatrix <- mae[["GeneExpressionMatrix"]]
rownames(GeneExpressionMatrix) <- rowData(GeneExpressionMatrix)$name
# define the order of hash_assigment
GeneExpressionMatrix$hash_assignment <-
factor(as.character(GeneExpressionMatrix$hash_assignment),
levels = c("HTO10_GATA6_UTR", "HTO2_GATA6_v2",
"HTO8_NKX2.1_UTR", "HTO3_NKX2.1_v2",
"HTO1_FOXA2_v2", "HTO4_mFOXA1_v2", "HTO6_hFOXA1_UTR",
"HTO5_NeonG_v2"))
# extract dimensional reduction matrix
reducedDimMatrix <- reducedDim(mae[['TileMatrix500']], "LSI_ATAC")
# transfer UMAP_combined from TileMatrix to GeneExpressionMatrix
reducedDim(GeneExpressionMatrix, "UMAP_Combined") <-
reducedDim(mae[['TileMatrix500']], "UMAP_Combined")Visualize singleCellExperiment by UMAP
scater::plotReducedDim(GeneExpressionMatrix,
dimred = "UMAP_Combined",
text_by = "Clusters",
colour_by = "Clusters")
Quick start
Retrieve bulk TF ChIP-seq binding sites
First, we retrieve a GRangesList object containing the
binding sites of all the transcription factors and co-regulators. These
binding sites are derived from bulk ChIP-seq data in the ChIP-Atlas and ENCODE databases. For the same
transcription factor, multiple ChIP-seq files from different cell lines
or tissues are merged. For further information on how these peaks are
derived, please refer to ?epiregulon::getTFMotifInfo.
Currently, human genomes hg19 and hg38 and mouse mm10 are supported.
grl <- getTFMotifInfo(genome = "hg38")
#> Retrieving chip-seq data, version 2
#> see ?scMultiome and browseVignettes('scMultiome') for documentation
#> downloading 1 resources
#> retrieving 1 resource
#> loading from cache
grl
#> GRangesList object of length 1377:
#> $AEBP2
#> GRanges object with 2700 ranges and 0 metadata columns:
#> seqnames ranges strand
#> <Rle> <IRanges> <Rle>
#> [1] chr1 9792-10446 *
#> [2] chr1 942105-942400 *
#> [3] chr1 984486-984781 *
#> [4] chr1 3068932-3069282 *
#> [5] chr1 3069411-3069950 *
#> ... ... ... ...
#> [2696] chrY 8465261-8465730 *
#> [2697] chrY 11721744-11722260 *
#> [2698] chrY 11747448-11747964 *
#> [2699] chrY 19302661-19303134 *
#> [2700] chrY 19985662-19985982 *
#> -------
#> seqinfo: 25 sequences from an unspecified genome; no seqlengths
#>
#> ...
#> <1376 more elements>We recommend the use of ChIP-seq data over motif for estimating TF
occupancy. However, if the user would like to start from motifs, it is
possible to switch to the motif mode. The user can provide
the peak regions as a GRanges object and the location of
the motifs will be annotated based on Cisbp from chromVARmotifs
(human_pwms_v2 and mouse_pwms_v2, version 0.2)
Link ATAC-seq peaks to target genes
Next, we try to link ATAC-seq peaks to their putative target genes. We assign a peak to a gene within a size window (default ±250kb) if the chromatin accessibility of the peak and expression of the target genes are highly correlated (default p-vale threshold 0.05). To compute correlations, we first create cell aggregates by performing k-means clustering on the reduced dimensionality matrix. Then we aggregate the counts of the gene expression and peak matrix and average across the number of cells. Correlations are computed on the averaged gene expression and chromatin accessibility.
Optimize the number of metacells (Optional)
The number of metacells used to aggregate cells into meta-cells is a
sensitive parameter, as it should strike the correct balance between
averaging out biological variation and reducing signal noise caused by
sparsity in single cells. Therefore, we provide a separate function,
optimizeMetacellNumber, to automatically select the optimal
value for this parameter. The algorithm samples different possible
values and calculates the mean empirical p-value of RE–TG connections
for each value. Then, a quadratic regression is fitted to the
relationship, and the minimum of the function is determined.
cellNum <- optimizeMetacellNumber(peakMatrix = PeakMatrix,
expMatrix = GeneExpressionMatrix,
reducedDim = reducedDimMatrix,
exp_assay = "normalizedCounts",
peak_assay = "counts",
BPPARAM = BiocParallel::SerialParam(progressbar = FALSE))
#> Warning in optimizeMetacellNumber(peakMatrix = PeakMatrix, expMatrix =
#> GeneExpressionMatrix, : Coefficient of determination of the regression model is
#> lower thant 0.7
#> An issue detected during estimation optimal number of metacells.
#> Consider at least one of the following actions:
#> 1. Change of the `cellNumMin` and `cellNumMax` paramaters
#> 2. Increasing the number of evaluation points (`n_evaluation_points` argument)
#> 3. Increasing the number of iterations (`n_iter` argument)
#> 4. Increasing the number of false connections used to compute p-value
#> null distribution (`nRandConns` argument)
#> Solution not found using quadratic regression. Using cluster size with the lowest mean p-value.We can plot the results to make sure that the relationship is correctly generalized.
Calculate Peak to Gene Links
The output of optimizeMetacellNumber function is then
passed to calculateP2G as cellNum argument.
Alternatively, users may skip optimizeMetacellNumber and
specify their own cell number in cellNum.
set.seed(1010)
p2g <- calculateP2G(peakMatrix = PeakMatrix,
expMatrix = GeneExpressionMatrix,
reducedDim = reducedDimMatrix,
exp_assay = "normalizedCounts",
peak_assay = "counts",
cellNum = cellNum,
BPPARAM = BiocParallel::SerialParam(progressbar = FALSE))
#> Using epiregulon to compute peak to gene links...
#> Creating metacells...
#> Looking for regulatory elements near target genes...
#> Computing correlations...
p2g
#> DataFrame with 28120 rows and 10 columns
#> idxATAC chr start end idxRNA target
#> <integer> <character> <integer> <integer> <integer> <array>
#> 1 1 chr1 817121 817621 15 LINC01409
#> 2 8 chr1 917255 917755 27 PLEKHN1
#> 3 10 chr1 920987 921487 28 PERM1
#> 4 14 chr1 932922 933422 18 LINC00115
#> 5 14 chr1 932922 933422 20 AL645608.6
#> ... ... ... ... ... ... ...
#> 28116 126579 chrX 154799320 154799820 36414 DKC1
#> 28117 126581 chrX 154917936 154918436 36420 FUNDC2
#> 28118 126589 chrX 155216677 155217177 36426 CLIC2
#> 28119 126590 chrX 155228844 155229344 36426 CLIC2
#> 28120 126591 chrX 155264314 155264814 36424 VBP1
#> Correlation p_val_peak_gene FDR_peak_gene distance
#> <matrix> <matrix> <matrix> <integer>
#> 1 0.670692 0.01973535 0.876821 38363
#> 2 -0.426836 0.03049020 0.893849 48725
#> 3 0.638859 0.02585148 0.892849 59540
#> 4 -0.576828 0.00344532 0.849255 105400
#> 5 -0.482178 0.01426327 0.874095 28088
#> ... ... ... ... ...
#> 28116 -0.498616 0.01140705 0.867484 36578
#> 28117 -0.501012 0.01110357 0.867484 108406
#> 28118 0.601789 0.03426402 0.893849 117435
#> 28119 0.803239 0.00582056 0.849255 105268
#> 28120 0.597658 0.03544632 0.893849 67307Add TF motif binding to peaks
The next step is to add the TF binding information by overlapping regions of the peak matrix with the bulk chip-seq database. The output is a data frame object with three columns:
idxATAC- index of the peak in the peak matrixidxTF- index in the gene expression matrix corresponding to the transcription factortf- name of the transcription factor
Generate regulons
A DataFrame, representing the inferred regulons, is then generated. The DataFrame consists of ten columns:
idxATAC- index of the peak in the peak matrixchr- chromosome numberstart- start position of the peakend- end position of the peakidxRNA- index in the gene expression matrix corresponding to the target genetarget- name of the target genedistance- distance between the transcription start site of the target gene and the middle of the peakidxTF- index in the gene expression matrix corresponding to the transcription factortf- name of the transcription factorcorr- correlation between target gene expression and the chromatin accessibility at the peak. if cluster labels are provided, this field is a matrix with columns names corresponding to correlation across all cells and for each of the clusters.
regulon <- getRegulon(p2g = p2g,
overlap = overlap)
regulon
#> DataFrame with 5265039 rows and 12 columns
#> idxATAC chr start end idxRNA target corr
#> <integer> <character> <integer> <integer> <integer> <array> <matrix>
#> 1 1 chr1 817121 817621 15 LINC01409 0.670692
#> 2 1 chr1 817121 817621 15 LINC01409 0.670692
#> 3 1 chr1 817121 817621 15 LINC01409 0.670692
#> 4 1 chr1 817121 817621 15 LINC01409 0.670692
#> 5 1 chr1 817121 817621 15 LINC01409 0.670692
#> ... ... ... ... ... ... ... ...
#> 5265035 126591 chrX 155264314 155264814 36424 VBP1 0.597658
#> 5265036 126591 chrX 155264314 155264814 36424 VBP1 0.597658
#> 5265037 126591 chrX 155264314 155264814 36424 VBP1 0.597658
#> 5265038 126591 chrX 155264314 155264814 36424 VBP1 0.597658
#> 5265039 126591 chrX 155264314 155264814 36424 VBP1 0.597658
#> p_val_peak_gene FDR_peak_gene distance idxTF tf
#> <matrix> <matrix> <integer> <integer> <character>
#> 1 0.0197353 0.876821 38363 16 ATF1
#> 2 0.0197353 0.876821 38363 17 ATF2
#> 3 0.0197353 0.876821 38363 18 ATF3
#> 4 0.0197353 0.876821 38363 21 ATF7
#> 5 0.0197353 0.876821 38363 35 BRCA2
#> ... ... ... ... ... ...
#> 5265035 0.0354463 0.893849 67307 1316 ZNF692
#> 5265036 0.0354463 0.893849 67307 1357 ZNF843
#> 5265037 0.0354463 0.893849 67307 1366 ZSCAN21
#> 5265038 0.0354463 0.893849 67307 1370 ZSCAN30
#> 5265039 0.0354463 0.893849 67307 1376 ZXDBNetwork pruning (highly recommended)
Epiregulon prunes the network by performing tests of independence on the observed number of cells jointly expressing transcription factor (TF), regulatory element (RE) and target gene (TG) vs the expected number of cells if TF/RE and TG are independently expressed. The users can choose between two tests, the binomial test and the chi-square test. In the binomial test, the expected probability is P(TF, RE) * P(TG), and the number of trials is the total number of cells, and the observed successes is the number of cells jointly expressing all three elements. In the chi-square test, the expected probability for having all 3 elements active is also P(TF, RE) * P(TG) and the probability otherwise is 1- P(TF, RE) * P(TG). The observed cell count for the category of “active TF” is the number of cells jointly expressing all three elements, and the cell count for the inactive category is n - n_triple.
We calculate cluster-specific p-values if users supply cluster labels. This is useful if we are interested in cluster-specific networks. The pruned regulons can then be used to visualize differential networks for transcription factors of interest.
pruned.regulon <- pruneRegulon(expMatrix = GeneExpressionMatrix,
exp_assay = "normalizedCounts",
peakMatrix = PeakMatrix,
peak_assay = "counts",
test = "chi.sq",
regulon = regulon[regulon$tf %in%
c("NKX2-1","GATA6","FOXA1","FOXA2", "AR"),],
clusters = GeneExpressionMatrix$Clusters,
prune_value = "pval",
regulon_cutoff = 0.05
)
pruned.regulonAdd Weights
While the pruneRegulon function provides statistics on
the joint occurrence of TF-RE-TG, we would like to further estimate the
strength of regulation. Biologically, this can be interpreted as the
magnitude of gene expression changes induced by transcription factor
activity. Epiregulon estimates the regulatory potential using one of the
three measures: 1) correlation between TF and target gene expression, 2)
mutual information between the TF and target gene expression and 3)
Wilcoxon test statistics of target gene expression in cells jointly
expressing all 3 elements vs cells that do not.
Two of the measures (correlation and Wilcoxon statistics) give both the magnitude and directionality of changes whereas mutual information is always positive. The correlation and mutual information statistics are computed on pseudobulks aggregated by user-supplied cluster labels, whereas the Wilcoxon method groups cells into two categories: 1) the active category of cells jointly expressing TF, RE and TG and 2) the inactive category consisting of the remaining cells.
We calculate cluster-specific weights if users supply cluster labels.
Annotate with TF motifs (Optional)
So far the gene regulatory network was constructed from TF ChIP-seq
exclusively. Some users would prefer to further annotate regulatory
elements with the presence of motifs. We provide an option to annotate
peaks with motifs from the Cisbp database. If no motifs are present for
this particular factor (as in the case of co-factors or chromatin
modifiers), we return NA. If motifs are available for a factor and the
RE contains a motif, we return 1. If motifs are available and the RE
does not contain a motif, we return 0. The users can also provide their
own motif annotation through the pwms argument.
regulon.w.motif <- addMotifScore(regulon = regulon.w,
peaks = rowRanges(PeakMatrix),
species = "human",
genome = "hg38")
#> annotating peaks with motifs
#> see ?scMultiome and browseVignettes('scMultiome') for documentation
#> downloading 1 resources
#> retrieving 1 resource
#> loading from cache
#>
#> Attaching package: 'Biostrings'
#> The following object is masked from 'package:base':
#>
#> strsplit
#>
#> Attaching package: 'rtracklayer'
#> The following object is masked from 'package:AnnotationHub':
#>
#> hubUrl
# if desired, set weight to 0 if no motif is found
regulon.w.motif$weight[regulon.w.motif$motif == 0] <- 0
regulon.w.motif
#> DataFrame with 12405 rows and 17 columns
#> idxATAC chr start end idxRNA target corr
#> <integer> <character> <integer> <integer> <integer> <array> <matrix>
#> 1 10 chr1 920987 921487 28 PERM1 0.638859
#> 2 14 chr1 932922 933422 25 NOC2L -0.395829
#> 3 14 chr1 932922 933422 20 AL645608.6 -0.482178
#> 4 14 chr1 932922 933422 28 PERM1 -0.388740
#> 5 37 chr1 1020509 1021009 33 AGRN 0.639086
#> ... ... ... ... ... ... ... ...
#> 12401 123380 chr9 128635245 128635745 35034 SPTAN1 0.843048
#> 12402 123444 chr9 129139814 129140314 35053 SH3GLB2 -0.399388
#> 12403 124120 chr9 137240872 137241372 35274 NELFB -0.394052
#> 12404 124405 chrX 15737784 15738284 35402 AP1S2 -0.389650
#> 12405 124978 chrX 47541057 47541557 35594 ZNF41 -0.452951
#> p_val_peak_gene FDR_peak_gene distance idxTF tf
#> <matrix> <matrix> <integer> <integer> <character>
#> 1 0.0258515 0.892849 59540 485 AR
#> 2 0.0436110 0.893849 25832 485 AR
#> 3 0.0142633 0.874095 28088 485 AR
#> 4 0.0469135 0.893849 47605 485 AR
#> 5 0.0258060 0.892849 389 485 AR
#> ... ... ... ... ... ...
#> 12401 0.00356964 0.849255 82687 723 NKX2-1
#> 12402 0.04196865 0.893849 111629 723 NKX2-1
#> 12403 0.04450355 0.893849 13953 723 NKX2-1
#> 12404 0.04650291 0.893849 116469 723 NKX2-1
#> 12405 0.02158235 0.880129 58472 723 NKX2-1
#> pval stats qval
#> <matrix> <matrix> <matrix>
#> 1 0.103465:0.880435:1:... 2.651304:0.0226256:0:... 1:1:1:...
#> 2 0.280507:0.843914:1:... 1.164630:0.0387653:0:... 1:1:1:...
#> 3 0.182226:0.509635:1:... 1.779378:0.4348165:0:... 1:1:1:...
#> 4 0.559820:0.787860:1:... 0.340016:0.0724101:0:... 1:1:1:...
#> 5 0.105800:0.564073:1:... 2.615861:0.3327009:0:... 1:1:1:...
#> ... ... ... ...
#> 12401 4.51202e-02:1:1:... 4.01414:0:0:... 1.000000:1:1:...
#> 12402 1.79623e-01:1:1:... 1.80074:0:0:... 1.000000:1:1:...
#> 12403 1.07516e-01:1:1:... 2.59036:0:0:... 1.000000:1:1:...
#> 12404 1.65845e-02:1:1:... 5.73982:0:0:... 1.000000:1:1:...
#> 12405 9.49637e-06:1:1:... 19.61014:0:0:... 0.680861:1:1:...
#> weight motif
#> <matrix> <numeric>
#> 1 0:0:0:... 0
#> 2 0:0:0:... 0
#> 3 0:0:0:... 0
#> 4 0:0:0:... 0
#> 5 0:0:0:... 0
#> ... ... ...
#> 12401 0:0:0:... 0
#> 12402 0:0:0:... 0
#> 12403 0:0:0:... 0
#> 12404 0:0:0:... 0
#> 12405 0:0:0:... 0Annotate with log fold changes (Optional)
It is sometimes helpful to filter the regulons based on gene
expression changes between two conditions. The addLogFC
function is a wrapper around scran::findMarkers and adds
extra columns of log changes to the regulon DataFrame. The users can
specify the reference condition in logFC_ref and conditions
for comparison in logFC_condition. If these are not
provided, log fold changes are calculated for every condition in the
cluster labels against the rest of the conditions.
# create logcounts
GeneExpressionMatrix <- scuttle::logNormCounts(GeneExpressionMatrix)
# add log fold changes which are calculated by taking the difference of the log counts
regulon.w <- addLogFC(regulon = regulon.w,
clusters = GeneExpressionMatrix$hash_assignment,
expMatrix = GeneExpressionMatrix,
assay.type = "logcounts",
pval.type = "any",
logFC_condition = c("HTO10_GATA6_UTR",
"HTO2_GATA6_v2",
"HTO8_NKX2.1_UTR"),
logFC_ref = "HTO5_NeonG_v2")
regulon.w
#> DataFrame with 12405 rows and 25 columns
#> idxATAC chr start end idxRNA target corr
#> <integer> <character> <integer> <integer> <integer> <array> <matrix>
#> 1 10 chr1 920987 921487 28 PERM1 0.638859
#> 2 14 chr1 932922 933422 25 NOC2L -0.395829
#> 3 14 chr1 932922 933422 20 AL645608.6 -0.482178
#> 4 14 chr1 932922 933422 28 PERM1 -0.388740
#> 5 37 chr1 1020509 1021009 33 AGRN 0.639086
#> ... ... ... ... ... ... ... ...
#> 12401 123380 chr9 128635245 128635745 35034 SPTAN1 0.843048
#> 12402 123444 chr9 129139814 129140314 35053 SH3GLB2 -0.399388
#> 12403 124120 chr9 137240872 137241372 35274 NELFB -0.394052
#> 12404 124405 chrX 15737784 15738284 35402 AP1S2 -0.389650
#> 12405 124978 chrX 47541057 47541557 35594 ZNF41 -0.452951
#> p_val_peak_gene FDR_peak_gene distance idxTF tf
#> <matrix> <matrix> <integer> <integer> <character>
#> 1 0.0258515 0.892849 59540 485 AR
#> 2 0.0436110 0.893849 25832 485 AR
#> 3 0.0142633 0.874095 28088 485 AR
#> 4 0.0469135 0.893849 47605 485 AR
#> 5 0.0258060 0.892849 389 485 AR
#> ... ... ... ... ... ...
#> 12401 0.00356964 0.849255 82687 723 NKX2-1
#> 12402 0.04196865 0.893849 111629 723 NKX2-1
#> 12403 0.04450355 0.893849 13953 723 NKX2-1
#> 12404 0.04650291 0.893849 116469 723 NKX2-1
#> 12405 0.02158235 0.880129 58472 723 NKX2-1
#> pval stats qval
#> <matrix> <matrix> <matrix>
#> 1 0.103465:0.880435:1:... 2.651304:0.0226256:0:... 1:1:1:...
#> 2 0.280507:0.843914:1:... 1.164630:0.0387653:0:... 1:1:1:...
#> 3 0.182226:0.509635:1:... 1.779378:0.4348165:0:... 1:1:1:...
#> 4 0.559820:0.787860:1:... 0.340016:0.0724101:0:... 1:1:1:...
#> 5 0.105800:0.564073:1:... 2.615861:0.3327009:0:... 1:1:1:...
#> ... ... ... ...
#> 12401 4.51202e-02:1:1:... 4.01414:0:0:... 1.000000:1:1:...
#> 12402 1.79623e-01:1:1:... 1.80074:0:0:... 1.000000:1:1:...
#> 12403 1.07516e-01:1:1:... 2.59036:0:0:... 1.000000:1:1:...
#> 12404 1.65845e-02:1:1:... 5.73982:0:0:... 1.000000:1:1:...
#> 12405 9.49637e-06:1:1:... 19.61014:0:0:... 0.680861:1:1:...
#> weight HTO10_GATA6_UTR.vs.HTO5_NeonG_v2.FDR
#> <matrix> <numeric>
#> 1 0.02638820:-0.00723681:0:... 1.0000000
#> 2 0.01967612: 0.00569181:0:... 0.9752600
#> 3 0.03590731:-0.03273486:0:... 0.3913742
#> 4 0.00967161:-0.01328838:0:... 1.0000000
#> 5 0.02420994:-0.02931996:0:... 0.0333213
#> ... ... ...
#> 12401 0.0277381:0:0:... 0.891051
#> 12402 0.0211504:0:0:... 1.000000
#> 12403 0.0246483:0:0:... 1.000000
#> 12404 0.0397841:0:0:... 1.000000
#> 12405 0.0696895:0:0:... 1.000000
#> HTO10_GATA6_UTR.vs.HTO5_NeonG_v2.p.value
#> <numeric>
#> 1 -0.306482
#> 2 -1.087602
#> 3 -3.630700
#> 4 -0.306482
#> 5 -6.908244
#> ... ...
#> 12401 -2.088809
#> 12402 -0.875472
#> 12403 -0.346989
#> 12404 -0.817361
#> 12405 -1.048695
#> HTO10_GATA6_UTR.vs.HTO5_NeonG_v2.logFC HTO2_GATA6_v2.vs.HTO5_NeonG_v2.FDR
#> <numeric> <numeric>
#> 1 0.00267018 0.95051180
#> 2 -0.02672751 1.00000000
#> 3 -0.02908575 1.00000000
#> 4 0.00267018 0.95051180
#> 5 0.06228930 0.00813329
#> ... ... ...
#> 12401 0.05722383 0.0780338
#> 12402 -0.01947302 1.0000000
#> 12403 -0.00696269 1.0000000
#> 12404 -0.01490592 0.9505118
#> 12405 0.01652101 1.0000000
#> HTO2_GATA6_v2.vs.HTO5_NeonG_v2.p.value
#> <numeric>
#> 1 -1.145473
#> 2 -0.764604
#> 3 -0.613957
#> 4 -1.145473
#> 5 -8.744274
#> ... ...
#> 12401 -5.984106
#> 12402 -0.685001
#> 12403 -0.430542
#> 12404 -1.533481
#> 12405 -0.434791
#> HTO2_GATA6_v2.vs.HTO5_NeonG_v2.logFC HTO8_NKX2.1_UTR.vs.HTO5_NeonG_v2.FDR
#> <numeric> <numeric>
#> 1 -0.00410095 1.000000
#> 2 -0.01947775 1.000000
#> 3 -0.00903561 1.000000
#> 4 -0.00410095 1.000000
#> 5 0.06470266 0.968083
#> ... ... ...
#> 12401 0.11104599 0.992412
#> 12402 -0.01469769 1.000000
#> 12403 -0.00758876 1.000000
#> 12404 -0.02184636 0.978457
#> 12405 0.00696425 0.978457
#> HTO8_NKX2.1_UTR.vs.HTO5_NeonG_v2.p.value
#> <numeric>
#> 1 -0.125877
#> 2 -0.877433
#> 3 -0.318033
#> 4 -0.125877
#> 5 -2.479073
#> ... ...
#> 12401 -1.122331
#> 12402 -0.154245
#> 12403 -0.784077
#> 12404 -1.855533
#> 12405 -1.356691
#> HTO8_NKX2.1_UTR.vs.HTO5_NeonG_v2.logFC
#> <numeric>
#> 1 -0.000708047
#> 2 -0.021300165
#> 3 0.005164618
#> 4 -0.000708047
#> 5 0.024761627
#> ... ...
#> 12401 0.03146638
#> 12402 0.00395594
#> 12403 -0.01222574
#> 12404 -0.02439659
#> 12405 0.01700461Calculate TF activity
Finally, the activities for a specific TF in each cell are computed by averaging expressions of target genes linked to the TF weighted by the test statistics of choice, chosen from either correlation, mutual information or the Wilcoxon test statistics. \[y=\frac{1}{n}\sum_{i=1}^{n} x_i * weights_i\] where \(y\) is the activity of a TF for a cell, \(n\) is the total number of targets for a TF, \(x_i\) is the log count expression of target \(i\) where \(i\) in {1,2,…,n} and \(weights_i\) is the weight of TF - target \(i\)
score.combine <- calculateActivity(expMatrix = GeneExpressionMatrix,
regulon = regulon.w,
mode = "weight",
method = "weightedMean",
exp_assay = "normalizedCounts",
normalize = FALSE)
#> Warning in calculateActivity(expMatrix = GeneExpressionMatrix, regulon =
#> regulon.w, : Argument 'method' to calculateActivity was deprecated as of
#> epiregulon version 2.0.0
#> calculating TF activity from regulon using weightedMean
#> Warning in calculateActivity(expMatrix = GeneExpressionMatrix, regulon = regulon.w, : The weight column contains multiple subcolumns but no cluster information was provided.
#> Using first column to compute activity...
#> aggregating regulons...
#> creating weight matrix...
#> calculating activity scores...
#> normalize by the number of targets...
score.combine[1:5,1:5]
#> 5 x 5 sparse Matrix of class "dgCMatrix"
#> reprogram#TTAGGAACAAGGTACG-1 reprogram#GAGCGGTCAACCTGGT-1
#> AR 0.01748754 0.01902210
#> FOXA1 0.01594352 0.01562493
#> FOXA2 0.01668285 0.02000509
#> GATA6 0.01590999 0.05706049
#> NKX2-1 0.02949717 0.01737281
#> reprogram#TTATAGCCACCCTCAC-1 reprogram#TGGTGATTCCTGTTCA-1
#> AR 0.010467290 0.010765099
#> FOXA1 0.010635861 0.010349299
#> FOXA2 0.012503277 0.014421759
#> GATA6 0.009035979 0.007895991
#> NKX2-1 0.012772370 0.011795167
#> reprogram#TCGGTTCTCACTAGGT-1
#> AR 0.01775971
#> FOXA1 0.01581682
#> FOXA2 0.01439649
#> GATA6 0.01188049
#> NKX2-1 0.02613054Session Info
sessionInfo()
#> R version 4.5.2 (2025-10-31)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.3 LTS
#>
#> Matrix products: default
#> BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
#> LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_US.UTF-8 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: Etc/UTC
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] BSgenome.Hsapiens.UCSC.hg38_1.4.5 BSgenome_1.78.0
#> [3] rtracklayer_1.70.0 BiocIO_1.20.0
#> [5] Biostrings_2.78.0 XVector_0.50.0
#> [7] GenomeInfoDb_1.46.0 scMultiome_1.10.0
#> [9] MultiAssayExperiment_1.36.0 ExperimentHub_3.0.0
#> [11] AnnotationHub_4.0.0 BiocFileCache_3.0.0
#> [13] dbplyr_2.5.1 epiregulon_2.0.1
#> [15] SingleCellExperiment_1.32.0 SummarizedExperiment_1.40.0
#> [17] Biobase_2.70.0 GenomicRanges_1.62.0
#> [19] Seqinfo_1.0.0 IRanges_2.44.0
#> [21] S4Vectors_0.48.0 BiocGenerics_0.56.0
#> [23] generics_0.1.4 MatrixGenerics_1.22.0
#> [25] matrixStats_1.5.0 BiocStyle_2.38.0
#>
#> loaded via a namespace (and not attached):
#> [1] RColorBrewer_1.1-3 sys_3.4.3
#> [3] jsonlite_2.0.0 magrittr_2.0.4
#> [5] ggbeeswarm_0.7.2 farver_2.1.2
#> [7] rmarkdown_2.30 vctrs_0.6.5
#> [9] memoise_2.0.1 Rsamtools_2.26.0
#> [11] RCurl_1.98-1.17 htmltools_0.5.8.1
#> [13] S4Arrays_1.10.0 BiocBaseUtils_1.12.0
#> [15] curl_7.0.0 BiocNeighbors_2.4.0
#> [17] Rhdf5lib_1.32.0 SparseArray_1.10.1
#> [19] rhdf5_2.54.0 sass_0.4.10
#> [21] bslib_0.9.0 httr2_1.2.1
#> [23] cachem_1.1.0 buildtools_1.0.0
#> [25] GenomicAlignments_1.46.0 igraph_2.2.1
#> [27] lifecycle_1.0.4 pkgconfig_2.0.3
#> [29] rsvd_1.0.5 Matrix_1.7-4
#> [31] R6_2.6.1 fastmap_1.2.0
#> [33] digest_0.6.37 TFMPvalue_0.0.9
#> [35] AnnotationDbi_1.72.0 scater_1.38.0
#> [37] dqrng_0.4.1 irlba_2.3.5.1
#> [39] RSQLite_2.4.4 beachmat_2.26.0
#> [41] seqLogo_1.76.0 filelock_1.0.3
#> [43] labeling_0.4.3 httr_1.4.7
#> [45] abind_1.4-8 compiler_4.5.2
#> [47] bit64_4.6.0-1 withr_3.0.2
#> [49] S7_0.2.0 backports_1.5.0
#> [51] BiocParallel_1.44.0 viridis_0.6.5
#> [53] DBI_1.2.3 HDF5Array_1.38.0
#> [55] rappdirs_0.3.3 DelayedArray_0.36.0
#> [57] bluster_1.20.0 rjson_0.2.23
#> [59] gtools_3.9.5 caTools_1.18.3
#> [61] tools_4.5.2 vipor_0.4.7
#> [63] beeswarm_0.4.0 glue_1.8.0
#> [65] h5mread_1.2.0 restfulr_0.0.16
#> [67] rhdf5filters_1.22.0 grid_4.5.2
#> [69] checkmate_2.3.3 cluster_2.1.8.1
#> [71] TFBSTools_1.48.0 gtable_0.3.6
#> [73] metapod_1.18.0 BiocSingular_1.26.0
#> [75] ScaledMatrix_1.18.0 ggrepel_0.9.6
#> [77] BiocVersion_3.23.1 pillar_1.11.1
#> [79] motifmatchr_1.32.0 limma_3.66.0
#> [81] dplyr_1.1.4 lattice_0.22-7
#> [83] bit_4.6.0 tidyselect_1.2.1
#> [85] DirichletMultinomial_1.52.0 locfit_1.5-9.12
#> [87] maketools_1.3.2 scuttle_1.20.0
#> [89] knitr_1.50 gridExtra_2.3
#> [91] scrapper_1.4.0 edgeR_4.8.0
#> [93] xfun_0.54 statmod_1.5.1
#> [95] UCSC.utils_1.6.0 yaml_2.3.10
#> [97] evaluate_1.0.5 codetools_0.2-20
#> [99] cigarillo_1.0.0 tibble_3.3.0
#> [101] BiocManager_1.30.26 cli_3.6.5
#> [103] jquerylib_0.1.4 Rcpp_1.1.0
#> [105] png_0.1-8 XML_3.99-0.20
#> [107] parallel_4.5.2 ggplot2_4.0.0
#> [109] blob_1.2.4 scran_1.38.0
#> [111] bitops_1.0-9 pwalign_1.6.0
#> [113] viridisLite_0.4.2 scales_1.4.0
#> [115] purrr_1.2.0 crayon_1.5.3
#> [117] rlang_1.1.6 KEGGREST_1.50.0