Condition_1 <- params$Condition_1
Condition_2 <- params$Condition_2

Top GO Analysis for AhHyd Agonist

1. Environment Set Up

library(RNASeqBulkExploratory)
library(DT)
library(ggplot2)
library(AnnotationDbi)
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library(topGO)
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library(SummarizedExperiment)
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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), 76 genes are selected: 20 up-regulated genes and 56 down-regulated genes.


3. RESULTS NAVIGATION: Interactive Table

An interactive table show the results for the top 500 DEGs (ranked according to FDR).

DEGsTable(SE_DEA, FdrTh=0.01, logFcTh=1, maxGenes=500, saveDEGs=TRUE, outDir=OutputFolder)

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 76 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: 0,1

Therefore:

  • universe genes: 14305 genes
  • modulated genes: 76 genes
  • down-regulated genes: 56 genes of interest
  • up-regulated genes: 20 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: 76 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 1745 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 18:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 17:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 16:  1 nodes to be scored    (18 eliminated genes)
## 
##   Level 15:  6 nodes to be scored    (23 eliminated genes)
## 
##   Level 14:  8 nodes to be scored    (25 eliminated genes)
## 
##   Level 13:  17 nodes to be scored   (196 eliminated genes)
## 
##   Level 12:  42 nodes to be scored   (464 eliminated genes)
## 
##   Level 11:  72 nodes to be scored   (2187 eliminated genes)
## 
##   Level 10:  133 nodes to be scored  (3801 eliminated genes)
## 
##   Level 9:   194 nodes to be scored  (4751 eliminated genes)
## 
##   Level 8:   237 nodes to be scored  (5954 eliminated genes)
## 
##   Level 7:   295 nodes to be scored  (7849 eliminated genes)
## 
##   Level 6:   294 nodes to be scored  (9721 eliminated genes)
## 
##   Level 5:   236 nodes to be scored  (11090 eliminated genes)
## 
##   Level 4:   133 nodes to be scored  (11955 eliminated genes)
## 
##   Level 3:   59 nodes to be scored   (12336 eliminated genes)
## 
##   Level 2:   15 nodes to be scored   (12463 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12521 eliminated genes)

Biological Process Analysis for DOWN-REGULATED genes: 56 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$GoPvalTh, minTerms=12, geneTh=4,
                          saveRes=TRUE, outDir=paste0(OutputFolder), fileName='BPDown')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 11416 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 14877 GO terms and 33562 relations. )
## 
## Annotating nodes ...............
##  ( 12665 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 1475 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 18:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 17:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 16:  1 nodes to be scored    (18 eliminated genes)
## 
##   Level 15:  5 nodes to be scored    (23 eliminated genes)
## 
##   Level 14:  8 nodes to be scored    (25 eliminated genes)
## 
##   Level 13:  15 nodes to be scored   (139 eliminated genes)
## 
##   Level 12:  34 nodes to be scored   (464 eliminated genes)
## 
##   Level 11:  57 nodes to be scored   (2180 eliminated genes)
## 
##   Level 10:  103 nodes to be scored  (3692 eliminated genes)
## 
##   Level 9:   152 nodes to be scored  (4613 eliminated genes)
## 
##   Level 8:   184 nodes to be scored  (5635 eliminated genes)
## 
##   Level 7:   243 nodes to be scored  (6783 eliminated genes)
## 
##   Level 6:   263 nodes to be scored  (8776 eliminated genes)
## 
##   Level 5:   219 nodes to be scored  (10581 eliminated genes)
## 
##   Level 4:   120 nodes to be scored  (11787 eliminated genes)
## 
##   Level 3:   53 nodes to be scored   (12270 eliminated genes)
## 
##   Level 2:   15 nodes to be scored   (12452 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12515 eliminated genes)
GOTable(ResBPDown$ResSel, maxGO=20)

Biological Process Analysis for UP-REGULATED genes: 20 genes

ResBPUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=BPann, ontology='BP', 
                        desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher', 
                        EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
                        saveRes=TRUE, outDir=OutputFolder, fileName='BPUp')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 11416 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 14877 GO terms and 33562 relations. )
## 
## Annotating nodes ...............
##  ( 12665 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 814 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 15:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 14:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 13:  6 nodes to be scored    (85 eliminated genes)
## 
##   Level 12:  15 nodes to be scored   (155 eliminated genes)
## 
##   Level 11:  27 nodes to be scored   (1910 eliminated genes)
## 
##   Level 10:  47 nodes to be scored   (3294 eliminated genes)
## 
##   Level 9:   65 nodes to be scored   (3849 eliminated genes)
## 
##   Level 8:   94 nodes to be scored   (4611 eliminated genes)
## 
##   Level 7:   129 nodes to be scored  (6074 eliminated genes)
## 
##   Level 6:   146 nodes to be scored  (7843 eliminated genes)
## 
##   Level 5:   139 nodes to be scored  (9232 eliminated genes)
## 
##   Level 4:   82 nodes to be scored   (11053 eliminated genes)
## 
##   Level 3:   45 nodes to be scored   (12003 eliminated genes)
## 
##   Level 2:   14 nodes to be scored   (12436 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12519 eliminated genes)
GOTable(ResBPUp$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: 76 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 230 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  3 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  4 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   11 nodes to be scored   (99 eliminated genes)
## 
##   Level 8:   18 nodes to be scored   (1055 eliminated genes)
## 
##   Level 7:   23 nodes to be scored   (2847 eliminated genes)
## 
##   Level 6:   34 nodes to be scored   (3178 eliminated genes)
## 
##   Level 5:   53 nodes to be scored   (3875 eliminated genes)
## 
##   Level 4:   52 nodes to be scored   (6137 eliminated genes)
## 
##   Level 3:   23 nodes to be scored   (9083 eliminated genes)
## 
##   Level 2:   8 nodes to be scored    (10570 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12786 eliminated genes)

Molecular Function Enrichment for DOWN-REGULATED genes: 56 genes

ResMFDown <- topGOResults(Genes=GeneVectors$DEGenesDown, gene2GO=MFann, ontology='MF', 
                          desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher', 
                          EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
                          saveRes=TRUE, outDir=OutputFolder, fileName='MFDown')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 4060 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 4530 GO terms and 5903 relations. )
## 
## Annotating nodes ...............
##  ( 12999 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 205 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  4 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   11 nodes to be scored   (65 eliminated genes)
## 
##   Level 8:   18 nodes to be scored   (1055 eliminated genes)
## 
##   Level 7:   21 nodes to be scored   (2847 eliminated genes)
## 
##   Level 6:   29 nodes to be scored   (3178 eliminated genes)
## 
##   Level 5:   46 nodes to be scored   (3844 eliminated genes)
## 
##   Level 4:   43 nodes to be scored   (5902 eliminated genes)
## 
##   Level 3:   22 nodes to be scored   (8806 eliminated genes)
## 
##   Level 2:   8 nodes to be scored    (10299 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12779 eliminated genes)
GOTable(ResMFDown$ResSel, maxGO=20)

Molecular Function Analysis for UP-REGULATED genes: 20 genes

ResMFUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=MFann, ontology='MF', 
                        desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher', 
                        EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
                        saveRes=TRUE, outDir=OutputFolder, fileName='MFUp')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 4060 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 4530 GO terms and 5903 relations. )
## 
## Annotating nodes ...............
##  ( 12999 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 91 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   2 nodes to be scored    (34 eliminated genes)
## 
##   Level 8:   1 nodes to be scored    (928 eliminated genes)
## 
##   Level 7:   5 nodes to be scored    (1112 eliminated genes)
## 
##   Level 6:   12 nodes to be scored   (1184 eliminated genes)
## 
##   Level 5:   18 nodes to be scored   (1816 eliminated genes)
## 
##   Level 4:   29 nodes to be scored   (3309 eliminated genes)
## 
##   Level 3:   16 nodes to be scored   (6610 eliminated genes)
## 
##   Level 2:   5 nodes to be scored    (9255 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12498 eliminated genes)
GOTable(ResMFUp$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: 76 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 203 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  10 nodes to be scored   (0 eliminated genes)
## 
##   Level 9:   15 nodes to be scored   (175 eliminated genes)
## 
##   Level 8:   24 nodes to be scored   (787 eliminated genes)
## 
##   Level 7:   36 nodes to be scored   (2160 eliminated genes)
## 
##   Level 6:   32 nodes to be scored   (6981 eliminated genes)
## 
##   Level 5:   35 nodes to be scored   (8881 eliminated genes)
## 
##   Level 4:   23 nodes to be scored   (11417 eliminated genes)
## 
##   Level 3:   23 nodes to be scored   (12642 eliminated genes)
## 
##   Level 2:   2 nodes to be scored    (13050 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13144 eliminated genes)

#write.table(ResCCAll$ResAll, file=paste0(OutputFolder, 'TopGO/CCAllResults.txt'), sep='\t', row.names=FALSE)

Cellular Component Enrichment for DOWN-REGULATED genes: 56 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$GoPvalTh, minTerms=12, geneTh=4,
                          saveRes=TRUE, outDir=OutputFolder, fileName='CCDown')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 1732 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 1926 GO terms and 3253 relations. )
## 
## Annotating nodes ...............
##  ( 13224 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 183 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  8 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   13 nodes to be scored   (109 eliminated genes)
## 
##   Level 8:   20 nodes to be scored   (539 eliminated genes)
## 
##   Level 7:   30 nodes to be scored   (2121 eliminated genes)
## 
##   Level 6:   29 nodes to be scored   (6912 eliminated genes)
## 
##   Level 5:   34 nodes to be scored   (8708 eliminated genes)
## 
##   Level 4:   22 nodes to be scored   (11397 eliminated genes)
## 
##   Level 3:   23 nodes to be scored   (12642 eliminated genes)
## 
##   Level 2:   2 nodes to be scored    (13050 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13144 eliminated genes)
GOTable(ResCCDown$ResSel, maxGO=20)

Cellular Component Analysis for UP-REGULATED genes: 20 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$GoPvalTh, minTerms=12, geneTh=4,
                        saveRes=TRUE, outDir=OutputFolder, fileName='CCUp')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 1732 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 1926 GO terms and 3253 relations. )
## 
## Annotating nodes ...............
##  ( 13224 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 91 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   4 nodes to be scored    (66 eliminated genes)
## 
##   Level 8:   9 nodes to be scored    (275 eliminated genes)
## 
##   Level 7:   15 nodes to be scored   (758 eliminated genes)
## 
##   Level 6:   15 nodes to be scored   (5021 eliminated genes)
## 
##   Level 5:   16 nodes to be scored   (7288 eliminated genes)
## 
##   Level 4:   13 nodes to be scored   (10161 eliminated genes)
## 
##   Level 3:   13 nodes to be scored   (12533 eliminated genes)
## 
##   Level 2:   2 nodes to be scored    (13039 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13139 eliminated genes)
GOTable(ResCCUp$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_0.4.2
Date
## [1] "Fri Jul 18 20:06:58 2025"