TopGO - CTL04
Date: July 18, 2025
Condition_1 <- params$Condition_1
Condition_2 <- params$Condition_2Top GO Analysis for Estr AgvsInh
1. Environment Set Up
library(RNASeqBulkExploratory)
library(DT)
library(ggplot2)
library(AnnotationDbi)
## 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':
##
## anyDuplicated, aperm, append, as.data.frame, basename, cbind,
## colnames, dirname, do.call, duplicated, eval, evalq, Filter, Find,
## get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
## match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
## Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
## table, tapply, union, unique, unsplit, which.max, which.min
## Loading required package: Biobase
## Welcome to Bioconductor
##
## Vignettes contain introductory material; view with
## 'browseVignettes()'. To cite Bioconductor, see
## 'citation("Biobase")', and for packages 'citation("pkgname")'.
## Loading required package: IRanges
## Loading required package: S4Vectors
##
## Attaching package: 'S4Vectors'
## The following objects are masked from 'package:base':
##
## expand.grid, I, unname
library(org.Hs.eg.db)
##
library(gridExtra)
##
## Attaching package: 'gridExtra'
## The following object is masked from 'package:Biobase':
##
## combine
## The following object is masked from 'package:BiocGenerics':
##
## combine
library(RColorBrewer)
library(viridis)
## Loading required package: viridisLite
library(topGO)
## Loading required package: graph
## Loading required package: GO.db
##
## Loading required package: SparseM
##
## Attaching package: 'SparseM'
## The following object is masked from 'package:base':
##
## backsolve
##
## groupGOTerms: GOBPTerm, GOMFTerm, GOCCTerm environments built.
##
## Attaching package: 'topGO'
## The following object is masked from 'package:IRanges':
##
## members
library(data.table)
##
## Attaching package: 'data.table'
## The following object is masked from 'package:IRanges':
##
## shift
## The following objects are masked from 'package:S4Vectors':
##
## first, second
library(tidyr)
##
## Attaching package: 'tidyr'
## The following object is masked from 'package:S4Vectors':
##
## expand
library(dplyr)
##
## Attaching package: 'dplyr'
## The following objects are masked from 'package:data.table':
##
## between, first, last
## The following object is masked from 'package:graph':
##
## union
## The following object is masked from 'package:gridExtra':
##
## combine
## The following object is masked from 'package:AnnotationDbi':
##
## select
## The following objects are masked from 'package:IRanges':
##
## collapse, desc, intersect, setdiff, slice, union
## The following objects are masked from 'package:S4Vectors':
##
## first, intersect, rename, setdiff, setequal, union
## The following object is masked from 'package:Biobase':
##
## combine
## The following objects are masked from 'package:BiocGenerics':
##
## combine, intersect, setdiff, union
## The following objects are masked from 'package:stats':
##
## filter, lag
## The following objects are masked from 'package:base':
##
## intersect, setdiff, setequal, union
library(SummarizedExperiment)
## Loading required package: MatrixGenerics
## Loading required package: matrixStats
##
## Attaching package: 'matrixStats'
## The following object is masked from 'package:dplyr':
##
## count
## The following objects are masked from 'package:Biobase':
##
## anyMissing, rowMedians
##
## Attaching package: 'MatrixGenerics'
## The following objects are masked from 'package:matrixStats':
##
## colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse,
## colCounts, colCummaxs, colCummins, colCumprods, colCumsums,
## colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs,
## colMads, colMaxs, colMeans2, colMedians, colMins, colOrderStats,
## colProds, colQuantiles, colRanges, colRanks, colSdDiffs, colSds,
## colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads,
## colWeightedMeans, colWeightedMedians, colWeightedSds,
## colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet,
## rowCollapse, rowCounts, rowCummaxs, rowCummins, rowCumprods,
## rowCumsums, rowDiffs, rowIQRDiffs, rowIQRs, rowLogSumExps,
## rowMadDiffs, rowMads, rowMaxs, rowMeans2, rowMedians, rowMins,
## rowOrderStats, rowProds, rowQuantiles, rowRanges, rowRanks,
## rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs, rowVars,
## rowWeightedMads, rowWeightedMeans, rowWeightedMedians,
## rowWeightedSds, rowWeightedVars
## The following object is masked from 'package:Biobase':
##
## rowMedians
## Loading required package: GenomicRanges
## Loading required package: GenomeInfoDb
library(sechm)
source("../../plotGenesInTerm_v2.R")Dataset <- params$Dataset
logFcTh <- params$logFcTh
FdrTh <- params$FdrTh
OutputFolder <- ifelse(is.null(params$OutputFolder), getwd(), params$OutputFolder)
if (dir.exists(OutputFolder) == FALSE) {
dir.create(OutputFolder, recursive=TRUE)
}2. Data Upload
- Summarized Experiment object containing expression data used for DEA
and gene and sample metadata
- DEA object, containing results of the differential expression
2.1 Load Data from DEA
# List with differential expression results
DEA <- readRDS(params$DEAFile)
#SE object coming from DEA, but not containing specific contrast results
SE_DEA <- readRDS(params$SEFile)2.2 Add DEA results to SE
if(! identical(rownames(SE_DEA), row.names(DEA[[Condition_1]][[Condition_2]]$Res))){
stop('Expression data in SE and results from differential espression analysis are inconsistent.')
}
## Loading required package: DESeq2
rowData(SE_DEA) <- cbind(rowData(SE_DEA)[,1:6], DEA[[Condition_1]][[Condition_2]]$Res)
# Column names must be set to be compliant with the required format to be recognized by ORA
names(rowData(SE_DEA))[which(names(rowData(SE_DEA))=='log2FoldChange')] <- 'logFC'
names(rowData(SE_DEA))[which(names(rowData(SE_DEA))=='padj')] <- 'FDR'
#metadata(SE_DEA_Prel)$annotation <- 'hsa'14305 genes in 21 samples have been testes for differential expression.
Imposing a threshold of 1 on the Log2FC and 0.01 on the FDR (as specified in parameters), 58 genes are selected: 32 up-regulated genes and 26 down-regulated genes.
4. RESULTS VISUALIZATION
4.1 Volcano plot
The results of the differential expression analysis are visualized by Volcano plot. An interactive version is included in the html (only genes with FDR < threshold), while a static version is saved.
plotVolcanoSE(SE=SE_DEA, FdrTh=FdrTh, logFcTh=logFcTh, FdrCeil=1e-10, logFcCeil=4)4.3 Heatmap for significant genes
Heatmaps for DEGs, showing scaled vst values.
DEGs <- dplyr::filter(data.frame(rowData(SE_DEA)), FDR < FdrTh & abs(logFC) > logFcTh)
ScaledCols <- c('darkblue', "purple","white","lightgoldenrod1", 'goldenrod1')
colData(SE_DEA)$Condition <- factor(colData(SE_DEA)$Condition, levels=c("CTL", "DMSO", "AhHyd_Ag", "AhHyd_Inh", "Andr_Ag", "Andr_Inh", "Estr_Ag", "Estr_Inh", "GC_Ag", "GC_Inh", "LivX_Ag", "LivX_Inh", "Ret_Ag", "Ret_Inh", "Thyr_Ag", "Thyr_Inh" ))
metadata(SE_DEA)$anno_colors <- list(Condition = c('DMSO' = 'grey30', 'CTL' = 'azure3',
'AhHyd_Ag'='#F8766D', 'AhHyd_Inh'='#F8766D50',
'Andr_Ag'='#fccb17', 'Andr_Inh'='#C49A0050',
"Estr_Ag"= '#53B400', "Estr_Inh"= '#53B40050',
'GC_Ag' = '#00C094', 'GC_Inh' = '#00C09450',
'LivX_Ag' = '#00B6EB', 'LivX_Inh' = '#00B6EB50',
'Ret_Ag' = '#A58AFF', 'Ret_Inh' = '#A58AFF50',
'Thyr_Ag' = '#FB61D7', 'Thyr_Inh' = '#FB61D750'
))
sechm(SE_DEA, features=DEGs$GeneName, assayName="vst", gaps_at="Condition", show_rownames=FALSE,
top_annotation=c('Condition'), hmcols=ScaledCols, show_colnames=TRUE,
do.scale=TRUE, breaks=0.85)
5. TOPGO for Gene Ontology Enrichment analysis
Gene ontology enrichment analysis is performed on the set of 58 genes using TopGO with Fisher statistics and weight01 algorithm.
For each specified domain of the ontology:
- Enrichment analysis on all DEGs or splitted in down- and up-regulated
5.1 Selection of modulated genes and generation of gene vectors
I generate vectors for the gene universe, all modulated genes, up-regulated genes and down-regulated genes in the format required by TopGo.
GeneVectors <- topGOGeneVectors(SE_DEA, FdrTh=FdrTh, logFcTh=logFcTh)## Gene vector contains levels: 0,1
## Gene vector contains levels: 0,1
## Gene vector contains levels: 0,1Therefore:
- universe genes: 14305 genes
- modulated genes: 58 genes
- down-regulated genes: 26 genes of interest
- up-regulated genes: 32 genes of interest
Then I set parameters according to the gene ontology domains to be evaluated. By default, Biological Process and Molecular Function domains are interrogated.
BpEval <- ifelse(length(grep('BP', params$TopGO))!=0, TRUE, FALSE)
MfEval <- ifelse(length(grep('MF', params$TopGO))!=0, TRUE, FALSE)
CcEval <- ifelse(length(grep('CC', params$TopGO))!=0, TRUE, FALSE)5.2 TopGO analysis: Biological Process
On the basis of the analysis settings, the enrichment for Biological Process IS performed.
Biological Process Analysis for ALL modulated genes: 58 genes
BPann <- topGO::annFUN.org(whichOnto="BP", feasibleGenes=names(GeneVectors$DEGenes),
mapping="org.Hs.eg.db", ID="symbol") %>% inverseList()
# Wrapper function for topGO analysis
ResBPAll <- topGOResults(Genes=GeneVectors$DEGenes, gene2GO=BPann, ontology='BP',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=paste0(OutputFolder), fileName='BPAll')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 11416 GO terms found. )
##
## Build GO DAG topology ..........
## ( 14877 GO terms and 33562 relations. )
##
## Annotating nodes ...............
## ( 12665 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 1547 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 15: 5 nodes to be scored (0 eliminated genes)
##
## Level 14: 6 nodes to be scored (0 eliminated genes)
##
## Level 13: 13 nodes to be scored (194 eliminated genes)
##
## Level 12: 28 nodes to be scored (502 eliminated genes)
##
## Level 11: 53 nodes to be scored (2178 eliminated genes)
##
## Level 10: 105 nodes to be scored (3659 eliminated genes)
##
## Level 9: 147 nodes to be scored (4422 eliminated genes)
##
## Level 8: 202 nodes to be scored (5615 eliminated genes)
##
## Level 7: 273 nodes to be scored (6810 eliminated genes)
##
## Level 6: 298 nodes to be scored (8971 eliminated genes)
##
## Level 5: 216 nodes to be scored (10503 eliminated genes)
##
## Level 4: 126 nodes to be scored (11856 eliminated genes)
##
## Level 3: 58 nodes to be scored (12315 eliminated genes)
##
## Level 2: 16 nodes to be scored (12459 eliminated genes)
##
## Level 1: 1 nodes to be scored (12542 eliminated genes)Biological Process Analysis for DOWN-REGULATED genes: 26 genes
# Wrapper function for topGO analysis
ResBPDown <- topGOResults(Genes=GeneVectors$DEGenesDown, gene2GO=BPann, ontology='BP',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=paste0(OutputFolder), fileName='BPDown')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 11416 GO terms found. )
##
## Build GO DAG topology ..........
## ( 14877 GO terms and 33562 relations. )
##
## Annotating nodes ...............
## ( 12665 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 1115 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 15: 2 nodes to be scored (0 eliminated genes)
##
## Level 14: 2 nodes to be scored (0 eliminated genes)
##
## Level 13: 5 nodes to be scored (120 eliminated genes)
##
## Level 12: 14 nodes to be scored (198 eliminated genes)
##
## Level 11: 34 nodes to be scored (1976 eliminated genes)
##
## Level 10: 69 nodes to be scored (3322 eliminated genes)
##
## Level 9: 95 nodes to be scored (4101 eliminated genes)
##
## Level 8: 139 nodes to be scored (5083 eliminated genes)
##
## Level 7: 184 nodes to be scored (6036 eliminated genes)
##
## Level 6: 218 nodes to be scored (7538 eliminated genes)
##
## Level 5: 179 nodes to be scored (9761 eliminated genes)
##
## Level 4: 105 nodes to be scored (11512 eliminated genes)
##
## Level 3: 53 nodes to be scored (12253 eliminated genes)
##
## Level 2: 15 nodes to be scored (12436 eliminated genes)
##
## Level 1: 1 nodes to be scored (12540 eliminated genes)GOTable(ResBPDown$ResSel, maxGO=20)Biological Process Analysis for UP-REGULATED genes: 32 genes
ResBPUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=BPann, ontology='BP',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='BPUp')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 11416 GO terms found. )
##
## Build GO DAG topology ..........
## ( 14877 GO terms and 33562 relations. )
##
## Annotating nodes ...............
## ( 12665 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 997 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 15: 3 nodes to be scored (0 eliminated genes)
##
## Level 14: 4 nodes to be scored (0 eliminated genes)
##
## Level 13: 12 nodes to be scored (83 eliminated genes)
##
## Level 12: 22 nodes to be scored (407 eliminated genes)
##
## Level 11: 29 nodes to be scored (2106 eliminated genes)
##
## Level 10: 57 nodes to be scored (3563 eliminated genes)
##
## Level 9: 81 nodes to be scored (4133 eliminated genes)
##
## Level 8: 107 nodes to be scored (4992 eliminated genes)
##
## Level 7: 176 nodes to be scored (6048 eliminated genes)
##
## Level 6: 190 nodes to be scored (8228 eliminated genes)
##
## Level 5: 161 nodes to be scored (9974 eliminated genes)
##
## Level 4: 96 nodes to be scored (11430 eliminated genes)
##
## Level 3: 45 nodes to be scored (12227 eliminated genes)
##
## Level 2: 13 nodes to be scored (12409 eliminated genes)
##
## Level 1: 1 nodes to be scored (12515 eliminated genes)GOTable(ResBPUp$ResSel, maxGO=20)Result visualization: Barplot
topGOBarplotAll(TopGOResAll=ResBPAll$ResSel, TopGOResDown=ResBPDown$ResSel, TopGOResUp=ResBPUp$ResSel,
terms=8, pvalTh=0.01, plotTitle=NULL)
Top Terms associated Genes
All
plotGenesInTerm_v2(ResBPAll$ResSel, ResBPAll$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle=NULL, Interactive=FALSE)
Down
plotGenesInTerm_v2(ResBPDown$ResSel, ResBPDown$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle='Genes in Term - Down DEGs', Interactive=FALSE, fillCol='blue')
Up
plotGenesInTerm_v2(ResBPUp$ResSel, ResBPUp$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle='Genes in Term - Up DEGs', Interactive=FALSE, fillCol='red')
5.3 TopGO analysis: Molecular Function
On the basis of the analysis settings, the enrichment for Molecular Function IS performed.
Molecular Function Enrichment for ALL modulated genes: 58 genes
MFann <- topGO::annFUN.org(whichOnto='MF', feasibleGenes=names(GeneVectors$DEGenes),
mapping='org.Hs.eg.db', ID='symbol') %>% inverseList()
# Wrapper function for topGO analysis
ResMFAll <- topGOResults(Genes=GeneVectors$DEGenes, gene2GO=MFann, ontology='MF',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='MFAll')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 4060 GO terms found. )
##
## Build GO DAG topology ..........
## ( 4530 GO terms and 5903 relations. )
##
## Annotating nodes ...............
## ( 12999 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 216 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 11: 1 nodes to be scored (0 eliminated genes)
##
## Level 10: 3 nodes to be scored (0 eliminated genes)
##
## Level 9: 7 nodes to be scored (34 eliminated genes)
##
## Level 8: 9 nodes to be scored (968 eliminated genes)
##
## Level 7: 17 nodes to be scored (2443 eliminated genes)
##
## Level 6: 36 nodes to be scored (2768 eliminated genes)
##
## Level 5: 51 nodes to be scored (3832 eliminated genes)
##
## Level 4: 57 nodes to be scored (6014 eliminated genes)
##
## Level 3: 25 nodes to be scored (9038 eliminated genes)
##
## Level 2: 9 nodes to be scored (10189 eliminated genes)
##
## Level 1: 1 nodes to be scored (12787 eliminated genes)Molecular Function Enrichment for DOWN-REGULATED genes: 26 genes
ResMFDown <- topGOResults(Genes=GeneVectors$DEGenesDown, gene2GO=MFann, ontology='MF',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='MFDown')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 4060 GO terms found. )
##
## Build GO DAG topology ..........
## ( 4530 GO terms and 5903 relations. )
##
## Annotating nodes ...............
## ( 12999 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 129 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 11: 1 nodes to be scored (0 eliminated genes)
##
## Level 10: 1 nodes to be scored (0 eliminated genes)
##
## Level 9: 4 nodes to be scored (34 eliminated genes)
##
## Level 8: 3 nodes to be scored (928 eliminated genes)
##
## Level 7: 7 nodes to be scored (2366 eliminated genes)
##
## Level 6: 19 nodes to be scored (2495 eliminated genes)
##
## Level 5: 28 nodes to be scored (3361 eliminated genes)
##
## Level 4: 36 nodes to be scored (4770 eliminated genes)
##
## Level 3: 21 nodes to be scored (7912 eliminated genes)
##
## Level 2: 8 nodes to be scored (9450 eliminated genes)
##
## Level 1: 1 nodes to be scored (12622 eliminated genes)GOTable(ResMFDown$ResSel, maxGO=20)Molecular Function Analysis for UP-REGULATED genes: 32 genes
ResMFUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=MFann, ontology='MF',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='MFUp')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 4060 GO terms found. )
##
## Build GO DAG topology ..........
## ( 4530 GO terms and 5903 relations. )
##
## Annotating nodes ...............
## ( 12999 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 169 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 10: 3 nodes to be scored (0 eliminated genes)
##
## Level 9: 6 nodes to be scored (0 eliminated genes)
##
## Level 8: 8 nodes to be scored (968 eliminated genes)
##
## Level 7: 15 nodes to be scored (2441 eliminated genes)
##
## Level 6: 27 nodes to be scored (2757 eliminated genes)
##
## Level 5: 41 nodes to be scored (3770 eliminated genes)
##
## Level 4: 40 nodes to be scored (5537 eliminated genes)
##
## Level 3: 20 nodes to be scored (8683 eliminated genes)
##
## Level 2: 8 nodes to be scored (9731 eliminated genes)
##
## Level 1: 1 nodes to be scored (12710 eliminated genes)GOTable(ResMFUp$ResSel, maxGO=20)Result visualization: Barplot
topGOBarplotAll(TopGOResAll=ResMFAll$ResSel, TopGOResDown=ResMFDown$ResSel, TopGOResUp=ResMFUp$ResSel,
terms=8, pvalTh=0.01, plotTitle=NULL)
Top Terms associated Genes
All
plotGenesInTerm_v2(ResMFAll$ResSel, ResMFAll$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle=NULL, Interactive=FALSE)
Down
plotGenesInTerm_v2(ResMFDown$ResSel, ResMFDown$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle='Genes in Term - Down DEGs', Interactive=FALSE, fillCol='blue')
Up
plotGenesInTerm_v2(ResMFUp$ResSel, ResMFUp$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle='Genes in Term - Up DEGs', Interactive=FALSE, fillCol='red')
5.4 TopGO analysis: Cellular Component
On the basis of the analysis settings, the enrichment for Cellular Component IS performed.
Cellular Component Enrichment for ALL modulated genes: 58 genes
CCann <- topGO::annFUN.org(whichOnto='CC', feasibleGenes=names(GeneVectors$DEGenes),
mapping='org.Hs.eg.db', ID='symbol') %>% inverseList()
# Wrapper function for topGO analysis
ResCCAll <- topGOResults(Genes=GeneVectors$DEGenes, gene2GO=CCann, ontology='CC',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='CCAll')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 1732 GO terms found. )
##
## Build GO DAG topology ..........
## ( 1926 GO terms and 3253 relations. )
##
## Annotating nodes ...............
## ( 13224 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 216 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 11: 2 nodes to be scored (0 eliminated genes)
##
## Level 10: 13 nodes to be scored (0 eliminated genes)
##
## Level 9: 21 nodes to be scored (214 eliminated genes)
##
## Level 8: 29 nodes to be scored (1016 eliminated genes)
##
## Level 7: 31 nodes to be scored (2361 eliminated genes)
##
## Level 6: 35 nodes to be scored (7495 eliminated genes)
##
## Level 5: 33 nodes to be scored (9137 eliminated genes)
##
## Level 4: 24 nodes to be scored (11453 eliminated genes)
##
## Level 3: 25 nodes to be scored (12652 eliminated genes)
##
## Level 2: 2 nodes to be scored (13068 eliminated genes)
##
## Level 1: 1 nodes to be scored (13164 eliminated genes)
#write.table(ResCCAll$ResAll, file=paste0(OutputFolder, 'TopGO/CCAllResults.txt'), sep='\t', row.names=FALSE)Cellular Component Enrichment for DOWN-REGULATED genes: 26 genes
# Wrapper function for topGO analysis
ResCCDown <- topGOResults(Genes=GeneVectors$DEGenesDown, gene2GO=CCann, ontology='CC',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='CCDown')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 1732 GO terms found. )
##
## Build GO DAG topology ..........
## ( 1926 GO terms and 3253 relations. )
##
## Annotating nodes ...............
## ( 13224 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 148 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 11: 1 nodes to be scored (0 eliminated genes)
##
## Level 10: 7 nodes to be scored (0 eliminated genes)
##
## Level 9: 13 nodes to be scored (105 eliminated genes)
##
## Level 8: 21 nodes to be scored (703 eliminated genes)
##
## Level 7: 18 nodes to be scored (1891 eliminated genes)
##
## Level 6: 25 nodes to be scored (7172 eliminated genes)
##
## Level 5: 21 nodes to be scored (8791 eliminated genes)
##
## Level 4: 19 nodes to be scored (11320 eliminated genes)
##
## Level 3: 20 nodes to be scored (12536 eliminated genes)
##
## Level 2: 2 nodes to be scored (13021 eliminated genes)
##
## Level 1: 1 nodes to be scored (13162 eliminated genes)GOTable(ResCCDown$ResSel, maxGO=20)Cellular Component Analysis for UP-REGULATED genes: 32 genes
# Wrapper function for topGO analysis
ResCCUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=CCann, ontology='CC',
desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher',
EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
saveRes=TRUE, outDir=OutputFolder, fileName='CCUp')
## Gene vector contains levels: 0,1
##
## Building most specific GOs .....
## ( 1732 GO terms found. )
##
## Build GO DAG topology ..........
## ( 1926 GO terms and 3253 relations. )
##
## Annotating nodes ...............
## ( 13224 genes annotated to the GO terms. )
##
## -- Weight01 Algorithm --
##
## the algorithm is scoring 164 nontrivial nodes
## parameters:
## test statistic: fisher
##
## Level 11: 1 nodes to be scored (0 eliminated genes)
##
## Level 10: 7 nodes to be scored (0 eliminated genes)
##
## Level 9: 13 nodes to be scored (109 eliminated genes)
##
## Level 8: 18 nodes to be scored (510 eliminated genes)
##
## Level 7: 25 nodes to be scored (1749 eliminated genes)
##
## Level 6: 29 nodes to be scored (6885 eliminated genes)
##
## Level 5: 28 nodes to be scored (8648 eliminated genes)
##
## Level 4: 20 nodes to be scored (10904 eliminated genes)
##
## Level 3: 20 nodes to be scored (12645 eliminated genes)
##
## Level 2: 2 nodes to be scored (13068 eliminated genes)
##
## Level 1: 1 nodes to be scored (13163 eliminated genes)GOTable(ResCCUp$ResSel, maxGO=20)Result visualization: Barplot
topGOBarplotAll(TopGOResAll=ResCCAll$ResSel, TopGOResDown=ResCCDown$ResSel, TopGOResUp=ResCCUp$ResSel,
terms=8, pvalTh=0.01, plotTitle=NULL)
Top Terms associated Genes
All
plotGenesInTerm_v2(ResCCAll$ResSel, ResCCAll$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle=NULL, Interactive=FALSE)
Down
plotGenesInTerm_v2(ResCCDown$ResSel, ResCCDown$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle='Genes in Term - Down DEGs', Interactive=FALSE, fillCol='blue')
Up
plotGenesInTerm_v2(ResCCUp$ResSel, ResCCUp$GOdata, SE_DEA, nterms=8, ngenes=12, plotTitle='Genes in Term - Up DEGs', Interactive=FALSE, fillCol='red')
6. Savings
SessionInfo <- sessionInfo()
Date <- date()
#
save.image(paste0(OutputFolder, Dataset, 'FunctionalAnalysisWorkspace.RData'))SessionInfo## R version 4.2.1 (2022-06-23)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.4 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.9.0
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.9.0
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
## [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
##
## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods
## [8] base
##
## other attached packages:
## [1] DESeq2_1.38.3 sechm_1.6.0
## [3] SummarizedExperiment_1.28.0 GenomicRanges_1.50.2
## [5] GenomeInfoDb_1.34.9 MatrixGenerics_1.10.0
## [7] matrixStats_0.63.0 dplyr_1.1.0
## [9] tidyr_1.3.0 data.table_1.14.8
## [11] topGO_2.50.0 SparseM_1.81
## [13] GO.db_3.16.0 graph_1.76.0
## [15] viridis_0.6.2 viridisLite_0.4.1
## [17] RColorBrewer_1.1-3 gridExtra_2.3
## [19] org.Hs.eg.db_3.16.0 AnnotationDbi_1.60.0
## [21] IRanges_2.32.0 S4Vectors_0.36.1
## [23] Biobase_2.58.0 BiocGenerics_0.44.0
## [25] ggplot2_3.4.1 DT_0.27
## [27] RNASeqBulkExploratory_0.2.1
##
## loaded via a namespace (and not attached):
## [1] Rtsne_0.16 colorspace_2.1-0 rjson_0.2.21
## [4] ellipsis_0.3.2 circlize_0.4.15 XVector_0.38.0
## [7] GlobalOptions_0.1.2 clue_0.3-64 rstudioapi_0.14
## [10] farver_2.1.1 bit64_4.0.5 fansi_1.0.4
## [13] codetools_0.2-19 doParallel_1.0.17 cachem_1.0.7
## [16] geneplotter_1.76.0 knitr_1.42 jsonlite_1.8.4
## [19] annotate_1.76.0 cluster_2.1.4 png_0.1-8
## [22] compiler_4.2.1 httr_1.4.5 lazyeval_0.2.2
## [25] Matrix_1.5-3 fastmap_1.1.1 cli_3.6.1
## [28] htmltools_0.5.4 tools_4.2.1 gtable_0.3.1
## [31] glue_1.6.2 GenomeInfoDbData_1.2.9 V8_4.2.2
## [34] Rcpp_1.0.10 jquerylib_0.1.4 vctrs_0.6.2
## [37] Biostrings_2.66.0 iterators_1.0.14 crosstalk_1.2.0
## [40] xfun_0.37 stringr_1.5.0 lifecycle_1.0.3
## [43] XML_3.99-0.13 ca_0.71.1 zlibbioc_1.44.0
## [46] scales_1.2.1 TSP_1.2-2 parallel_4.2.1
## [49] ComplexHeatmap_2.14.0 yaml_2.3.7 curl_5.0.0
## [52] memoise_2.0.1 sass_0.4.5 stringi_1.7.12
## [55] RSQLite_2.3.0 highr_0.10 randomcoloR_1.1.0.1
## [58] foreach_1.5.2 seriation_1.4.1 BiocParallel_1.32.5
## [61] shape_1.4.6 rlang_1.1.1 pkgconfig_2.0.3
## [64] bitops_1.0-7 evaluate_0.20 lattice_0.20-45
## [67] purrr_1.0.1 labeling_0.4.2 htmlwidgets_1.6.1
## [70] bit_4.0.5 tidyselect_1.2.0 magrittr_2.0.3
## [73] R6_2.5.1 generics_0.1.3 DelayedArray_0.24.0
## [76] DBI_1.1.3 pillar_1.8.1 withr_2.5.0
## [79] KEGGREST_1.38.0 RCurl_1.98-1.10 tibble_3.2.1
## [82] crayon_1.5.2 utf8_1.2.3 plotly_4.10.1
## [85] rmarkdown_2.20 GetoptLong_1.0.5 locfit_1.5-9.7
## [88] grid_4.2.1 blob_1.2.3 digest_0.6.31
## [91] xtable_1.8-4 munsell_0.5.0 registry_0.5-1
## [94] bslib_0.4.2Date## [1] "Fri Jul 18 19:31:36 2025"