egsea-main: Core functions to perform ensemble of gene set enrichment...
In EGSEA: Ensemble of Gene Set Enrichment Analyses
Description
Usage
Arguments
Details
Value
References
See Also
Examples
Description
egsea is the main function to carry out gene set enrichment
analysis using the
EGSEA algorithm. This function is aimed to extend the limma-voom pipeline
of RNA-seq analysis.
egsea.cnt is the main function to carry out gene set
enrichment analysis using the EGSEA algorithm. This function is aimed to
use raw RNASeq count matrix to perform the EGSEA analysis.
egsea.ora is the main function to carry out
over-representation analysis (ORA) on gene set collections using
a list of genes.
egsea.ma is the main function to carry out gene set
enrichment analysis using the EGSEA algorithm. This function is aimed to
use a microarray expression matrix to perform the EGSEA analysis.
Usage
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egsea(voom.results, contrasts = NULL, logFC = NULL, gs.annots,
symbolsMap = NULL, baseGSEAs = egsea.base(), minSize = 2,
display.top = 20, combineMethod = "wilkinson", combineWeights = NULL,
sort.by = "p.adj", report.dir = NULL, kegg.dir = NULL,
logFC.cutoff = 0, fdr.cutoff = 0.05, sum.plot.axis = "p.adj",
sum.plot.cutoff = NULL, vote.bin.width = 5, num.threads = 4,
report = TRUE, interactive = FALSE, keep.base = FALSE,
verbose = FALSE, keep.limma = TRUE, keep.set.scores = FALSE)
egsea.cnt(counts, group, design = NULL, contrasts = NULL, logFC = NULL,
gs.annots, symbolsMap = NULL, baseGSEAs = egsea.base(), minSize = 2,
display.top = 20, combineMethod = "wilkinson", combineWeights = NULL,
sort.by = "p.adj", report.dir = NULL, kegg.dir = NULL,
logFC.cutoff = 0, fdr.cutoff = 0.05, sum.plot.axis = "p.adj",
sum.plot.cutoff = NULL, vote.bin.width = 5, num.threads = 4,
report = TRUE, interactive = FALSE, keep.base = FALSE,
verbose = FALSE, keep.limma = TRUE, keep.set.scores = FALSE)
egsea.ora(geneIDs, universe = NULL, logFC = NULL, title = NULL, gs.annots,
symbolsMap = NULL, minSize = 2, display.top = 20, sort.by = "p.adj",
report.dir = NULL, kegg.dir = NULL, sum.plot.axis = "p.adj",
sum.plot.cutoff = NULL, num.threads = 4, report = TRUE,
interactive = FALSE, verbose = FALSE)
egsea.ma(expr, group, probe.annot, probeMap.method = "avg", design = NULL,
contrasts = NULL, logFC = NULL, gs.annots, baseGSEAs = egsea.base(),
minSize = 2, display.top = 20, combineMethod = "wilkinson",
combineWeights = NULL, sort.by = "p.adj", report.dir = NULL,
kegg.dir = NULL, logFC.cutoff = 0, fdr.cutoff = 0.05,
sum.plot.axis = "p.adj", sum.plot.cutoff = NULL, vote.bin.width = 5,
num.threads = 4, report = TRUE, interactive = FALSE,
keep.base = FALSE, verbose = FALSE, keep.limma = TRUE,
keep.set.scores = FALSE)
Arguments
voom.results
list, an EList object generated using the
voom function, which it has at least
three elements: E log2 normalized expression values,
design design matrix, targets sample information,
which must have a column named group. If a matrix
weights is provided, it will be used in limma-based
methods. Entrez Gene IDs should be used as row names.
contrasts
double, an N x L matrix indicates the contrasts of the
linear model coefficients for which the test is required if the
design matrix does not have an intercept. N is number
of columns of the design matrix and L is number of contrasts. This
matrix should be based on the primary factor of the design matrix.
If the design matrix includes an intercept, this parameter can be
a vector of integers that specify the columns of the design matrix.
If this parameter is NULL, all pairwise comparisons based on
group or voom.results$targets$group are created,
assuming that group is the primary factor in the design
matrix. Likewise, all the coefficients of the primary factor are used
if the design matrix has an intercept.
logFC
double, an K x L matrix indicates the log2 fold change of each
gene for each contrast. K is the number of genes included in the analysis.
If logFC=NULL, the logFC values are
estimated using the ebayes for each contrast.
For egsea.ora, it can be a matrix or vector of the same
length of entrezIDs. If logFC=NULL, 1 is used as a default value. Then, the
regulation direction in heatmaps and pathway maps is not indicative of the
gene regulation direction.
gs.annots
list, list of objects of class GSCollectionIndex. It is generated
using one of these functions:
buildIdx, buildMSigDBIdx,
buildKEGGIdx,
buildGeneSetDBIdx, and buildCustomIdx.
symbolsMap
dataframe, an K x 2 matrix stores the gene symbol of each
Entrez Gene ID. The first column must be the Entrez Gene IDs and the
second column must be the Gene Symbols. It is used for the heatmap visualization.
In egsea and egsea.cnt, the number of rows should match that
of the voom.results and counts, respectively.
Default symbolsMap=NULL.
baseGSEAs
character, a vector of the gene set tests that should be
included in the
ensemble. Type egsea.base to see the supported GSE methods.
By default, all
supported methods are used.
minSize
integer, the minimum size of a gene set to be included in the
analysis.
Default minSize= 2.
display.top
integer, the number of top gene sets to be displayed in
the EGSEA report.
You can always access the list of all tested gene sets using the returned
gsa list.
Default is 20.
combineMethod
character, determines how to combine p-values from
different
GSEA method. Type egsea.combine() to see supported methods.
combineWeights
double, a vector determines how different GSEA methods
will be weighted.
Its values should range between 0 and 1. This option is not supported
currently.
sort.by
character, determines how to order the analysis results in
the stats table. Type
egsea.sort() to see all available options.
For egsea.ora, it takes "p.value", "p.adj" or "Significance".
report.dir
character, directory into which the analysis results are
written out.
kegg.dir
character, the directory of KEGG pathway data file (.xml)
and image file (.png).
Default kegg.dir=paste0(report.dir, "/kegg-dir/").
logFC.cutoff
numeric, cut-off threshold of logFC and is used for
the calculation of Sginificance Score
and Regulation Direction. Default logFC.cutoff=0.
fdr.cutoff
numeric, cut-off threshold of DE genes and is used
for the calculation of Significance Score and Regulation Direction.
Default fdr.cutoff = 0.05.
sum.plot.axis
character, the x-axis of the summary plot. All the
values accepted by the
sort.by parameter can be used. Default sum.plot.axis="p.value".
sum.plot.cutoff
numeric, cut-off threshold to filter the gene sets of
the summary plots
based on the values of the sum.plot.axis. Default
sum.plot.cutoff=NULL.
vote.bin.width
numeric, the bin width of the vote ranking. Default
vote.bin.width=5.
num.threads
numeric, number of CPU cores to be used. Default
num.threads=2.
report
logical, whether to generate the EGSEA interactive report. It
takes longer time
to run. Default is True.
interactive
logical, whether to generate interactive tables and plots.
Note this might dramatically increase the size of the EGSEA report.
keep.base
logical, whether to write out the results of the
individual GSE methods.
Default FALSE.
verbose
logical, whether to print out progress messages and warnings.
keep.limma
logical, whether to store the results of the limma analysis
in the EGSEAResults object.
keep.set.scores
logical, whether to calculate the gene set enrichment scores
per sample for the methods that support this option, i.e., "ssgsea".
counts
double, an K x M numeric matrix of read counts where genes are
the rows and samples are the columns. In this case, TMM normalization is used
to calculate the normalization factors. counts can be also a
DGEList object. In this case, the normalization factors can be
calculated by the user prior to invoking this method. This is REQUIRED.
group
character, vector or factor giving the experimental
group/condition for each sample/library. This is REQUIRED.
design
double, an M x N numeric matrix giving the design matrix of
the linear model fitting. N is the number of coefficients in model.
If this parameter is
NULL, model.matrix(~0+group) is used to create a deisgn matrix.
geneIDs
character, a vector of Gene IDs to be tested for ORA. They
must be Entrez IDs if EGSEAdata collections are used.
universe
character, a vector of Enterz IDs to be used as a background
list. If universe=NULL, the background list is created from the
AnnotationDbi package.
title
character, a short description of the experimental contrast.
expr
double, an K x M numeric matrix of intensities where genes
are the rows and samples are the columns. In this case, it is assumed
that the expression values are already normalized log2 intensities
and filtered, i.e. rows corresponding to control and low-quality
probes removed. Row names of expr must match the first column in
probe.annot. This is REQUIRED.
probe.annot
double, an K x n numeric matrix where rows are
probes, the first column
contains probe IDs, the second column contains Entrez Gene IDs,
and the third column (optional) contains the Gene Symbols.
Symbols are used for the heatmap visualization
Additional annotation columns can be added for
the probes, e.g., Chromosome, QualityScore, etc. These
additional columns are not used. This is REQUIRED.
probeMap.method
character, the method to be used in mapping the
Probe IDs into Enterz Gene IDs. Accepted methods include:
"avg", "med", "var", "sum" and "iqr".
EGSEA selects the probe with the highest
average, median, variance, sum or IQR of expression, respectively,
as a representative for each expressed gene.
Details
EGSEA, an acronym for Ensemble of Gene Set Enrichment
Analyses, utilizes the
analysis results of eleven prominent GSE algorithms from the literature to
calculate
collective significance scores for gene sets. These methods include:
ora,
globaltest, plage, safe, zscore, gage,
ssgsea,
roast, fry, padog, camera and gsva.
The ora, gage, camera and gsva methods depend on a competitive null
hypothesis while the
remaining seven methods are based on a self-contained hypothesis.
Conveniently, the
algorithm proposed here is not limited to these twelve GSE methods and new
GSE tests
can be easily integrated into the framework. This function takes the voom
object and
the contrast matrix as parameters.
The results of EGSEA can be seen using the topSets function.
EGSEA report is an interactive HTML report that is generated if report=TRUE to
enable a swift navigation through the results of an EGSEA analysis. The following pages
are generated for each gene set collection and contrast/comparison:
1. Stats Table page shows the detailed statistics of the EGSEA analysis for the
display.top gene sets. It shows the EGSEA scores, individual rankings and
additional annotation for each gene set. Hyperlinks to the source of each gene set
can be seen in this table when they are available. The "Direction" column shows the regulation
direction of a gene set which is calculated based on the logFC, which is
either calculated from the limma differential expression analysis or provided by the user.
The logFC.cutoff and fdr.cutoff are applied for this calculation.
The calculations of the EGSEA
scores can be seen in the references section. The method topSets can be used to
generate custom Stats Table.
2. Heatmaps page shows the heatmaps of the gene fold changes for the gene sets that are
presented in the Stats Table page. Red indicates up-regulation
while blue indicates down-regulation. Only genes that appear in the input expression/count
matrix are visualized in the heat map. Gene names are coloured based on their
statistical significance in the limma differential expression analysis.
The "Interpret Results" link below each heat map allows the user to download the
original heat map values along with additional statistics from limma DE analysis (
if available) so that they can be used to perform further analysis in R, e.g., customizing
the heat map visualization. Additional heat maps can be generated and customized
using the method plotHeatmap.
3. Summary Plots page shows the methods ranking plot along with the summary plots of
EGSEA analysis. The method plot uses multidimensional scaling (MDS) to visualize the
ranking of individual methods on a given gene set collection. The summary plots are
bubble plots that visualize the distribution of gene sets based on the EGSEA
Significance Score and another EGSEA score (default, p-value).
Two summary plots are generated: ranking and directional plots. Each gene set is
reprersented with a bubble which is coloured based on the EGSEA ranking (in ranking
plots ) or gene set regulation direction (in directional plots) and sized based on the
gene set cardinality (in ranking plots) or EGSEA Significance score (in directional plots).
Since the EGSEA "Significance Score" is proportional to the p-value and the
absolute fold changes, it could be useful to highlight gene sets that
have high Significance scores. The blue labels on the summary plot indicate
gene sets that do not appear in the top 10 list of gene sets based on the "sort.by"
argument (black labels) yet they appear in the top 5 list of gene sets based on
the EGSEA "Significance Score". If two contrasts are provided, the rank is calculated
based on the "comparison" analysis results and the "Significance Score" is calculated
as the mean. If sort.by = NULL, the slot sort.by of the object
is used to order gene sets.
The method plotSummary can be used to customize the Summary plots by
changing the x-axis score
and filtering bubbles based on the values of the x-axis. The method plotMethods can be
used to generate Methods plots.
4. Pathways page shows the KEGG pathways for the gene sets that are presented in the
Stats Table of a KEGG gene set collection. The gene fold changes are overlaid on the
pathway maps and coloured based on the gene regulation direction: blue for down-regulation
and red for up-regulation. The method plotPathway can be used to generate
additional pathway maps. Note that this page only appears if a KEGG gene set collection
is used in the EGSEA analysis.
5. Go Graphs page shows the Gene Ontology graphs for top 5 GO terms in each of
three GO categories: Biological Processes (BP), Molecular Functions (MF),
and Cellular Components (CC). Nodes are coloured based on the default sort.by
score where red indicates high significance and yellow indicates low significance.
The method plotGOGraph can be used to customize GO graphs by
changing the default sorting score and the number of significance nodes that can be
visualized. It is recommended that a small number of nodes is selected. Note that
this page only appears if a Gene Ontology gene set collection is used, i.e., for
the c5 collection from MSigDB or the gsdbgo collection from GeneSetDB.
Finally, the "Interpret Results" hyperlink in the EGSEA report allows the user to download
the fold changes and limma analysis results and thus improve the interpretation of the results.
Note that the running time of this function significantly increseas when
report = TRUE. For example, the analysis in the example section below
was conducted on the $203$ signaling and disease KEGG pathways using a MacBook Pro
machine that had a 2.8 GHz Intel Core i7 CPU and 16 GB of RAM. The execution time
varied between 23.1 seconds (single thread) to 7.9 seconds (16 threads) when the HTML
report generation was disabled. The execution time took 145.5 seconds when the report
generation was enabled using 16 threads.
egsea.ora takes a list of gene IDs and uses the
gene set collections from EGSEAdata or a custom-built collection to
find over-represented gene sets in this list. It takes the advantage of
the existing EGSEA reporting capabilities and generate an interative report
for the ORA analysis.
The results can be explored using the topSets function.
Value
egsea returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results for each contrast and the comparative analysis results.
egsea.cnt returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results for each contrast and the comparative analysis results.
egsea.ora returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results.
egsea.ma returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results for each contrast and the comparative analysis results.
References
Monther Alhamdoosh, Milica Ng, Nicholas J. Wilson, Julie M. Sheridan, Huy Huynh,
Michael J. Wilson, Matthew E. Ritchie; Combining multiple tools outperforms
individual methods in gene set enrichment analyses. Bioinformatics 2017;
33 (3): 414-424. doi: 10.1093/bioinformatics/btw623
See Also
topSets, egsea.base,
egsea.sort,
buildIdx, buildMSigDBIdx,
buildKEGGIdx,
buildGeneSetDBIdx, and buildCustomIdx
Examples
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# Example of egsea
library(EGSEAdata)
data(il13.data)
v = il13.data$voom
contrasts = il13.data$contra
gs.annots = buildIdx(entrezIDs=rownames(v$E), species="human",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea(voom.results=v, contrasts=contrasts, gs.annots=gs.annots,
symbolsMap=v$genes, baseGSEAs=egsea.base()[-c(2,5,6,9,12)],
display.top = 5, sort.by="avg.rank",
report.dir="./il13-egsea-report",
num.threads = 2, report = FALSE)
topSets(gsa)
# Example of egsea.cnt
library(EGSEAdata)
data(il13.data.cnt)
cnt = il13.data.cnt$counts
group = il13.data.cnt$group
design = il13.data.cnt$design
contrasts = il13.data.cnt$contra
genes = il13.data.cnt$genes
gs.annots = buildIdx(entrezIDs=rownames(cnt), species="human",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea.cnt(counts=cnt, group=group, design=design, contrasts=contrasts,
gs.annots=gs.annots,
symbolsMap=genes, baseGSEAs=egsea.base()[-c(2,5,6,9,12)],
display.top = 5,
sort.by="avg.rank",
report.dir="./il13-egsea-cnt-report",
num.threads = 2, report = FALSE)
topSets(gsa)
# Example of egsea.ora
library(EGSEAdata)
data(il13.data)
voom.results = il13.data$voom
contrast = il13.data$contra
library(limma)
vfit = lmFit(voom.results, voom.results$design)
vfit = contrasts.fit(vfit, contrast)
vfit = eBayes(vfit)
top.Table = topTable(vfit, coef=1, number=Inf, p.value=0.05, lfc=1)
deGenes = as.character(top.Table$FeatureID)
logFC = top.Table$logFC
names(logFC) = deGenes
gs.annots = buildIdx(entrezIDs=deGenes, species="human",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea.ora(geneIDs=deGenes, universe=
as.character(voom.results$genes[,1]),
logFC =logFC, title="X24IL13-X24",
gs.annots=gs.annots,
symbolsMap=top.Table[, c(1,2)], display.top = 5,
report.dir="./il13-egsea-ora-report", num.threads = 2,
report = FALSE)
topSets(gsa)
# Example of egsea.ma
library(Glimma)
data(arraydata)
expr = arraydata$arrays$E
group = as.factor(arraydata$targets$Condition)
levels(group) = c("DPcreEzh2", "DPev", "LumcreEzh2", "Lumev")
probe.annot = arraydata$arrays$genes[-1]
design <- model.matrix(~0+ group + as.factor(arraydata$targets$Experiment))
colnames(design)[1:4] = levels(group)
colnames(design)[5:6] = c("Exp2", "Exp3")
contr = makeContrasts("DPEzh2KOvsWT" = DPcreEzh2-DPev,
"LumEzh2KOvsWT" = LumcreEzh2-Lumev,
levels=colnames(design))
gs.annots = buildIdx(entrezIDs=unique(probe.annot[, 2]),
species="mouse",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea.ma(expr=expr, group=group,
probe.annot = probe.annot,
design=design, contrasts=contr,
gs.annots=gs.annots,
baseGSEAs=egsea.base()[-c(2,5,6,9,12)],
display.top = 5,
sort.by="avg.rank",
report.dir="./ezh2-egsea-ma-report",
num.threads = 2, report = FALSE)
topSets(gsa)
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Description Usage Arguments Details Value References See Also Examples
Description
egsea is the main function to carry out gene set enrichment
analysis using the
EGSEA algorithm. This function is aimed to extend the limma-voom pipeline
of RNA-seq analysis.
egsea.cnt is the main function to carry out gene set
enrichment analysis using the EGSEA algorithm. This function is aimed to
use raw RNASeq count matrix to perform the EGSEA analysis.
egsea.ora is the main function to carry out
over-representation analysis (ORA) on gene set collections using
a list of genes.
egsea.ma is the main function to carry out gene set
enrichment analysis using the EGSEA algorithm. This function is aimed to
use a microarray expression matrix to perform the EGSEA analysis.
Usage
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 | egsea(voom.results, contrasts = NULL, logFC = NULL, gs.annots,
symbolsMap = NULL, baseGSEAs = egsea.base(), minSize = 2,
display.top = 20, combineMethod = "wilkinson", combineWeights = NULL,
sort.by = "p.adj", report.dir = NULL, kegg.dir = NULL,
logFC.cutoff = 0, fdr.cutoff = 0.05, sum.plot.axis = "p.adj",
sum.plot.cutoff = NULL, vote.bin.width = 5, num.threads = 4,
report = TRUE, interactive = FALSE, keep.base = FALSE,
verbose = FALSE, keep.limma = TRUE, keep.set.scores = FALSE)
egsea.cnt(counts, group, design = NULL, contrasts = NULL, logFC = NULL,
gs.annots, symbolsMap = NULL, baseGSEAs = egsea.base(), minSize = 2,
display.top = 20, combineMethod = "wilkinson", combineWeights = NULL,
sort.by = "p.adj", report.dir = NULL, kegg.dir = NULL,
logFC.cutoff = 0, fdr.cutoff = 0.05, sum.plot.axis = "p.adj",
sum.plot.cutoff = NULL, vote.bin.width = 5, num.threads = 4,
report = TRUE, interactive = FALSE, keep.base = FALSE,
verbose = FALSE, keep.limma = TRUE, keep.set.scores = FALSE)
egsea.ora(geneIDs, universe = NULL, logFC = NULL, title = NULL, gs.annots,
symbolsMap = NULL, minSize = 2, display.top = 20, sort.by = "p.adj",
report.dir = NULL, kegg.dir = NULL, sum.plot.axis = "p.adj",
sum.plot.cutoff = NULL, num.threads = 4, report = TRUE,
interactive = FALSE, verbose = FALSE)
egsea.ma(expr, group, probe.annot, probeMap.method = "avg", design = NULL,
contrasts = NULL, logFC = NULL, gs.annots, baseGSEAs = egsea.base(),
minSize = 2, display.top = 20, combineMethod = "wilkinson",
combineWeights = NULL, sort.by = "p.adj", report.dir = NULL,
kegg.dir = NULL, logFC.cutoff = 0, fdr.cutoff = 0.05,
sum.plot.axis = "p.adj", sum.plot.cutoff = NULL, vote.bin.width = 5,
num.threads = 4, report = TRUE, interactive = FALSE,
keep.base = FALSE, verbose = FALSE, keep.limma = TRUE,
keep.set.scores = FALSE)
|
Arguments
voom.results |
list, an EList object generated using the
|
contrasts |
double, an N x L matrix indicates the contrasts of the
linear model coefficients for which the test is required if the
design matrix does not have an intercept. N is number
of columns of the design matrix and L is number of contrasts. This
matrix should be based on the primary factor of the design matrix.
If the design matrix includes an intercept, this parameter can be
a vector of integers that specify the columns of the design matrix.
If this parameter is |
logFC |
double, an K x L matrix indicates the log2 fold change of each
gene for each contrast. K is the number of genes included in the analysis.
If logFC=NULL, the logFC values are
estimated using the |
gs.annots |
list, list of objects of class GSCollectionIndex. It is generated
using one of these functions:
|
symbolsMap |
dataframe, an K x 2 matrix stores the gene symbol of each
Entrez Gene ID. The first column must be the Entrez Gene IDs and the
second column must be the Gene Symbols. It is used for the heatmap visualization.
In |
baseGSEAs |
character, a vector of the gene set tests that should be
included in the
ensemble. Type |
minSize |
integer, the minimum size of a gene set to be included in the analysis. Default minSize= 2. |
display.top |
integer, the number of top gene sets to be displayed in the EGSEA report. You can always access the list of all tested gene sets using the returned gsa list. Default is 20. |
combineMethod |
character, determines how to combine p-values from
different
GSEA method. Type |
combineWeights |
double, a vector determines how different GSEA methods will be weighted. Its values should range between 0 and 1. This option is not supported currently. |
sort.by |
character, determines how to order the analysis results in
the stats table. Type
|
report.dir |
character, directory into which the analysis results are written out. |
kegg.dir |
character, the directory of KEGG pathway data file (.xml) and image file (.png). Default kegg.dir=paste0(report.dir, "/kegg-dir/"). |
logFC.cutoff |
numeric, cut-off threshold of logFC and is used for the calculation of Sginificance Score and Regulation Direction. Default logFC.cutoff=0. |
fdr.cutoff |
numeric, cut-off threshold of DE genes and is used for the calculation of Significance Score and Regulation Direction. Default fdr.cutoff = 0.05. |
sum.plot.axis |
character, the x-axis of the summary plot. All the values accepted by the sort.by parameter can be used. Default sum.plot.axis="p.value". |
sum.plot.cutoff |
numeric, cut-off threshold to filter the gene sets of the summary plots based on the values of the sum.plot.axis. Default sum.plot.cutoff=NULL. |
vote.bin.width |
numeric, the bin width of the vote ranking. Default vote.bin.width=5. |
num.threads |
numeric, number of CPU cores to be used. Default num.threads=2. |
report |
logical, whether to generate the EGSEA interactive report. It takes longer time to run. Default is True. |
interactive |
logical, whether to generate interactive tables and plots. Note this might dramatically increase the size of the EGSEA report. |
keep.base |
logical, whether to write out the results of the individual GSE methods. Default FALSE. |
verbose |
logical, whether to print out progress messages and warnings. |
keep.limma |
logical, whether to store the results of the limma analysis in the EGSEAResults object. |
keep.set.scores |
logical, whether to calculate the gene set enrichment scores per sample for the methods that support this option, i.e., "ssgsea". |
counts |
double, an K x M numeric matrix of read counts where genes are
the rows and samples are the columns. In this case, TMM normalization is used
to calculate the normalization factors. |
group |
character, vector or factor giving the experimental group/condition for each sample/library. This is REQUIRED. |
design |
double, an M x N numeric matrix giving the design matrix of
the linear model fitting. N is the number of coefficients in model.
If this parameter is
|
geneIDs |
character, a vector of Gene IDs to be tested for ORA. They must be Entrez IDs if EGSEAdata collections are used. |
universe |
character, a vector of Enterz IDs to be used as a background list. If universe=NULL, the background list is created from the AnnotationDbi package. |
title |
character, a short description of the experimental contrast. |
expr |
double, an K x M numeric matrix of intensities where genes
are the rows and samples are the columns. In this case, it is assumed
that the expression values are already normalized log2 intensities
and filtered, i.e. rows corresponding to control and low-quality
probes removed. Row names of |
probe.annot |
double, an K x n numeric matrix where rows are probes, the first column contains probe IDs, the second column contains Entrez Gene IDs, and the third column (optional) contains the Gene Symbols. Symbols are used for the heatmap visualization Additional annotation columns can be added for the probes, e.g., Chromosome, QualityScore, etc. These additional columns are not used. This is REQUIRED. |
probeMap.method |
character, the method to be used in mapping the Probe IDs into Enterz Gene IDs. Accepted methods include: "avg", "med", "var", "sum" and "iqr". EGSEA selects the probe with the highest average, median, variance, sum or IQR of expression, respectively, as a representative for each expressed gene. |
Details
EGSEA, an acronym for Ensemble of Gene Set Enrichment
Analyses, utilizes the
analysis results of eleven prominent GSE algorithms from the literature to
calculate
collective significance scores for gene sets. These methods include:
ora,
globaltest, plage, safe, zscore, gage,
ssgsea,
roast, fry, padog, camera and gsva.
The ora, gage, camera and gsva methods depend on a competitive null
hypothesis while the
remaining seven methods are based on a self-contained hypothesis.
Conveniently, the
algorithm proposed here is not limited to these twelve GSE methods and new
GSE tests
can be easily integrated into the framework. This function takes the voom
object and
the contrast matrix as parameters.
The results of EGSEA can be seen using the topSets function.
EGSEA report is an interactive HTML report that is generated if report=TRUE to
enable a swift navigation through the results of an EGSEA analysis. The following pages
are generated for each gene set collection and contrast/comparison:
1. Stats Table page shows the detailed statistics of the EGSEA analysis for the
display.top gene sets. It shows the EGSEA scores, individual rankings and
additional annotation for each gene set. Hyperlinks to the source of each gene set
can be seen in this table when they are available. The "Direction" column shows the regulation
direction of a gene set which is calculated based on the logFC, which is
either calculated from the limma differential expression analysis or provided by the user.
The logFC.cutoff and fdr.cutoff are applied for this calculation.
The calculations of the EGSEA
scores can be seen in the references section. The method topSets can be used to
generate custom Stats Table.
2. Heatmaps page shows the heatmaps of the gene fold changes for the gene sets that are
presented in the Stats Table page. Red indicates up-regulation
while blue indicates down-regulation. Only genes that appear in the input expression/count
matrix are visualized in the heat map. Gene names are coloured based on their
statistical significance in the limma differential expression analysis.
The "Interpret Results" link below each heat map allows the user to download the
original heat map values along with additional statistics from limma DE analysis (
if available) so that they can be used to perform further analysis in R, e.g., customizing
the heat map visualization. Additional heat maps can be generated and customized
using the method plotHeatmap.
3. Summary Plots page shows the methods ranking plot along with the summary plots of
EGSEA analysis. The method plot uses multidimensional scaling (MDS) to visualize the
ranking of individual methods on a given gene set collection. The summary plots are
bubble plots that visualize the distribution of gene sets based on the EGSEA
Significance Score and another EGSEA score (default, p-value).
Two summary plots are generated: ranking and directional plots. Each gene set is
reprersented with a bubble which is coloured based on the EGSEA ranking (in ranking
plots ) or gene set regulation direction (in directional plots) and sized based on the
gene set cardinality (in ranking plots) or EGSEA Significance score (in directional plots).
Since the EGSEA "Significance Score" is proportional to the p-value and the
absolute fold changes, it could be useful to highlight gene sets that
have high Significance scores. The blue labels on the summary plot indicate
gene sets that do not appear in the top 10 list of gene sets based on the "sort.by"
argument (black labels) yet they appear in the top 5 list of gene sets based on
the EGSEA "Significance Score". If two contrasts are provided, the rank is calculated
based on the "comparison" analysis results and the "Significance Score" is calculated
as the mean. If sort.by = NULL, the slot sort.by of the object
is used to order gene sets.
The method plotSummary can be used to customize the Summary plots by
changing the x-axis score
and filtering bubbles based on the values of the x-axis. The method plotMethods can be
used to generate Methods plots.
4. Pathways page shows the KEGG pathways for the gene sets that are presented in the
Stats Table of a KEGG gene set collection. The gene fold changes are overlaid on the
pathway maps and coloured based on the gene regulation direction: blue for down-regulation
and red for up-regulation. The method plotPathway can be used to generate
additional pathway maps. Note that this page only appears if a KEGG gene set collection
is used in the EGSEA analysis.
5. Go Graphs page shows the Gene Ontology graphs for top 5 GO terms in each of
three GO categories: Biological Processes (BP), Molecular Functions (MF),
and Cellular Components (CC). Nodes are coloured based on the default sort.by
score where red indicates high significance and yellow indicates low significance.
The method plotGOGraph can be used to customize GO graphs by
changing the default sorting score and the number of significance nodes that can be
visualized. It is recommended that a small number of nodes is selected. Note that
this page only appears if a Gene Ontology gene set collection is used, i.e., for
the c5 collection from MSigDB or the gsdbgo collection from GeneSetDB.
Finally, the "Interpret Results" hyperlink in the EGSEA report allows the user to download
the fold changes and limma analysis results and thus improve the interpretation of the results.
Note that the running time of this function significantly increseas when
report = TRUE. For example, the analysis in the example section below
was conducted on the $203$ signaling and disease KEGG pathways using a MacBook Pro
machine that had a 2.8 GHz Intel Core i7 CPU and 16 GB of RAM. The execution time
varied between 23.1 seconds (single thread) to 7.9 seconds (16 threads) when the HTML
report generation was disabled. The execution time took 145.5 seconds when the report
generation was enabled using 16 threads.
egsea.ora takes a list of gene IDs and uses the
gene set collections from EGSEAdata or a custom-built collection to
find over-represented gene sets in this list. It takes the advantage of
the existing EGSEA reporting capabilities and generate an interative report
for the ORA analysis.
The results can be explored using the topSets function.
Value
egsea returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results for each contrast and the comparative analysis results.
egsea.cnt returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results for each contrast and the comparative analysis results.
egsea.ora returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results.
egsea.ma returns an object of the class EGSEAResults, which
stores the top gene sets and the detailed analysis
results for each contrast and the comparative analysis results.
References
Monther Alhamdoosh, Milica Ng, Nicholas J. Wilson, Julie M. Sheridan, Huy Huynh, Michael J. Wilson, Matthew E. Ritchie; Combining multiple tools outperforms individual methods in gene set enrichment analyses. Bioinformatics 2017; 33 (3): 414-424. doi: 10.1093/bioinformatics/btw623
See Also
topSets, egsea.base,
egsea.sort,
buildIdx, buildMSigDBIdx,
buildKEGGIdx,
buildGeneSetDBIdx, and buildCustomIdx
Examples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 | # Example of egsea
library(EGSEAdata)
data(il13.data)
v = il13.data$voom
contrasts = il13.data$contra
gs.annots = buildIdx(entrezIDs=rownames(v$E), species="human",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea(voom.results=v, contrasts=contrasts, gs.annots=gs.annots,
symbolsMap=v$genes, baseGSEAs=egsea.base()[-c(2,5,6,9,12)],
display.top = 5, sort.by="avg.rank",
report.dir="./il13-egsea-report",
num.threads = 2, report = FALSE)
topSets(gsa)
# Example of egsea.cnt
library(EGSEAdata)
data(il13.data.cnt)
cnt = il13.data.cnt$counts
group = il13.data.cnt$group
design = il13.data.cnt$design
contrasts = il13.data.cnt$contra
genes = il13.data.cnt$genes
gs.annots = buildIdx(entrezIDs=rownames(cnt), species="human",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea.cnt(counts=cnt, group=group, design=design, contrasts=contrasts,
gs.annots=gs.annots,
symbolsMap=genes, baseGSEAs=egsea.base()[-c(2,5,6,9,12)],
display.top = 5,
sort.by="avg.rank",
report.dir="./il13-egsea-cnt-report",
num.threads = 2, report = FALSE)
topSets(gsa)
# Example of egsea.ora
library(EGSEAdata)
data(il13.data)
voom.results = il13.data$voom
contrast = il13.data$contra
library(limma)
vfit = lmFit(voom.results, voom.results$design)
vfit = contrasts.fit(vfit, contrast)
vfit = eBayes(vfit)
top.Table = topTable(vfit, coef=1, number=Inf, p.value=0.05, lfc=1)
deGenes = as.character(top.Table$FeatureID)
logFC = top.Table$logFC
names(logFC) = deGenes
gs.annots = buildIdx(entrezIDs=deGenes, species="human",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea.ora(geneIDs=deGenes, universe=
as.character(voom.results$genes[,1]),
logFC =logFC, title="X24IL13-X24",
gs.annots=gs.annots,
symbolsMap=top.Table[, c(1,2)], display.top = 5,
report.dir="./il13-egsea-ora-report", num.threads = 2,
report = FALSE)
topSets(gsa)
# Example of egsea.ma
library(Glimma)
data(arraydata)
expr = arraydata$arrays$E
group = as.factor(arraydata$targets$Condition)
levels(group) = c("DPcreEzh2", "DPev", "LumcreEzh2", "Lumev")
probe.annot = arraydata$arrays$genes[-1]
design <- model.matrix(~0+ group + as.factor(arraydata$targets$Experiment))
colnames(design)[1:4] = levels(group)
colnames(design)[5:6] = c("Exp2", "Exp3")
contr = makeContrasts("DPEzh2KOvsWT" = DPcreEzh2-DPev,
"LumEzh2KOvsWT" = LumcreEzh2-Lumev,
levels=colnames(design))
gs.annots = buildIdx(entrezIDs=unique(probe.annot[, 2]),
species="mouse",
msigdb.gsets="none",
kegg.updated=FALSE, kegg.exclude = c("Metabolism"))
# set report = TRUE to generate the EGSEA interactive report
gsa = egsea.ma(expr=expr, group=group,
probe.annot = probe.annot,
design=design, contrasts=contr,
gs.annots=gs.annots,
baseGSEAs=egsea.base()[-c(2,5,6,9,12)],
display.top = 5,
sort.by="avg.rank",
report.dir="./ezh2-egsea-ma-report",
num.threads = 2, report = FALSE)
topSets(gsa)
|
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