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

Top GO Analysis for GC Inhibitor

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'

14850 genes in 20 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), 351 genes are selected: 48 up-regulated genes and 303 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

plotVolcanoSE(SE=SE_DEA, FdrTh=FdrTh, logFcTh=logFcTh, FdrCeil=1e-10, logFcCeil=4)

4.2 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'
                 ), SeqRun = c("20210310" = "orange", "20210724"  = "green4", "20220422" = 
                                          "#66ACB7"))

sechm(SE_DEA, features=DEGs$GeneName, assayName="vst", gaps_at="Condition", show_rownames=FALSE,
      top_annotation=c('Condition', 'SeqRun'), hmcols=ScaledCols, show_colnames=TRUE,
      do.scale=TRUE, breaks=0.85, column_title = "Scaled Vst Values")

5. TOPGO for Gene Ontology Enrichment analysis

Gene ontology enrichment analysis is performed on the set of 351 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

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: 14850 genes
  • modulated genes: 351 genes
  • down-regulated genes: 303 genes of interest
  • up-regulated genes: 48 genes of interest
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: 351 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 .....
##  ( 11591 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 15045 GO terms and 33988 relations. )
## 
## Annotating nodes ...............
##  ( 13128 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 3282 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 18:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 17:  3 nodes to be scored    (0 eliminated genes)
## 
##   Level 16:  6 nodes to be scored    (18 eliminated genes)
## 
##   Level 15:  10 nodes to be scored   (50 eliminated genes)
## 
##   Level 14:  25 nodes to be scored   (130 eliminated genes)
## 
##   Level 13:  56 nodes to be scored   (284 eliminated genes)
## 
##   Level 12:  76 nodes to be scored   (1007 eliminated genes)
## 
##   Level 11:  172 nodes to be scored  (2937 eliminated genes)
## 
##   Level 10:  291 nodes to be scored  (4590 eliminated genes)
## 
##   Level 9:   427 nodes to be scored  (6068 eliminated genes)
## 
##   Level 8:   485 nodes to be scored  (7723 eliminated genes)
## 
##   Level 7:   578 nodes to be scored  (9952 eliminated genes)
## 
##   Level 6:   522 nodes to be scored  (11318 eliminated genes)
## 
##   Level 5:   336 nodes to be scored  (12180 eliminated genes)
## 
##   Level 4:   191 nodes to be scored  (12645 eliminated genes)
## 
##   Level 3:   85 nodes to be scored   (12854 eliminated genes)
## 
##   Level 2:   17 nodes to be scored   (12955 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13002 eliminated genes)

Biological Process Analysis for DOWN-REGULATED genes: 303 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$GoPvalTh, minTerms=12, geneTh=4,
                          saveRes=TRUE, outDir=paste0(OutputFolder), fileName='BPDown')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 11591 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 15045 GO terms and 33988 relations. )
## 
## Annotating nodes ...............
##  ( 13128 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 3176 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 18:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 17:  3 nodes to be scored    (0 eliminated genes)
## 
##   Level 16:  6 nodes to be scored    (18 eliminated genes)
## 
##   Level 15:  10 nodes to be scored   (50 eliminated genes)
## 
##   Level 14:  25 nodes to be scored   (130 eliminated genes)
## 
##   Level 13:  51 nodes to be scored   (284 eliminated genes)
## 
##   Level 12:  72 nodes to be scored   (1007 eliminated genes)
## 
##   Level 11:  159 nodes to be scored  (2817 eliminated genes)
## 
##   Level 10:  273 nodes to be scored  (4351 eliminated genes)
## 
##   Level 9:   411 nodes to be scored  (5809 eliminated genes)
## 
##   Level 8:   465 nodes to be scored  (7461 eliminated genes)
## 
##   Level 7:   561 nodes to be scored  (9859 eliminated genes)
## 
##   Level 6:   515 nodes to be scored  (11236 eliminated genes)
## 
##   Level 5:   333 nodes to be scored  (12170 eliminated genes)
## 
##   Level 4:   188 nodes to be scored  (12643 eliminated genes)
## 
##   Level 3:   85 nodes to be scored   (12853 eliminated genes)
## 
##   Level 2:   17 nodes to be scored   (12955 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13002 eliminated genes)
GOTable(ResBPDown$ResSel, maxGO=20)

Biological Process Analysis for UP-REGULATED genes: 48 genes

ResBPUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=BPann, ontology='BP', 
                        desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher', 
                        EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
                        saveRes=TRUE, outDir=OutputFolder, fileName='BPUp')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 11591 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 15045 GO terms and 33988 relations. )
## 
## Annotating nodes ...............
##  ( 13128 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 941 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 15:  1 nodes to be scored    (0 eliminated genes)
## 
##   Level 14:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 13:  12 nodes to be scored   (49 eliminated genes)
## 
##   Level 12:  21 nodes to be scored   (260 eliminated genes)
## 
##   Level 11:  40 nodes to be scored   (2060 eliminated genes)
## 
##   Level 10:  67 nodes to be scored   (3704 eliminated genes)
## 
##   Level 9:   76 nodes to be scored   (4503 eliminated genes)
## 
##   Level 8:   99 nodes to be scored   (5535 eliminated genes)
## 
##   Level 7:   142 nodes to be scored  (6790 eliminated genes)
## 
##   Level 6:   167 nodes to be scored  (8995 eliminated genes)
## 
##   Level 5:   155 nodes to be scored  (10771 eliminated genes)
## 
##   Level 4:   96 nodes to be scored   (12185 eliminated genes)
## 
##   Level 3:   49 nodes to be scored   (12748 eliminated genes)
## 
##   Level 2:   13 nodes to be scored   (12895 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (12990 eliminated genes)
GOTable(ResBPUp$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: 351 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 .....
##  ( 4114 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 4579 GO terms and 5967 relations. )
## 
## Annotating nodes ...............
##  ( 13476 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 465 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  5 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  7 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   16 nodes to be scored   (109 eliminated genes)
## 
##   Level 8:   24 nodes to be scored   (1163 eliminated genes)
## 
##   Level 7:   45 nodes to be scored   (3066 eliminated genes)
## 
##   Level 6:   80 nodes to be scored   (3491 eliminated genes)
## 
##   Level 5:   117 nodes to be scored  (4721 eliminated genes)
## 
##   Level 4:   116 nodes to be scored  (7779 eliminated genes)
## 
##   Level 3:   43 nodes to be scored   (10553 eliminated genes)
## 
##   Level 2:   11 nodes to be scored   (11502 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13345 eliminated genes)

Molecular Function Enrichment for DOWN-REGULATED genes: 303 genes

ResMFDown <- topGOResults(Genes=GeneVectors$DEGenesDown, gene2GO=MFann, ontology='MF', 
                          desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher', 
                          EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
                          saveRes=TRUE, outDir=OutputFolder, fileName='MFDown')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 4114 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 4579 GO terms and 5967 relations. )
## 
## Annotating nodes ...............
##  ( 13476 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 445 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  4 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  7 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   15 nodes to be scored   (90 eliminated genes)
## 
##   Level 8:   24 nodes to be scored   (1163 eliminated genes)
## 
##   Level 7:   42 nodes to be scored   (3064 eliminated genes)
## 
##   Level 6:   75 nodes to be scored   (3491 eliminated genes)
## 
##   Level 5:   114 nodes to be scored  (4704 eliminated genes)
## 
##   Level 4:   112 nodes to be scored  (7693 eliminated genes)
## 
##   Level 3:   41 nodes to be scored   (10521 eliminated genes)
## 
##   Level 2:   10 nodes to be scored   (11443 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13345 eliminated genes)
GOTable(ResMFDown$ResSel, maxGO=20)

Molecular Function Analysis for UP-REGULATED genes: 48 genes

ResMFUp <- topGOResults(Genes=GeneVectors$DEGenesUp, gene2GO=MFann, ontology='MF', 
                        desc=NULL, nodeSize=15, algorithm='weight01', statistic='fisher', 
                        EnTh=params$GoEnTh, PvalTh=params$GoPvalTh, minTerms=12, geneTh=4,
                        saveRes=TRUE, outDir=OutputFolder, fileName='MFUp')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 4114 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 4579 GO terms and 5967 relations. )
## 
## Annotating nodes ...............
##  ( 13476 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 187 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   9 nodes to be scored    (39 eliminated genes)
## 
##   Level 8:   10 nodes to be scored   (1037 eliminated genes)
## 
##   Level 7:   15 nodes to be scored   (2669 eliminated genes)
## 
##   Level 6:   25 nodes to be scored   (2980 eliminated genes)
## 
##   Level 5:   38 nodes to be scored   (4046 eliminated genes)
## 
##   Level 4:   49 nodes to be scored   (5437 eliminated genes)
## 
##   Level 3:   26 nodes to be scored   (8796 eliminated genes)
## 
##   Level 2:   10 nodes to be scored   (10249 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13286 eliminated genes)
GOTable(ResMFUp$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: 351 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 .....
##  ( 1740 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 1936 GO terms and 3272 relations. )
## 
## Annotating nodes ...............
##  ( 13716 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 396 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  18 nodes to be scored   (0 eliminated genes)
## 
##   Level 10:  36 nodes to be scored   (0 eliminated genes)
## 
##   Level 9:   48 nodes to be scored   (644 eliminated genes)
## 
##   Level 8:   58 nodes to be scored   (2133 eliminated genes)
## 
##   Level 7:   64 nodes to be scored   (4361 eliminated genes)
## 
##   Level 6:   56 nodes to be scored   (8245 eliminated genes)
## 
##   Level 5:   47 nodes to be scored   (10016 eliminated genes)
## 
##   Level 4:   32 nodes to be scored   (11954 eliminated genes)
## 
##   Level 3:   34 nodes to be scored   (13136 eliminated genes)
## 
##   Level 2:   2 nodes to be scored    (13552 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13659 eliminated genes)

Cellular Component Enrichment for DOWN-REGULATED genes: 303 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$GoPvalTh, minTerms=12, geneTh=4,
                          saveRes=TRUE, outDir=OutputFolder, fileName='CCDown')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 1740 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 1936 GO terms and 3272 relations. )
## 
## Annotating nodes ...............
##  ( 13716 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 378 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  17 nodes to be scored   (0 eliminated genes)
## 
##   Level 10:  35 nodes to be scored   (0 eliminated genes)
## 
##   Level 9:   48 nodes to be scored   (625 eliminated genes)
## 
##   Level 8:   54 nodes to be scored   (2132 eliminated genes)
## 
##   Level 7:   60 nodes to be scored   (4361 eliminated genes)
## 
##   Level 6:   54 nodes to be scored   (8219 eliminated genes)
## 
##   Level 5:   46 nodes to be scored   (9996 eliminated genes)
## 
##   Level 4:   29 nodes to be scored   (11950 eliminated genes)
## 
##   Level 3:   32 nodes to be scored   (13136 eliminated genes)
## 
##   Level 2:   2 nodes to be scored    (13551 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13659 eliminated genes)
GOTable(ResCCDown$ResSel, maxGO=20)

Cellular Component Analysis for UP-REGULATED genes: 48 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$GoPvalTh, minTerms=12, geneTh=4,
                        saveRes=TRUE, outDir=OutputFolder, fileName='CCUp')
## Gene vector contains levels: 0,1
## 
## Building most specific GOs .....
##  ( 1740 GO terms found. )
## 
## Build GO DAG topology ..........
##  ( 1936 GO terms and 3272 relations. )
## 
## Annotating nodes ...............
##  ( 13716 genes annotated to the GO terms. )
## 
##           -- Weight01 Algorithm -- 
## 
##       the algorithm is scoring 159 nontrivial nodes
##       parameters: 
##           test statistic: fisher
## 
##   Level 11:  2 nodes to be scored    (0 eliminated genes)
## 
##   Level 10:  6 nodes to be scored    (0 eliminated genes)
## 
##   Level 9:   8 nodes to be scored    (93 eliminated genes)
## 
##   Level 8:   19 nodes to be scored   (833 eliminated genes)
## 
##   Level 7:   24 nodes to be scored   (1873 eliminated genes)
## 
##   Level 6:   28 nodes to be scored   (6533 eliminated genes)
## 
##   Level 5:   27 nodes to be scored   (8889 eliminated genes)
## 
##   Level 4:   20 nodes to be scored   (11736 eliminated genes)
## 
##   Level 3:   22 nodes to be scored   (12998 eliminated genes)
## 
##   Level 2:   2 nodes to be scored    (13504 eliminated genes)
## 
##   Level 1:   1 nodes to be scored    (13633 eliminated genes)
GOTable(ResCCUp$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 18:30:16 2025"