SRP038101: Example2 gene set analysis: The case of Azacytidine
Mark Ziemann
2022-02-08
Source: https://github.com/markziemann/combined_enrichment
Intro
Here we are performing an analysis of some gene expression data to demonstrate the difference between ORA and FCS methods and to highlight the differences caused by improper background gene set use.
The dataset being used is SRP038101 and we are comparing the cells grown in normal condition (control) to those grown with addition of Azacitidine (case).
Data are obtained from http://dee2.io/
suppressPackageStartupMessages({
library("getDEE2")
library("DESeq2")
library("clusterProfiler")
library("mitch")
library("kableExtra")
library("eulerr")
library("gplots")
})Get expression data
I’m using some RNA-seq data looking at the effect of Azacytidine on AML3 cells.
name="SRP038101"
mdat<-getDEE2Metadata("hsapiens")
samplesheet <- mdat[grep("SRP038101",mdat$SRP_accession),]
samplesheet<-samplesheet[order(samplesheet$SRR_accession),]
samplesheet$trt<-as.factor(c(1,1,1,0,0,0))
s1 <- samplesheet
s1 %>% kbl(caption = "sample sheet") %>% kable_paper("hover", full_width = F)| SRR_accession | QC_summary | SRX_accession | SRS_accession | SRP_accession | Sample_name | GEO_series | Library_name | trt | |
|---|---|---|---|---|---|---|---|---|---|
| 239157 | SRR1171523 | PASS | SRX472607 | SRS559064 | SRP038101 | GSM1329859 | GSE55123 | 1 | |
| 239158 | SRR1171524 | WARN(3,4) | SRX472608 | SRS559066 | SRP038101 | GSM1329860 | GSE55123 | 1 | |
| 239159 | SRR1171525 | WARN(3,4) | SRX472609 | SRS559065 | SRP038101 | GSM1329861 | GSE55123 | 1 | |
| 239160 | SRR1171526 | WARN(3,4) | SRX472610 | SRS559068 | SRP038101 | GSM1329862 | GSE55123 | 0 | |
| 239161 | SRR1171527 | WARN(3,4) | SRX472611 | SRS559067 | SRP038101 | GSM1329863 | GSE55123 | 0 | |
| 239162 | SRR1171528 | WARN(3,4) | SRX472612 | SRS559069 | SRP038101 | GSM1329864 | GSE55123 | 0 |
w<-getDEE2("hsapiens",samplesheet$SRR_accession,metadata=mdat,legacy = TRUE)## For more information about DEE2 QC metrics, visit
## https://github.com/markziemann/dee2/blob/master/qc/qc_metrics.mdx<-Tx2Gene(w)
x<-x$Tx2Gene
# save the genetable for later
gt<-w$GeneInfo[,1,drop=FALSE]
gt$accession<-rownames(gt)
# counts
x1<-x[,which(colnames(x) %in% samplesheet$SRR_accession)]
# calculate RPM
rpm <- x1/colSums(x1) * 1000000
rownames(rpm) <- sapply(strsplit(rownames(rpm),"\\."),"[[",1)
# convert to gene symbols
rpm2 <- rpm
rpm2$symbol <- w$GeneInfo[match(rownames(rpm),rownames(w$GeneInfo)),"GeneSymbol"]
rpm2 <- aggregate(. ~ symbol, rpm2, sum)
rownames(rpm2) <- rpm2$symbol
rpm2$symbol=NULLHere show the number of genes in the annotation set, and those detected above the detection threshold.
# filter out lowly expressed genes
x1<-x1[which(rowSums(x1)/ncol(x1)>=(10)),]
nrow(x)## [1] 39297nrow(x1)## [1] 13926Now multidimensional scaling (MDS) plot to show the correlation between the datasets. If the control and case datasets are clustered separately, then it is likely that there will be many differentially expressed genes with FDR<0.05.
plot(cmdscale(dist(t(x1))), xlab="Coordinate 1", ylab="Coordinate 2", pch=19, col=s1$trt, main="MDS")
Differential expression
Now run DESeq2 for control vs case.
y <- DESeqDataSetFromMatrix(countData = round(x1), colData = s1, design = ~ trt)## converting counts to integer modey <- DESeq(y)## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testingde <- results(y)
de<-as.data.frame(de[order(de$pvalue),])
rownames(de)<-sapply(strsplit(rownames(de),"\\."),"[[",1)
head(de) %>% kbl() %>% kable_paper("hover", full_width = F)| baseMean | log2FoldChange | lfcSE | stat | pvalue | padj | |
|---|---|---|---|---|---|---|
| ENSG00000090382 | 14491.5013 | 1.686516 | 0.0460883 | 36.59317 | 0 | 0 |
| ENSG00000165949 | 1288.2858 | 3.326522 | 0.1053302 | 31.58185 | 0 | 0 |
| ENSG00000275214 | 911.4085 | 3.432709 | 0.1151394 | 29.81351 | 0 | 0 |
| ENSG00000115461 | 615.6738 | 5.004631 | 0.1746345 | 28.65774 | 0 | 0 |
| ENSG00000111331 | 2366.8131 | 2.649803 | 0.0944821 | 28.04556 | 0 | 0 |
| ENSG00000157601 | 1153.6028 | 2.820804 | 0.1016690 | 27.74499 | 0 | 0 |
Now let’s have a look at some of the charts showing differential expression. In particular, an MA plot and volcano plot.
maplot <- function(de,contrast_name) {
sig <-subset(de, padj < 0.05 )
up <-rownames(subset(de, padj < 0.05 & log2FoldChange > 0))
dn <-rownames(subset(de, padj < 0.05 & log2FoldChange < 0))
GENESUP <- length(up)
GENESDN <- length(dn)
DET=nrow(de)
SUBHEADER = paste(GENESUP, "up, ", GENESDN, "down", DET, "detected")
ns <-subset(de, padj > 0.05 )
plot(log2(de$baseMean),de$log2FoldChange,
xlab="log2 basemean", ylab="log2 foldchange",
pch=19, cex=0.5, col="dark gray",
main=contrast_name, cex.main=0.7)
points(log2(sig$baseMean),sig$log2FoldChange,
pch=19, cex=0.5, col="red")
mtext(SUBHEADER,cex = 0.7)
}
make_volcano <- function(de,name) {
sig <- subset(de,padj<0.05)
N_SIG=nrow(sig)
N_UP=nrow(subset(sig,log2FoldChange>0))
N_DN=nrow(subset(sig,log2FoldChange<0))
DET=nrow(de)
HEADER=paste(N_SIG,"@5%FDR,", N_UP, "up", N_DN, "dn", DET, "detected")
plot(de$log2FoldChange,-log10(de$padj),cex=0.5,pch=19,col="darkgray",
main=name, xlab="log2 FC", ylab="-log10 pval", xlim=c(-6,6))
mtext(HEADER)
grid()
points(sig$log2FoldChange,-log10(sig$padj),cex=0.5,pch=19,col="red")
}
make_volcano2 <- function(de,name,genes) {
myens <- rownames(w$GeneInfo)[match(genes,w$GeneInfo$GeneSymbol)]
sig <- de[which(rownames(de) %in% myens),]
N_SIG=nrow(subset(sig,padj<0.05))
N_UP=nrow(subset(sig,log2FoldChange>0 & padj<0.05))
N_DN=nrow(subset(sig,log2FoldChange<0 & padj<0.05))
DET=nrow(sig)
HEADER=paste(N_SIG,"@5%FDR,", N_UP, "up", N_DN, "dn", DET, "detected")
plot(de$log2FoldChange,-log10(de$padj),cex=0.5,pch=19,col="darkgray",
main=name, xlab="log2 FC", ylab="-log10 pval", xlim=c(-6,6))
mtext(HEADER)
grid()
points(sig$log2FoldChange,-log10(sig$padj),cex=0.5,pch=19,col="red")
}
maplot(de,name)
make_volcano(de,name)
Gene sets from Reactome
In order to perform gene set analysis, we need some gene sets.
if (! file.exists("ReactomePathways.gmt")) {
download.file("https://reactome.org/download/current/ReactomePathways.gmt.zip",
destfile="ReactomePathways.gmt.zip")
unzip("ReactomePathways.gmt.zip")
}
genesets<-gmt_import("ReactomePathways.gmt")FCS with Mitch
Mitch uses rank-ANOVA statistics for enrichment detection.
Here I’m using the standard approach
m <- mitch_import(de,DEtype = "DEseq2", geneTable = gt)## The input is a single dataframe; one contrast only. Converting
## it to a list for you.## Note: Mean no. genes in input = 13926## Note: no. genes in output = 12980## Note: estimated proportion of input genes in output = 0.932msep <- mitch_calc(m,genesets = genesets)## Note: When prioritising by significance (ie: small
## p-values), large effect sizes might be missed.ms_up <- subset(msep$enrichment_result,p.adjustANOVA<0.05 & s.dist > 0)[,1]
ms_dn <- subset(msep$enrichment_result,p.adjustANOVA<0.05 & s.dist < 0)[,1]
message(paste("Number of up-regulated pathways:",length(ms_up) ))## Number of up-regulated pathways: 192message(paste("Number of down-regulated pathways:",length(ms_dn) ))## Number of down-regulated pathways: 317head(msep$enrichment_result,10) #%>% kbl() %>% kable_paper("hover", full_width = F)## set setSize pANOVA s.dist
## 143 Cell Cycle 597 1.437842e-48 -0.3528846
## 601 Metabolism of RNA 655 3.431548e-43 -0.3178492
## 145 Cell Cycle, Mitotic 481 9.893334e-41 -0.3573001
## 144 Cell Cycle Checkpoints 251 1.852216e-30 -0.4210885
## 576 M Phase 341 3.588517e-29 -0.3543974
## 511 Innate Immune System 768 2.339749e-26 0.2276835
## 494 Immune System 1419 2.122194e-25 0.1688007
## 641 Mitotic Prometaphase 185 3.767721e-24 -0.4325959
## 640 Mitotic Metaphase and Anaphase 222 4.261274e-24 -0.3950088
## 637 Mitotic Anaphase 221 1.072557e-23 -0.3923537
## p.adjustANOVA
## 143 1.909454e-45
## 601 2.278548e-40
## 145 4.379449e-38
## 144 6.149357e-28
## 576 9.531100e-27
## 511 5.178645e-24
## 494 4.026106e-23
## 641 6.254417e-22
## 640 6.287746e-22
## 637 1.424355e-21#mitch_report(msep,outfile="mitch_separate.html",overwrite=TRUE)Here I’m using the combined approach.
mcom <- mitch_calc(abs(m),genesets = genesets)## Note: When prioritising by significance (ie: small
## p-values), large effect sizes might be missed.mc_up <- subset(mcom$enrichment_result,p.adjustANOVA<0.05 & s.dist > 0)[,1]
mc_dn <- subset(mcom$enrichment_result,p.adjustANOVA<0.05 & s.dist < 0)[,1]
message(paste("Number of up-regulated pathways:",length(mc_up) ))## Number of up-regulated pathways: 209message(paste("Number of down-regulated pathways:",length(mc_dn) ))## Number of down-regulated pathways: 23head(mcom$enrichment_result,10) #%>% kbl() %>% kable_paper("hover", full_width = F)## set setSize pANOVA s.dist
## 494 Immune System 1419 1.472928e-36 0.2044769
## 523 Interferon Signaling 157 1.680440e-29 0.5216180
## 213 Cytokine Signaling in Immune system 528 3.859906e-25 0.2651856
## 698 Neutrophil degranulation 390 8.596147e-22 0.2842736
## 143 Cell Cycle 597 4.249510e-21 0.2276749
## 511 Innate Immune System 768 1.570907e-20 0.1991816
## 524 Interferon alpha/beta signaling 51 4.376190e-19 0.7220900
## 145 Cell Cycle, Mitotic 481 5.331827e-19 0.2385536
## 1035 Signal Transduction 1694 1.066908e-17 0.1287007
## 69 Antiviral mechanism by IFN-stimulated genes 80 1.360598e-13 0.4786957
## p.adjustANOVA
## 494 1.956048e-33
## 523 1.115812e-26
## 213 1.708652e-22
## 698 2.853921e-19
## 143 1.128670e-18
## 511 3.476941e-18
## 524 8.302257e-17
## 145 8.850833e-17
## 1035 1.574282e-15
## 69 1.806874e-11#mitch_report(mcom,outfile="mitch_combined.html",overwrite=TRUE)Let’s look at the significant ones based on the combined analysis. There weren’t many gene sets classe d as significant. Let’s see how many have a direction agnostic enrichment score which is larger in magnitude than the direction informed enrichment score. There are only 11 such sets which would benefit from such combined analysis.
Euler diagram of the significant pathways found with each approach.
l0 <- list("sep up"=ms_up,"sep dn"=ms_dn,"comb up"=mc_up,"comb dn"=mc_dn)
par(cex.main=0.5)
plot(euler(l0),quantities = TRUE, edges = "gray", main="FCS: combined vs separated")
length(ms_up)## [1] 192length(ms_dn)## [1] 317length(ms_up)+length(ms_dn)## [1] 509length(mc_up)## [1] 209length(mc_dn)## [1] 23length(mc_up)+length(mc_dn)## [1] 232( length(ms_up)+length(ms_dn) ) / ( length(mc_up)+length(mc_dn) )## [1] 2.193966List gene sets which are specific to each approach.
ms <- c(ms_up,ms_dn)
# in sep but not comb
sep <- setdiff(ms,mc_up)
sep## [1] "Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell"
## [2] "Toll Like Receptor 4 (TLR4) Cascade"
## [3] "Toll-like Receptor Cascades"
## [4] "Degradation of the extracellular matrix"
## [5] "Signaling by Receptor Tyrosine Kinases"
## [6] "GPCR ligand binding"
## [7] "Integrin cell surface interactions"
## [8] "FCGR3A-mediated phagocytosis"
## [9] "Leishmania phagocytosis"
## [10] "Parasite infection"
## [11] "MyD88:MAL(TIRAP) cascade initiated on plasma membrane"
## [12] "Toll Like Receptor 2 (TLR2) Cascade"
## [13] "Toll Like Receptor TLR1:TLR2 Cascade"
## [14] "Toll Like Receptor TLR6:TLR2 Cascade"
## [15] "Interleukin-2 family signaling"
## [16] "Cell-Cell communication"
## [17] "Smooth Muscle Contraction"
## [18] "Muscle contraction"
## [19] "RHO GTPases Activate WASPs and WAVEs"
## [20] "Signaling by CSF3 (G-CSF)"
## [21] "Biological oxidations"
## [22] "Nucleotide-binding domain, leucine rich repeat containing receptor (NLR) signaling pathways"
## [23] "Cholesterol biosynthesis"
## [24] "MyD88 dependent cascade initiated on endosome"
## [25] "Toll Like Receptor 7/8 (TLR7/8) Cascade"
## [26] "Regulation of actin dynamics for phagocytic cup formation"
## [27] "Inactivation of CSF3 (G-CSF) signaling"
## [28] "Activated NOTCH1 Transmits Signal to the Nucleus"
## [29] "Collagen degradation"
## [30] "Death Receptor Signalling"
## [31] "MyD88-independent TLR4 cascade"
## [32] "TRIF(TICAM1)-mediated TLR4 signaling"
## [33] "TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation"
## [34] "Metabolism of lipids"
## [35] "Toll Like Receptor 3 (TLR3) Cascade"
## [36] "Nicotinamide salvaging"
## [37] "EPH-Ephrin signaling"
## [38] "Signaling by NOTCH3"
## [39] "Glycosphingolipid metabolism"
## [40] "IRAK4 deficiency (TLR2/4)"
## [41] "The NLRP3 inflammasome"
## [42] "Interleukin-3, Interleukin-5 and GM-CSF signaling"
## [43] "Anti-inflammatory response favouring Leishmania parasite infection"
## [44] "Leishmania parasite growth and survival"
## [45] "G alpha (q) signalling events"
## [46] "Cell recruitment (pro-inflammatory response)"
## [47] "Purinergic signaling in leishmaniasis infection"
## [48] "Cytochrome P450 - arranged by substrate type"
## [49] "NOTCH3 Intracellular Domain Regulates Transcription"
## [50] "Signaling by VEGF"
## [51] "MyD88 cascade initiated on plasma membrane"
## [52] "Toll Like Receptor 10 (TLR10) Cascade"
## [53] "Toll Like Receptor 5 (TLR5) Cascade"
## [54] "Signaling by NOTCH2"
## [55] "PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling"
## [56] "Sensory Perception"
## [57] "Developmental Biology"
## [58] "Endogenous sterols"
## [59] "MyD88 deficiency (TLR2/4)"
## [60] "RUNX3 regulates NOTCH signaling"
## [61] "Sphingolipid metabolism"
## [62] "Interleukin-15 signaling"
## [63] "Toll Like Receptor 9 (TLR9) Cascade"
## [64] "Negative regulation of the PI3K/AKT network"
## [65] "NOTCH2 Activation and Transmission of Signal to the Nucleus"
## [66] "NOTCH4 Intracellular Domain Regulates Transcription"
## [67] "Cell junction organization"
## [68] "TNFs bind their physiological receptors"
## [69] "Visual phototransduction"
## [70] "ECM proteoglycans"
## [71] "Antigen activates B Cell Receptor (BCR) leading to generation of second messengers"
## [72] "VEGFA-VEGFR2 Pathway"
## [73] "Signaling by NOTCH1"
## [74] "Glycosaminoglycan metabolism"
## [75] "Keratinization"
## [76] "p75NTR signals via NF-kB"
## [77] "Arachidonic acid metabolism"
## [78] "Neuronal System"
## [79] "Cargo recognition for clathrin-mediated endocytosis"
## [80] "G alpha (s) signalling events"
## [81] "Cell-cell junction organization"
## [82] "CDC42 GTPase cycle"
## [83] "Regulation of TLR by endogenous ligand"
## [84] "Synthesis of PIPs at the plasma membrane"
## [85] "Interleukin-17 signaling"
## [86] "EGFR downregulation"
## [87] "Constitutive Signaling by Aberrant PI3K in Cancer"
## [88] "Clathrin-mediated endocytosis"
## [89] "Phase I - Functionalization of compounds"
## [90] "Phase II - Conjugation of compounds"
## [91] "ZBP1(DAI) mediated induction of type I IFNs"
## [92] "HDMs demethylate histones"
## [93] "Notch-HLH transcription pathway"
## [94] "Metabolism of steroid hormones"
## [95] "Nuclear Events (kinase and transcription factor activation)"
## [96] "Caspase activation via Death Receptors in the presence of ligand"
## [97] "Adherens junctions interactions"
## [98] "Caspase-mediated cleavage of cytoskeletal proteins"
## [99] "Regulation of insulin secretion"
## [100] "Activation of Matrix Metalloproteinases"
## [101] "DDX58/IFIH1-mediated induction of interferon-alpha/beta"
## [102] "Cytosolic sulfonation of small molecules"
## [103] "Caspase activation via extrinsic apoptotic signalling pathway"
## [104] "The canonical retinoid cycle in rods (twilight vision)"
## [105] "Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells"
## [106] "TNFR1-induced NFkappaB signaling pathway"
## [107] "Defective EXT1 causes exostoses 1, TRPS2 and CHDS"
## [108] "Defective EXT2 causes exostoses 2"
## [109] "PI Metabolism"
## [110] "Chondroitin sulfate/dermatan sulfate metabolism"
## [111] "Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants"
## [112] "Constitutive Signaling by NOTCH1 PEST Domain Mutants"
## [113] "Signaling by NOTCH1 HD+PEST Domain Mutants in Cancer"
## [114] "Signaling by NOTCH1 PEST Domain Mutants in Cancer"
## [115] "Signaling by NOTCH1 in Cancer"
## [116] "Interleukin receptor SHC signaling"
## [117] "Nervous system development"
## [118] "Constitutive Signaling by NOTCH1 HD Domain Mutants"
## [119] "Signaling by NOTCH1 HD Domain Mutants in Cancer"
## [120] "Dopamine Neurotransmitter Release Cycle"
## [121] "Transmission across Chemical Synapses"
## [122] "Cardiac conduction"
## [123] "Biosynthesis of specialized proresolving mediators (SPMs)"
## [124] "Regulation of IFNA signaling"
## [125] "RAF-independent MAPK1/3 activation"
## [126] "Negative regulators of DDX58/IFIH1 signaling"
## [127] "MAP kinase activation"
## [128] "Nicotinate metabolism"
## [129] "Heparan sulfate/heparin (HS-GAG) metabolism"
## [130] "Diseases associated with O-glycosylation of proteins"
## [131] "Antimicrobial peptides"
## [132] "Peptide hormone metabolism"
## [133] "Protein-protein interactions at synapses"
## [134] "RIPK1-mediated regulated necrosis"
## [135] "Regulation of necroptotic cell death"
## [136] "NF-kB is activated and signals survival"
## [137] "GPVI-mediated activation cascade"
## [138] "Regulation of Complement cascade"
## [139] "Regulated Necrosis"
## [140] "Formation of the cornified envelope"
## [141] "Pregnenolone biosynthesis"
## [142] "Diseases associated with glycosaminoglycan metabolism"
## [143] "Interleukin-35 Signalling"
## [144] "ROS and RNS production in phagocytes"
## [145] "NOTCH4 Activation and Transmission of Signal to the Nucleus"
## [146] "Sialic acid metabolism"
## [147] "Signaling by PDGF"
## [148] "Signaling by FGFR1 in disease"
## [149] "HS-GAG biosynthesis"
## [150] "mRNA Splicing - Major Pathway"
## [151] "mRNA Splicing"
## [152] "rRNA modification in the nucleus and cytosol"
## [153] "tRNA processing"
## [154] "Homologous DNA Pairing and Strand Exchange"
## [155] "rRNA processing"
## [156] "rRNA processing in the nucleus and cytosol"
## [157] "HIV Infection"
## [158] "Presynaptic phase of homologous DNA pairing and strand exchange"
## [159] "Processing of DNA double-strand break ends"
## [160] "Major pathway of rRNA processing in the nucleolus and cytosol"
## [161] "HIV Life Cycle"
## [162] "Diseases of DNA repair"
## [163] "Late Phase of HIV Life Cycle"
## [164] "RNA Polymerase II Transcription"
## [165] "Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA)"
## [166] "Organelle biogenesis and maintenance"
## [167] "Nucleotide Excision Repair"
## [168] "Translation"
## [169] "Defective HDR through Homologous Recombination (HRR) due to BRCA1 loss-of-function"
## [170] "Defective HDR through Homologous Recombination (HRR) due to PALB2 loss of function"
## [171] "Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function"
## [172] "Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function"
## [173] "Diseases of DNA Double-Strand Break Repair"
## [174] "Resolution of D-Loop Structures"
## [175] "Global Genome Nucleotide Excision Repair (GG-NER)"
## [176] "Resolution of D-loop Structures through Holliday Junction Intermediates"
## [177] "Gap-filling DNA repair synthesis and ligation in TC-NER"
## [178] "RNA Polymerase I Promoter Escape"
## [179] "HDR through Single Strand Annealing (SSA)"
## [180] "Cilium Assembly"
## [181] "Epigenetic regulation of gene expression"
## [182] "Transcription-Coupled Nucleotide Excision Repair (TC-NER)"
## [183] "APC/C-mediated degradation of cell cycle proteins"
## [184] "Regulation of mitotic cell cycle"
## [185] "RNA Polymerase II Transcription Termination"
## [186] "Transcription of E2F targets under negative control by DREAM complex"
## [187] "Mitochondrial translation"
## [188] "Intra-Golgi and retrograde Golgi-to-ER traffic"
## [189] "Switching of origins to a post-replicative state"
## [190] "RNA Polymerase I Transcription Termination"
## [191] "Regulation of APC/C activators between G1/S and early anaphase"
## [192] "Activation of APC/C and APC/C:Cdc20 mediated degradation of mitotic proteins"
## [193] "Mitochondrial translation elongation"
## [194] "Assembly of the pre-replicative complex"
## [195] "TP53 Regulates Transcription of DNA Repair Genes"
## [196] "Golgi-to-ER retrograde transport"
## [197] "HCMV Infection"
## [198] "Nonhomologous End-Joining (NHEJ)"
## [199] "Meiotic recombination"
## [200] "APC/C:Cdc20 mediated degradation of mitotic proteins"
## [201] "HCMV Late Events"
## [202] "Mitochondrial translation termination"
## [203] "mRNA Splicing - Minor Pathway"
## [204] "APC:Cdc20 mediated degradation of cell cycle proteins prior to satisfation of the cell cycle checkpoint"
## [205] "Generic Transcription Pathway"
## [206] "Regulation of TP53 Activity through Phosphorylation"
## [207] "Negative epigenetic regulation of rRNA expression"
## [208] "Cdc20:Phospho-APC/C mediated degradation of Cyclin A"
## [209] "COPI-dependent Golgi-to-ER retrograde traffic"
## [210] "Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1"
## [211] "RNA Polymerase I Transcription Initiation"
## [212] "RNA Polymerase I Promoter Clearance"
## [213] "RNA Polymerase I Transcription"
## [214] "Mitochondrial translation initiation"
## [215] "Regulation of TP53 Activity"
## [216] "Dual incision in TC-NER"
## [217] "Formation of TC-NER Pre-Incision Complex"
## [218] "Processing of Capped Intronless Pre-mRNA"
## [219] "Kinesins"
## [220] "Mitochondrial protein import"
## [221] "Establishment of Sister Chromatid Cohesion"
## [222] "SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs"
## [223] "Anchoring of the basal body to the plasma membrane"
## [224] "Positive epigenetic regulation of rRNA expression"
## [225] "CDK-mediated phosphorylation and removal of Cdc6"
## [226] "Meiosis"
## [227] "Resolution of AP sites via the multiple-nucleotide patch replacement pathway"
## [228] "DNA Double Strand Break Response"
## [229] "APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1"
## [230] "Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex"
## [231] "NoRC negatively regulates rRNA expression"
## [232] "Recruitment of NuMA to mitotic centrosomes"
## [233] "Centrosome maturation"
## [234] "Recruitment of mitotic centrosome proteins and complexes"
## [235] "Formation of RNA Pol II elongation complex"
## [236] "RNA Polymerase II Transcription Elongation"
## [237] "SLBP independent Processing of Histone Pre-mRNAs"
## [238] "PCNA-Dependent Long Patch Base Excision Repair"
## [239] "Recognition of DNA damage by PCNA-containing replication complex"
## [240] "Orc1 removal from chromatin"
## [241] "Cyclin A:Cdk2-associated events at S phase entry"
## [242] "mRNA 3'-end processing"
## [243] "Inactivation of APC/C via direct inhibition of the APC/C complex"
## [244] "Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components"
## [245] "E2F mediated regulation of DNA replication"
## [246] "Antigen processing: Ubiquitination & Proteasome degradation"
## [247] "Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks"
## [248] "Influenza Infection"
## [249] "Base Excision Repair"
## [250] "RNA Polymerase II Pre-transcription Events"
## [251] "PERK regulates gene expression"
## [252] "Telomere C-strand (Lagging Strand) Synthesis"
## [253] "Formation of HIV-1 elongation complex containing HIV-1 Tat"
## [254] "HIV Transcription Elongation"
## [255] "Tat-mediated elongation of the HIV-1 transcript"
## [256] "Resolution of Abasic Sites (AP sites)"
## [257] "APC/C:Cdc20 mediated degradation of Securin"
## [258] "PKMTs methylate histone lysines"
## [259] "Formation of HIV elongation complex in the absence of HIV Tat"
## [260] "RNA Polymerase III Abortive And Retractive Initiation"
## [261] "RNA Polymerase III Transcription"
## [262] "Influenza Viral RNA Transcription and Replication"
## [263] "Aberrant regulation of mitotic cell cycle due to RB1 defects"
## [264] "Phosphorylation of the APC/C"
## [265] "Loss of Nlp from mitotic centrosomes"
## [266] "Loss of proteins required for interphase microtubule organization from the centrosome"
## [267] "The role of GTSE1 in G2/M progression after G2 checkpoint"
## [268] "Diseases of mitotic cell cycle"
## [269] "B-WICH complex positively regulates rRNA expression"
## [270] "Leading Strand Synthesis"
## [271] "Polymerase switching"
## [272] "Transcription of the HIV genome"
## [273] "tRNA modification in the nucleus and cytosol"
## [274] "Mismatch Repair"
## [275] "Transcriptional Regulation by E2F6"
## [276] "CDT1 association with the CDC6:ORC:origin complex"
## [277] "Mitochondrial calcium ion transport"
## [278] "Chromatin modifying enzymes"
## [279] "Chromatin organization"
## [280] "APC-Cdc20 mediated degradation of Nek2A"
## [281] "Reproduction"
## [282] "Dual Incision in GG-NER"
## [283] "mRNA Capping"
## [284] "mRNA decay by 3' to 5' exoribonuclease"
## [285] "Processive synthesis on the lagging strand"
## [286] "Autodegradation of Cdh1 by Cdh1:APC/C"
## [287] "Assembly of the ORC complex at the origin of replication"
## [288] "Neddylation"
## [289] "CDC6 association with the ORC:origin complex"
## [290] "Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha)"
## [291] "Gap-filling DNA repair synthesis and ligation in GG-NER"
## [292] "Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta)"
## [293] "HDR through MMEJ (alt-NHEJ)"
## [294] "RNA Polymerase III Transcription Initiation"
## [295] "RNA Polymerase III Transcription Initiation From Type 1 Promoter"
## [296] "Deadenylation-dependent mRNA decay"
## [297] "Regulation of PLK1 Activity at G2/M Transition"
## [298] "Formation of the Early Elongation Complex"
## [299] "Formation of the HIV-1 Early Elongation Complex"
## [300] "Regulation of PTEN stability and activity"
## [301] "Ubiquitin Mediated Degradation of Phosphorylated Cdc25A"
## [302] "p53-Independent DNA Damage Response"
## [303] "p53-Independent G1/S DNA damage checkpoint"
## [304] "Regulation of ornithine decarboxylase (ODC)"
## [305] "Disorders of transmembrane transporters"
## [306] "Factors involved in megakaryocyte development and platelet production"
## [307] "Removal of the Flap Intermediate"
## [308] "Retrograde transport at the Trans-Golgi-Network"
## [309] "Cellular responses to stress"
## [310] "Metabolism of polyamines"
## [311] "Translesion synthesis by REV1"
## [312] "RNA Polymerase III Transcription Initiation From Type 2 Promoter"
## [313] "Degradation of AXIN"
## [314] "RHOBTB GTPase Cycle"
## [315] "ATF4 activates genes in response to endoplasmic reticulum stress"
## [316] "DNA Damage Recognition in GG-NER"
## [317] "Glutamate and glutamine metabolism"
## [318] "Formation of Incision Complex in GG-NER"
## [319] "APC/C:Cdc20 mediated degradation of Cyclin B"
## [320] "RHOBTB1 GTPase cycle"
## [321] "FGFR2 alternative splicing"
## [322] "Vif-mediated degradation of APOBEC3G"
## [323] "Mitophagy"
## [324] "Cellular responses to stimuli"
## [325] "Transcriptional activation of mitochondrial biogenesis"
## [326] "RNA Pol II CTD phosphorylation and interaction with CE"
## [327] "RNA Pol II CTD phosphorylation and interaction with CE during HIV infection"
## [328] "Translesion synthesis by POLI"
## [329] "RNA Polymerase III Transcription Termination"
## [330] "Translesion synthesis by POLK"
## [331] "Mitochondrial biogenesis"
## [332] "Amino acid transport across the plasma membrane"
## [333] "Regulation of RUNX2 expression and activity"
## [334] "SCF-beta-TrCP mediated degradation of Emi1"
## [335] "Processive synthesis on the C-strand of the telomere"
## [336] "Carboxyterminal post-translational modifications of tubulin"
## [337] "AUF1 (hnRNP D0) binds and destabilizes mRNA"
## [338] "Regulation of activated PAK-2p34 by proteasome mediated degradation"
## [339] "Diseases associated with N-glycosylation of proteins"
## [340] "Regulation of Apoptosis"
## [341] "Processing of Intronless Pre-mRNAs"
## [342] "Telomere Extension By Telomerase"
## [343] "Initiation of Nuclear Envelope (NE) Reformation"
## [344] "FBXL7 down-regulates AURKA during mitotic entry and in early mitosis"
## [345] "DNA Damage/Telomere Stress Induced Senescence"
## [346] "Autodegradation of the E3 ubiquitin ligase COP1"
## [347] "tRNA Aminoacylation"
## [348] "Downstream signaling events of B Cell Receptor (BCR)"
## [349] "Vpu mediated degradation of CD4"
## [350] "UCH proteinases"
## [351] "HATs acetylate histones"
## [352] "SCF(Skp2)-mediated degradation of p27/p21"
## [353] "Interconversion of nucleotide di- and triphosphates"
## [354] "Aberrant regulation of mitotic exit in cancer due to RB1 defects"
## [355] "Ubiquitin-dependent degradation of Cyclin D"
## [356] "RNA Polymerase III Transcription Initiation From Type 3 Promoter"
## [357] "Inhibition of replication initiation of damaged DNA by RB1/E2F1"sep_up_only_df <- msep$enrichment_result[which(msep$enrichment_result$set %in% sep),]
sep_up_only_df <- head(subset(sep_up_only_df,s.dist>0),10)
sep_up_only_df %>% kbl(caption = "Upregulated sets identified in sep-DE only") %>% kable_paper("hover", full_width = F)| set | setSize | pANOVA | s.dist | p.adjustANOVA | |
|---|---|---|---|---|---|
| 495 | Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell | 58 | 2.0e-07 | 0.3952628 | 2.20e-06 |
| 1214 | Toll Like Receptor 4 (TLR4) Cascade | 117 | 8.0e-07 | 0.2649014 | 8.20e-06 |
| 1220 | Toll-like Receptor Cascades | 130 | 1.0e-06 | 0.2492870 | 1.00e-05 |
| 259 | Degradation of the extracellular matrix | 60 | 2.5e-06 | 0.3517286 | 2.42e-05 |
| 1108 | Signaling by Receptor Tyrosine Kinases | 365 | 4.7e-06 | 0.1402373 | 4.31e-05 |
| 408 | GPCR ligand binding | 127 | 6.4e-06 | 0.2323126 | 5.76e-05 |
| 518 | Integrin cell surface interactions | 41 | 8.0e-06 | 0.4032524 | 6.90e-05 |
| 346 | FCGR3A-mediated phagocytosis | 52 | 8.7e-06 | 0.3566855 | 7.35e-05 |
| 570 | Leishmania phagocytosis | 52 | 8.7e-06 | 0.3566855 | 7.35e-05 |
| 758 | Parasite infection | 52 | 8.7e-06 | 0.3566855 | 7.35e-05 |
sep_dn_only_df <- msep$enrichment_result[which(msep$enrichment_result$set %in% sep),]
sep_dn_only_df <- head(subset(sep_dn_only_df,s.dist<0),10)
sep_dn_only_df %>% kbl(caption = "Downregulated sets identified in sep-DE only") %>% kable_paper("hover", full_width = F)| set | setSize | pANOVA | s.dist | p.adjustANOVA | |
|---|---|---|---|---|---|
| 1304 | mRNA Splicing - Major Pathway | 177 | 0 | -0.3627765 | 0 |
| 1303 | mRNA Splicing | 185 | 0 | -0.3509669 | 0 |
| 1318 | rRNA modification in the nucleus and cytosol | 58 | 0 | -0.5377224 | 0 |
| 1325 | tRNA processing | 114 | 0 | -0.3832773 | 0 |
| 477 | Homologous DNA Pairing and Strand Exchange | 42 | 0 | -0.5834419 | 0 |
| 1319 | rRNA processing | 207 | 0 | -0.2635802 | 0 |
| 1321 | rRNA processing in the nucleus and cytosol | 186 | 0 | -0.2706460 | 0 |
| 458 | HIV Infection | 216 | 0 | -0.2430599 | 0 |
| 801 | Presynaptic phase of homologous DNA pairing and strand exchange | 39 | 0 | -0.5663673 | 0 |
| 805 | Processing of DNA double-strand break ends | 63 | 0 | -0.4419634 | 0 |
# in comb but not sep
comb <- setdiff(mc_up,ms)
comb## [1] "Antiviral mechanism by IFN-stimulated genes"
## [2] "Signaling by Rho GTPases, Miro GTPases and RHOBTB3"
## [3] "Signaling by Rho GTPases"
## [4] "Transcriptional regulation of granulopoiesis"
## [5] "TP53 Regulates Transcription of Cell Cycle Genes"
## [6] "Disease"
## [7] "Signaling by NOTCH"
## [8] "G-protein mediated events"
## [9] "PLC beta mediated events"
## [10] "TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest"
## [11] "Signaling by Nuclear Receptors"
## [12] "Aberrant regulation of mitotic G1/S transition in cancer due to RB1 defects"
## [13] "Defective binding of RB1 mutants to E2F1,(E2F2, E2F3)"
## [14] "Adaptive Immune System"
## [15] "Deubiquitination"
## [16] "Ca-dependent events"
## [17] "Cyclin D associated events in G1"
## [18] "G1 Phase"
## [19] "ESR-mediated signaling"
## [20] "Ub-specific processing proteases"
## [21] "FCGR3A-mediated IL10 synthesis"
## [22] "Diseases of signal transduction by growth factor receptors and second messengers"
## [23] "Regulation of cholesterol biosynthesis by SREBP (SREBF)"
## [24] "Oncogene Induced Senescence"
## [25] "CaM pathway"
## [26] "Calmodulin induced events"
## [27] "RHO GTPase cycle"
## [28] "Nuclear events stimulated by ALK signaling in cancer"
## [29] "Transcriptional regulation by RUNX1"
## [30] "Opioid Signalling"
## [31] "STING mediated induction of host immune responses"
## [32] "DAG and IP3 signaling"
## [33] "Transcriptional regulation by RUNX2"
## [34] "Metabolism of carbohydrates"
## [35] "RUNX1 interacts with co-factors whose precise effect on RUNX1 targets is not known"
## [36] "Signaling by TGF-beta Receptor Complex"
## [37] "Translesion synthesis by Y family DNA polymerases bypasses lesions on DNA template"
## [38] "Signaling by TGFB family members"
## [39] "Antigen processing-Cross presentation"
## [40] "Antigen Presentation: Folding, assembly and peptide loading of class I MHC"
## [41] "Protein folding"
## [42] "Cooperation of Prefoldin and TriC/CCT in actin and tubulin folding"
## [43] "Termination of translesion DNA synthesis"
## [44] "Signaling by ALK fusions and activated point mutants"
## [45] "Signaling by ALK in cancer"
## [46] "Chaperonin-mediated protein folding"
## [47] "FCERI mediated MAPK activation"
## [48] "Prefoldin mediated transfer of substrate to CCT/TriC"
## [49] "HS-GAG degradation"
## [50] "Metabolism of vitamins and cofactors"
## [51] "Prolactin receptor signaling"
## [52] "Sema3A PAK dependent Axon repulsion"
## [53] "Activation of gene expression by SREBF (SREBP)"
## [54] "Pre-NOTCH Expression and Processing"
## [55] "Defective B3GALT6 causes EDSP2 and SEMDJL1"
## [56] "Meiotic synapsis"
## [57] "Estrogen-dependent nuclear events downstream of ESR-membrane signaling"comb_only_df <- mcom$enrichment_result[which(mcom$enrichment_result$set %in% comb),]
comb_only_df %>% kbl(caption = "Dysregulated sets identified in all-DE only") %>% kable_paper("hover", full_width = F)| set | setSize | pANOVA | s.dist | p.adjustANOVA | |
|---|---|---|---|---|---|
| 69 | Antiviral mechanism by IFN-stimulated genes | 80 | 0.0000000 | 0.4786957 | 0.0000000 |
| 1111 | Signaling by Rho GTPases, Miro GTPases and RHOBTB3 | 551 | 0.0000000 | 0.1481631 | 0.0000003 |
| 1110 | Signaling by Rho GTPases | 536 | 0.0000000 | 0.1489638 | 0.0000003 |
| 1237 | Transcriptional regulation of granulopoiesis | 29 | 0.0000001 | 0.5683465 | 0.0000047 |
| 1178 | TP53 Regulates Transcription of Cell Cycle Genes | 47 | 0.0000003 | 0.4309823 | 0.0000109 |
| 270 | Disease | 1210 | 0.0000076 | 0.0780063 | 0.0001598 |
| 1085 | Signaling by NOTCH | 159 | 0.0000290 | 0.1925717 | 0.0005168 |
| 394 | G-protein mediated events | 42 | 0.0000361 | 0.3685452 | 0.0006125 |
| 752 | PLC beta mediated events | 38 | 0.0000422 | 0.3839966 | 0.0006840 |
| 1183 | TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest | 14 | 0.0000734 | 0.6119962 | 0.0010958 |
| 1099 | Signaling by Nuclear Receptors | 179 | 0.0001741 | 0.1630900 | 0.0023127 |
| 24 | Aberrant regulation of mitotic G1/S transition in cancer due to RB1 defects | 15 | 0.0002002 | 0.5545957 | 0.0025806 |
| 250 | Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) | 15 | 0.0002002 | 0.5545957 | 0.0025806 |
| 51 | Adaptive Immune System | 566 | 0.0003238 | 0.0892085 | 0.0039087 |
| 266 | Deubiquitination | 218 | 0.0003815 | 0.1400776 | 0.0044439 |
| 127 | Ca-dependent events | 28 | 0.0003940 | 0.3870500 | 0.0045500 |
| 209 | Cyclin D associated events in G1 | 45 | 0.0004800 | 0.3009973 | 0.0052679 |
| 396 | G1 Phase | 45 | 0.0004800 | 0.3009973 | 0.0052679 |
| 318 | ESR-mediated signaling | 133 | 0.0005703 | 0.1733514 | 0.0060108 |
| 1269 | Ub-specific processing proteases | 149 | 0.0005779 | 0.1637116 | 0.0060429 |
| 345 | FCGR3A-mediated IL10 synthesis | 29 | 0.0005850 | 0.3690063 | 0.0060696 |
| 284 | Diseases of signal transduction by growth factor receptors and second messengers | 351 | 0.0006201 | 0.1069169 | 0.0063838 |
| 951 | Regulation of cholesterol biosynthesis by SREBP (SREBF) | 54 | 0.0007002 | 0.2668266 | 0.0069387 |
| 727 | Oncogene Induced Senescence | 33 | 0.0010239 | 0.3304264 | 0.0093759 |
| 129 | CaM pathway | 26 | 0.0010308 | 0.3719433 | 0.0093759 |
| 130 | Calmodulin induced events | 26 | 0.0010308 | 0.3719433 | 0.0093759 |
| 838 | RHO GTPase cycle | 371 | 0.0010881 | 0.0993371 | 0.0097633 |
| 714 | Nuclear events stimulated by ALK signaling in cancer | 17 | 0.0011382 | 0.4558903 | 0.0101447 |
| 1232 | Transcriptional regulation by RUNX1 | 151 | 0.0013244 | 0.1517191 | 0.0114204 |
| 729 | Opioid Signalling | 70 | 0.0013671 | 0.2215005 | 0.0117131 |
| 1005 | STING mediated induction of host immune responses | 13 | 0.0014354 | 0.5106038 | 0.0119896 |
| 218 | DAG and IP3 signaling | 32 | 0.0016126 | 0.3222312 | 0.0131384 |
| 1233 | Transcriptional regulation by RUNX2 | 97 | 0.0017009 | 0.1846283 | 0.0136896 |
| 603 | Metabolism of carbohydrates | 219 | 0.0018077 | 0.1227567 | 0.0142896 |
| 895 | RUNX1 interacts with co-factors whose precise effect on RUNX1 targets is not known | 35 | 0.0020162 | 0.3017227 | 0.0153905 |
| 1113 | Signaling by TGF-beta Receptor Complex | 72 | 0.0022849 | 0.2081181 | 0.0171434 |
| 1249 | Translesion synthesis by Y family DNA polymerases bypasses lesions on DNA template | 38 | 0.0023793 | 0.2849556 | 0.0177512 |
| 1114 | Signaling by TGFB family members | 91 | 0.0024297 | 0.1841344 | 0.0180260 |
| 66 | Antigen processing-Cross presentation | 96 | 0.0024449 | 0.1791984 | 0.0180382 |
| 64 | Antigen Presentation: Folding, assembly and peptide loading of class I MHC | 23 | 0.0025631 | 0.3633524 | 0.0188053 |
| 814 | Protein folding | 82 | 0.0026165 | 0.1924745 | 0.0190920 |
| 203 | Cooperation of Prefoldin and TriC/CCT in actin and tubulin folding | 27 | 0.0030772 | 0.3291987 | 0.0222093 |
| 1200 | Termination of translesion DNA synthesis | 32 | 0.0031186 | 0.3020254 | 0.0223283 |
| 1040 | Signaling by ALK fusions and activated point mutants | 52 | 0.0035489 | 0.2338960 | 0.0249363 |
| 1041 | Signaling by ALK in cancer | 52 | 0.0035489 | 0.2338960 | 0.0249363 |
| 163 | Chaperonin-mediated protein folding | 76 | 0.0040420 | 0.1909404 | 0.0278124 |
| 343 | FCERI mediated MAPK activation | 28 | 0.0045604 | 0.3098087 | 0.0312178 |
| 798 | Prefoldin mediated transfer of substrate to CCT/TriC | 25 | 0.0048171 | 0.3258078 | 0.0325468 |
| 464 | HS-GAG degradation | 17 | 0.0054883 | 0.3890757 | 0.0358489 |
| 616 | Metabolism of vitamins and cofactors | 134 | 0.0061517 | 0.1373422 | 0.0396575 |
| 811 | Prolactin receptor signaling | 12 | 0.0062896 | 0.4555701 | 0.0400619 |
| 1023 | Sema3A PAK dependent Axon repulsion | 14 | 0.0063653 | 0.4212005 | 0.0400619 |
| 44 | Activation of gene expression by SREBF (SREBP) | 41 | 0.0068898 | 0.2440174 | 0.0427551 |
| 795 | Pre-NOTCH Expression and Processing | 47 | 0.0078890 | 0.2241388 | 0.0464036 |
| 237 | Defective B3GALT6 causes EDSP2 and SEMDJL1 | 14 | 0.0082304 | 0.4079461 | 0.0477294 |
| 596 | Meiotic synapsis | 26 | 0.0085644 | 0.2979596 | 0.0494503 |
| 333 | Estrogen-dependent nuclear events downstream of ESR-membrane signaling | 18 | 0.0086676 | 0.3574380 | 0.0497433 |
# intersection
intersect(mc_up,ms)## [1] "Immune System"
## [2] "Interferon Signaling"
## [3] "Cytokine Signaling in Immune system"
## [4] "Neutrophil degranulation"
## [5] "Cell Cycle"
## [6] "Innate Immune System"
## [7] "Interferon alpha/beta signaling"
## [8] "Cell Cycle, Mitotic"
## [9] "Signal Transduction"
## [10] "Interferon gamma signaling"
## [11] "Cell Cycle Checkpoints"
## [12] "ISG15 antiviral mechanism"
## [13] "M Phase"
## [14] "RHO GTPase Effectors"
## [15] "SUMOylation"
## [16] "Mitotic Metaphase and Anaphase"
## [17] "Mitotic Prometaphase"
## [18] "Mitotic Anaphase"
## [19] "Resolution of Sister Chromatid Cohesion"
## [20] "SUMO E3 ligases SUMOylate target proteins"
## [21] "Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs)"
## [22] "Signaling by Interleukins"
## [23] "Mitotic G1 phase and G1/S transition"
## [24] "Post-translational protein phosphorylation"
## [25] "Post-translational protein modification"
## [26] "SUMOylation of DNA replication proteins"
## [27] "EML4 and NUDC in mitotic spindle formation"
## [28] "Interactions of Rev with host cellular proteins"
## [29] "Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal"
## [30] "Amplification of signal from the kinetochores"
## [31] "RHO GTPases Activate Formins"
## [32] "SUMOylation of DNA damage response and repair proteins"
## [33] "DNA Repair"
## [34] "Interleukin-4 and Interleukin-13 signaling"
## [35] "Postmitotic nuclear pore complex (NPC) reformation"
## [36] "NS1 Mediated Effects on Host Pathways"
## [37] "G1/S Transition"
## [38] "Nuclear import of Rev protein"
## [39] "Separation of Sister Chromatids"
## [40] "S Phase"
## [41] "Rev-mediated nuclear export of HIV RNA"
## [42] "Mitotic Spindle Checkpoint"
## [43] "Extracellular matrix organization"
## [44] "Transport of the SLBP Dependant Mature mRNA"
## [45] "Interleukin-20 family signaling"
## [46] "Transport of the SLBP independent Mature mRNA"
## [47] "Hemostasis"
## [48] "Nuclear Pore Complex (NPC) Disassembly"
## [49] "Metabolism of proteins"
## [50] "Transport of Ribonucleoproteins into the Host Nucleus"
## [51] "DNA Replication"
## [52] "Mitotic Prophase"
## [53] "Export of Viral Ribonucleoproteins from Nucleus"
## [54] "NEP/NS2 Interacts with the Cellular Export Machinery"
## [55] "SUMOylation of ubiquitinylation proteins"
## [56] "Estrogen-dependent gene expression"
## [57] "G1/S-Specific Transcription"
## [58] "DNA Double-Strand Break Repair"
## [59] "Nuclear Envelope Breakdown"
## [60] "Transport of Mature mRNAs Derived from Intronless Transcripts"
## [61] "SUMOylation of chromatin organization proteins"
## [62] "G2/M Checkpoints"
## [63] "Nuclear Envelope (NE) Reassembly"
## [64] "Transport of Mature mRNA Derived from an Intronless Transcript"
## [65] "Gene Silencing by RNA"
## [66] "G0 and Early G1"
## [67] "Cellular response to heat stress"
## [68] "Transcriptional Regulation by TP53"
## [69] "Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC)"
## [70] "Regulation of Glucokinase by Glucokinase Regulatory Protein"
## [71] "SUMOylation of SUMOylation proteins"
## [72] "Glycolysis"
## [73] "Synthesis of DNA"
## [74] "DNA strand elongation"
## [75] "Signaling by GPCR"
## [76] "Host Interactions of HIV factors"
## [77] "Vpr-mediated nuclear import of PICs"
## [78] "HCMV Early Events"
## [79] "Interactions of Vpr with host cellular proteins"
## [80] "Growth hormone receptor signaling"
## [81] "G alpha (i) signalling events"
## [82] "Chromosome Maintenance"
## [83] "Binding and Uptake of Ligands by Scavenger Receptors"
## [84] "Glucose metabolism"
## [85] "Condensation of Prometaphase Chromosomes"
## [86] "GPCR downstream signalling"
## [87] "Interleukin-10 signaling"
## [88] "DNA Damage Bypass"
## [89] "TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest"
## [90] "Mitotic G2-G2/M phases"
## [91] "RHO GTPases Activate NADPH Oxidases"
## [92] "Gene expression (Transcription)"
## [93] "G2/M Transition"
## [94] "Mitotic Telophase/Cytokinesis"
## [95] "SUMOylation of RNA binding proteins"
## [96] "Condensation of Prophase Chromosomes"
## [97] "Transport of Mature mRNA derived from an Intron-Containing Transcript"
## [98] "Homology Directed Repair"
## [99] "Formation of tubulin folding intermediates by CCT/TriC"
## [100] "Transport of Mature Transcript to Cytoplasm"
## [101] "Metabolism of non-coding RNA"
## [102] "snRNP Assembly"
## [103] "Leishmania infection"
## [104] "TP53 Regulates Transcription of Cell Death Genes"
## [105] "G2/M DNA damage checkpoint"
## [106] "Metabolism of fat-soluble vitamins"
## [107] "tRNA processing in the nucleus"
## [108] "Regulation of mRNA stability by proteins that bind AU-rich elements"
## [109] "Polo-like kinase mediated events"
## [110] "HDR through Homologous Recombination (HRR) or Single Strand Annealing (SSA)"
## [111] "RHOA GTPase cycle"
## [112] "Regulation of HSF1-mediated heat shock response"
## [113] "Extension of Telomeres"
## [114] "Transcriptional regulation by small RNAs"
## [115] "Nucleotide biosynthesis"
## [116] "Inflammasomes"
## [117] "Folding of actin by CCT/TriC"
## [118] "FGFR1 mutant receptor activation"
## [119] "Retinoid metabolism and transport"
## [120] "Association of TriC/CCT with target proteins during biosynthesis"
## [121] "RAC1 GTPase cycle"
## [122] "SLC transporter disorders"
## [123] "Processing of Capped Intron-Containing Pre-mRNA"
## [124] "Cyclin A/B1/B2 associated events during G2/M transition"
## [125] "Activation of the pre-replicative complex"
## [126] "DNA Replication Pre-Initiation"
## [127] "Viral Messenger RNA Synthesis"
## [128] "Signaling by cytosolic FGFR1 fusion mutants"
## [129] "Unwinding of DNA"
## [130] "HDR through Homologous Recombination (HRR)"
## [131] "Deposition of new CENPA-containing nucleosomes at the centromere"
## [132] "Nucleosome assembly"
## [133] "Platelet activation, signaling and aggregation"
## [134] "Cohesin Loading onto Chromatin"
## [135] "Activation of ATR in response to replication stress"
## [136] "Diseases associated with the TLR signaling cascade"
## [137] "Diseases of Immune System"
## [138] "Response to elevated platelet cytosolic Ca2+"
## [139] "Class A/1 (Rhodopsin-like receptors)"
## [140] "Cyclin E associated events during G1/S transition"
## [141] "RMTs methylate histone arginines"
## [142] "Telomere Maintenance"
## [143] "Amyloid fiber formation"
## [144] "Metabolism of RNA"
## [145] "Signal regulatory protein family interactions"
## [146] "Endosomal/Vacuolar pathway"
## [147] "Platelet degranulation"
## [148] "Cell surface interactions at the vascular wall"
## [149] "Other interleukin signaling"
## [150] "Lagging Strand Synthesis"
## [151] "Fcgamma receptor (FCGR) dependent phagocytosis"
## [152] "AURKA Activation by TPX2"If we consider both strategies to be valid, then we can define the significant sets as dysregulated. We can calculate the percent sentitivity of both approaches.
all <- unique(c(ms_up,ms_dn,mc_up))
message("Sensitivity: separate only")## Sensitivity: separate only(length(ms_up)+length(ms_dn))/length(all)## [1] 0.8992933message("Sensitivity: combined only")## Sensitivity: combined onlylength(mc_up)/length(all)## [1] 0.369258Now use heatmap to visualise gene set regulation
comb_only_df## set
## 69 Antiviral mechanism by IFN-stimulated genes
## 1111 Signaling by Rho GTPases, Miro GTPases and RHOBTB3
## 1110 Signaling by Rho GTPases
## 1237 Transcriptional regulation of granulopoiesis
## 1178 TP53 Regulates Transcription of Cell Cycle Genes
## 270 Disease
## 1085 Signaling by NOTCH
## 394 G-protein mediated events
## 752 PLC beta mediated events
## 1183 TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest
## 1099 Signaling by Nuclear Receptors
## 24 Aberrant regulation of mitotic G1/S transition in cancer due to RB1 defects
## 250 Defective binding of RB1 mutants to E2F1,(E2F2, E2F3)
## 51 Adaptive Immune System
## 266 Deubiquitination
## 127 Ca-dependent events
## 209 Cyclin D associated events in G1
## 396 G1 Phase
## 318 ESR-mediated signaling
## 1269 Ub-specific processing proteases
## 345 FCGR3A-mediated IL10 synthesis
## 284 Diseases of signal transduction by growth factor receptors and second messengers
## 951 Regulation of cholesterol biosynthesis by SREBP (SREBF)
## 727 Oncogene Induced Senescence
## 129 CaM pathway
## 130 Calmodulin induced events
## 838 RHO GTPase cycle
## 714 Nuclear events stimulated by ALK signaling in cancer
## 1232 Transcriptional regulation by RUNX1
## 729 Opioid Signalling
## 1005 STING mediated induction of host immune responses
## 218 DAG and IP3 signaling
## 1233 Transcriptional regulation by RUNX2
## 603 Metabolism of carbohydrates
## 895 RUNX1 interacts with co-factors whose precise effect on RUNX1 targets is not known
## 1113 Signaling by TGF-beta Receptor Complex
## 1249 Translesion synthesis by Y family DNA polymerases bypasses lesions on DNA template
## 1114 Signaling by TGFB family members
## 66 Antigen processing-Cross presentation
## 64 Antigen Presentation: Folding, assembly and peptide loading of class I MHC
## 814 Protein folding
## 203 Cooperation of Prefoldin and TriC/CCT in actin and tubulin folding
## 1200 Termination of translesion DNA synthesis
## 1040 Signaling by ALK fusions and activated point mutants
## 1041 Signaling by ALK in cancer
## 163 Chaperonin-mediated protein folding
## 343 FCERI mediated MAPK activation
## 798 Prefoldin mediated transfer of substrate to CCT/TriC
## 464 HS-GAG degradation
## 616 Metabolism of vitamins and cofactors
## 811 Prolactin receptor signaling
## 1023 Sema3A PAK dependent Axon repulsion
## 44 Activation of gene expression by SREBF (SREBP)
## 795 Pre-NOTCH Expression and Processing
## 237 Defective B3GALT6 causes EDSP2 and SEMDJL1
## 596 Meiotic synapsis
## 333 Estrogen-dependent nuclear events downstream of ESR-membrane signaling
## setSize pANOVA s.dist p.adjustANOVA
## 69 80 1.360598e-13 0.47869574 1.806874e-11
## 1111 551 3.678861e-09 0.14816309 2.571330e-07
## 1110 536 4.854344e-09 0.14896378 3.223284e-07
## 1237 29 1.173278e-07 0.56834647 4.721557e-06
## 1178 47 3.214176e-07 0.43098226 1.094468e-05
## 270 1210 7.579987e-06 0.07800628 1.597813e-04
## 1085 159 2.902350e-05 0.19257174 5.168408e-04
## 394 42 3.609191e-05 0.36854522 6.125115e-04
## 752 38 4.223795e-05 0.38399662 6.840488e-04
## 1183 14 7.343661e-05 0.61199621 1.095773e-03
## 1099 179 1.741498e-04 0.16309000 2.312709e-03
## 24 15 2.001530e-04 0.55459571 2.580614e-03
## 250 15 2.001530e-04 0.55459571 2.580614e-03
## 51 566 3.237614e-04 0.08920852 3.908683e-03
## 266 218 3.814809e-04 0.14007755 4.443918e-03
## 127 28 3.940161e-04 0.38704999 4.550029e-03
## 209 45 4.799773e-04 0.30099729 5.267851e-03
## 396 45 4.799773e-04 0.30099729 5.267851e-03
## 318 133 5.702972e-04 0.17335137 6.010751e-03
## 1269 149 5.778939e-04 0.16371163 6.042859e-03
## 345 29 5.850199e-04 0.36900625 6.069581e-03
## 284 351 6.201140e-04 0.10691690 6.383809e-03
## 951 54 7.002035e-04 0.26682655 6.938724e-03
## 727 33 1.023867e-03 0.33042638 9.375869e-03
## 129 26 1.030781e-03 0.37194333 9.375869e-03
## 130 26 1.030781e-03 0.37194333 9.375869e-03
## 838 371 1.088080e-03 0.09933712 9.763309e-03
## 714 17 1.138221e-03 0.45589029 1.014468e-02
## 1232 151 1.324359e-03 0.15171907 1.142044e-02
## 729 70 1.367122e-03 0.22150050 1.171315e-02
## 1005 13 1.435385e-03 0.51060384 1.198959e-02
## 218 32 1.612619e-03 0.32223123 1.313839e-02
## 1233 97 1.700896e-03 0.18462835 1.368963e-02
## 603 219 1.807727e-03 0.12275666 1.428965e-02
## 895 35 2.016197e-03 0.30172267 1.539051e-02
## 1113 72 2.284931e-03 0.20811814 1.714344e-02
## 1249 38 2.379300e-03 0.28495555 1.775118e-02
## 1114 91 2.429713e-03 0.18413435 1.802602e-02
## 66 96 2.444936e-03 0.17919836 1.803820e-02
## 64 23 2.563073e-03 0.36335236 1.880531e-02
## 814 82 2.616526e-03 0.19247454 1.909201e-02
## 203 27 3.077190e-03 0.32919873 2.220929e-02
## 1200 32 3.118639e-03 0.30202541 2.232830e-02
## 1040 52 3.548923e-03 0.23389602 2.493635e-02
## 1041 52 3.548923e-03 0.23389602 2.493635e-02
## 163 76 4.042010e-03 0.19094039 2.781238e-02
## 343 28 4.560437e-03 0.30980874 3.121784e-02
## 798 25 4.817055e-03 0.32580780 3.254683e-02
## 464 17 5.488325e-03 0.38907570 3.584886e-02
## 616 134 6.151684e-03 0.13734225 3.965746e-02
## 811 12 6.289638e-03 0.45557012 4.006190e-02
## 1023 14 6.365257e-03 0.42120050 4.006190e-02
## 44 41 6.889756e-03 0.24401743 4.275512e-02
## 795 47 7.889013e-03 0.22413881 4.640359e-02
## 237 14 8.230439e-03 0.40794606 4.772936e-02
## 596 26 8.564435e-03 0.29795964 4.945030e-02
## 333 18 8.667551e-03 0.35743798 4.974334e-02lapply(1:nrow(comb_only_df),function(i) {
message(i)
myset <- comb_only_df$set[i]
genes <- genesets[[which(names(genesets) == myset)]]
mymx <- rpm2[which(rownames(rpm2) %in% genes),]
mymx2 <- mymx / rowMeans(mymx)
mymx2 <- mymx2[which(!is.na(mymx2[,1])),]
colfunc <- colorRampPalette(c("blue", "white", "red"))
heatmap.2(as.matrix(mymx2),main=myset,trace="none",scale="row",col=colfunc(25),margin=c(6,12),Colv="none",dendrogram="row",cexCol=0.8,cexRow=0.5)
make_volcano2(de,myset,genes)
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## NULLORA with clusterprofiler
Clusterprofiler uses a hypergeometric test. Firstly I will conduct the analysis separately for up and down regulated genes and with the correct backgound (as intended by the developers).
genesets2 <- read.gmt("ReactomePathways.gmt")
de_up <- rownames(subset(de, padj<0.05 & log2FoldChange > 0))
de_up <- unique(gt[which(rownames(gt) %in% de_up),1])
de_dn <- rownames(subset(de, padj<0.05 & log2FoldChange < 0))
de_dn <- unique(gt[which(rownames(gt) %in% de_dn),1])
de_bg <- rownames(de)
de_bg <- unique(gt[which(rownames(gt) %in% de_bg),1])
o_up <- as.data.frame(enricher(gene = de_up, universe = de_bg, maxGSSize = 5000, TERM2GENE = genesets2, pAdjustMethod="fdr"))
o_up <- rownames(subset(o_up, p.adjust < 0.05))
o_dn <- as.data.frame(enricher(gene = de_dn, universe = de_bg, maxGSSize = 5000, TERM2GENE = genesets2, pAdjustMethod="fdr"))
o_dn <- rownames(subset(o_dn, p.adjust < 0.05))
o_com <- as.data.frame(enricher(gene = union(de_up,de_dn), universe = de_bg, maxGSSize = 5000, TERM2GENE = genesets2, pAdjustMethod="fdr"))
o_com <- rownames(subset(o_com, p.adjust < 0.05))
length(o_up)## [1] 68length(o_dn)## [1] 191length(o_up) + length(o_dn)## [1] 259length(o_com)## [1] 83( length(o_up) + length(o_dn) ) / length(o_com)## [1] 3.120482all <- unique(c(o_up,o_dn,o_com))
message("Sensitivity: separate only")## Sensitivity: separate only(length(o_up)+length(o_dn))/length(all)## [1] 1message("Sensitivity: combined only")## Sensitivity: combined onlylength(o_com)/length(all)## [1] 0.3204633Euler diagram of the significant pathways found with each approach.
l2 <- list("sep up"=o_up,"sep dn"=o_dn,"comb"=o_com)
plot(euler(l2),quantities = TRUE, edges = "gray", main="ORA: combined vs separated")
List gene sets which are specific to each approach.
o_sep <- c(o_up,o_dn)
# in sep but not comb
setdiff(o_sep,o_com)## [1] "Platelet activation, signaling and aggregation"
## [2] "Hemostasis"
## [3] "Response to elevated platelet cytosolic Ca2+"
## [4] "Platelet degranulation"
## [5] "Interleukin-2 family signaling"
## [6] "GPCR downstream signalling"
## [7] "Leishmania infection"
## [8] "Signaling by GPCR"
## [9] "Degradation of the extracellular matrix"
## [10] "Cell surface interactions at the vascular wall"
## [11] "Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell"
## [12] "RHO GTPases Activate NADPH Oxidases"
## [13] "Cell-Cell communication"
## [14] "Interleukin-3, Interleukin-5 and GM-CSF signaling"
## [15] "Integrin cell surface interactions"
## [16] "G alpha (i) signalling events"
## [17] "Collagen degradation"
## [18] "Inactivation of CSF3 (G-CSF) signaling"
## [19] "Growth hormone receptor signaling"
## [20] "Smooth Muscle Contraction"
## [21] "RAC1 GTPase cycle"
## [22] "Signaling by Receptor Tyrosine Kinases"
## [23] "PLC beta mediated events"
## [24] "Class A/1 (Rhodopsin-like receptors)"
## [25] "FGFR1 mutant receptor activation"
## [26] "Other interleukin signaling"
## [27] "Cell junction organization"
## [28] "Antigen activates B Cell Receptor (BCR) leading to generation of second messengers"
## [29] "Interleukin-15 signaling"
## [30] "G-protein mediated events"
## [31] "Activation of Matrix Metalloproteinases"
## [32] "Anti-inflammatory response favouring Leishmania parasite infection"
## [33] "Leishmania parasite growth and survival"
## [34] "Retinoid metabolism and transport"
## [35] "Activated NOTCH1 Transmits Signal to the Nucleus"
## [36] "Adherens junctions interactions"
## [37] "MyD88 deficiency (TLR2/4)"
## [38] "Fcgamma receptor (FCGR) dependent phagocytosis"
## [39] "Constitutive Signaling by Aberrant PI3K in Cancer"
## [40] "Signaling by SCF-KIT"
## [41] "Inflammasomes"
## [42] "Cholesterol biosynthesis"
## [43] "Diseases associated with the TLR signaling cascade"
## [44] "Diseases of Immune System"
## [45] "GPCR ligand binding"
## [46] "HS-GAG degradation"
## [47] "Signaling by cytosolic FGFR1 fusion mutants"
## [48] "Amyloid fiber formation"
## [49] "Metabolism of RNA"
## [50] "Separation of Sister Chromatids"
## [51] "DNA Repair"
## [52] "Processing of Capped Intron-Containing Pre-mRNA"
## [53] "G2/M Checkpoints"
## [54] "Transport of Mature mRNA derived from an Intron-Containing Transcript"
## [55] "Transport of Mature Transcript to Cytoplasm"
## [56] "HDR through Homologous Recombination (HRR) or Single Strand Annealing (SSA)"
## [57] "S Phase"
## [58] "G2/M DNA damage checkpoint"
## [59] "Host Interactions of HIV factors"
## [60] "Transcriptional regulation by small RNAs"
## [61] "mRNA Splicing - Major Pathway"
## [62] "Regulation of HSF1-mediated heat shock response"
## [63] "Viral Messenger RNA Synthesis"
## [64] "mRNA Splicing"
## [65] "HCMV Early Events"
## [66] "HDR through Homologous Recombination (HRR)"
## [67] "DNA Replication"
## [68] "Homologous DNA Pairing and Strand Exchange"
## [69] "Mitotic G1 phase and G1/S transition"
## [70] "HIV Life Cycle"
## [71] "G1/S Transition"
## [72] "Chromosome Maintenance"
## [73] "Synthesis of DNA"
## [74] "Processing of DNA double-strand break ends"
## [75] "tRNA processing"
## [76] "Presynaptic phase of homologous DNA pairing and strand exchange"
## [77] "HIV Infection"
## [78] "Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA)"
## [79] "Global Genome Nucleotide Excision Repair (GG-NER)"
## [80] "Defective HDR through Homologous Recombination (HRR) due to BRCA1 loss-of-function"
## [81] "Defective HDR through Homologous Recombination (HRR) due to PALB2 loss of function"
## [82] "Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function"
## [83] "Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function"
## [84] "Diseases of DNA Double-Strand Break Repair"
## [85] "Diseases of DNA repair"
## [86] "HCMV Late Events"
## [87] "rRNA modification in the nucleus and cytosol"
## [88] "DNA strand elongation"
## [89] "Gene expression (Transcription)"
## [90] "Late Phase of HIV Life Cycle"
## [91] "TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest"
## [92] "Nonhomologous End-Joining (NHEJ)"
## [93] "Nucleotide Excision Repair"
## [94] "Deposition of new CENPA-containing nucleosomes at the centromere"
## [95] "Nucleosome assembly"
## [96] "G2/M Transition"
## [97] "Transcriptional Regulation by TP53"
## [98] "Resolution of AP sites via the multiple-nucleotide patch replacement pathway"
## [99] "HCMV Infection"
## [100] "PCNA-Dependent Long Patch Base Excision Repair"
## [101] "Mitotic G2-G2/M phases"
## [102] "Extension of Telomeres"
## [103] "Cyclin A/B1/B2 associated events during G2/M transition"
## [104] "Resolution of D-Loop Structures"
## [105] "Activation of ATR in response to replication stress"
## [106] "HDR through Single Strand Annealing (SSA)"
## [107] "Resolution of Abasic Sites (AP sites)"
## [108] "Polo-like kinase mediated events"
## [109] "Mitotic Telophase/Cytokinesis"
## [110] "Recognition of DNA damage by PCNA-containing replication complex"
## [111] "RMTs methylate histone arginines"
## [112] "Metabolism of proteins"
## [113] "Amino acid transport across the plasma membrane"
## [114] "Formation of tubulin folding intermediates by CCT/TriC"
## [115] "Lagging Strand Synthesis"
## [116] "Mitochondrial calcium ion transport"
## [117] "Gap-filling DNA repair synthesis and ligation in TC-NER"
## [118] "Establishment of Sister Chromatid Cohesion"
## [119] "rRNA processing in the nucleus and cytosol"
## [120] "Condensation of Prophase Chromosomes"
## [121] "Nucleotide biosynthesis"
## [122] "rRNA processing"
## [123] "Regulation of TP53 Activity through Phosphorylation"
## [124] "Activation of the pre-replicative complex"
## [125] "Resolution of D-loop Structures through Holliday Junction Intermediates"
## [126] "Post-translational protein modification"
## [127] "Gap-filling DNA repair synthesis and ligation in GG-NER"
## [128] "DNA Replication Pre-Initiation"
## [129] "Base Excision Repair"
## [130] "Dual Incision in GG-NER"
## [131] "Protein folding"
## [132] "Transcription-Coupled Nucleotide Excision Repair (TC-NER)"
## [133] "Transcription of E2F targets under negative control by DREAM complex"
## [134] "DNA Damage Bypass"
## [135] "TP53 Regulates Transcription of DNA Repair Genes"
## [136] "Telomere Maintenance"
## [137] "Major pathway of rRNA processing in the nucleolus and cytosol"
## [138] "Early Phase of HIV Life Cycle"
## [139] "APC/C-mediated degradation of cell cycle proteins"
## [140] "Regulation of mitotic cell cycle"
## [141] "AURKA Activation by TPX2"
## [142] "Cohesin Loading onto Chromatin"
## [143] "SLBP independent Processing of Histone Pre-mRNAs"
## [144] "Chaperonin-mediated protein folding"
## [145] "Regulation of APC/C activators between G1/S and early anaphase"
## [146] "Regulation of mRNA stability by proteins that bind AU-rich elements"
## [147] "Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta)"
## [148] "Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha)"
## [149] "Organelle biogenesis and maintenance"
## [150] "Dual incision in TC-NER"
## [151] "SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs"
## [152] "Cilium Assembly"
## [153] "Influenza Infection"
## [154] "Telomere C-strand (Lagging Strand) Synthesis"
## [155] "PERK regulates gene expression"
## [156] "mRNA Splicing - Minor Pathway"
## [157] "Formation of Incision Complex in GG-NER"
## [158] "Disorders of transmembrane transporters"
## [159] "Retrograde transport at the Trans-Golgi-Network"
## [160] "Aberrant regulation of mitotic G1/S transition in cancer due to RB1 defects"
## [161] "Defective binding of RB1 mutants to E2F1,(E2F2, E2F3)"
## [162] "Metabolism of folate and pterines"
## [163] "Mismatch Repair"
## [164] "Processive synthesis on the lagging strand"
## [165] "Positive epigenetic regulation of rRNA expression"
## [166] "Kinesins"
## [167] "Processive synthesis on the C-strand of the telomere"
## [168] "Cooperation of Prefoldin and TriC/CCT in actin and tubulin folding"
## [169] "DNA Double Strand Break Response"
## [170] "Regulation of TP53 Activity"
## [171] "Activation of APC/C and APC/C:Cdc20 mediated degradation of mitotic proteins"
## [172] "mRNA decay by 3' to 5' exoribonuclease"
## [173] "Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1"
## [174] "ESR-mediated signaling"
## [175] "Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding"
## [176] "RNA Polymerase I Promoter Escape"# in comb but not sep
setdiff(o_com,o_sep)## [1] "TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest"# intersection
intersect(mc_up,ms)## [1] "Immune System"
## [2] "Interferon Signaling"
## [3] "Cytokine Signaling in Immune system"
## [4] "Neutrophil degranulation"
## [5] "Cell Cycle"
## [6] "Innate Immune System"
## [7] "Interferon alpha/beta signaling"
## [8] "Cell Cycle, Mitotic"
## [9] "Signal Transduction"
## [10] "Interferon gamma signaling"
## [11] "Cell Cycle Checkpoints"
## [12] "ISG15 antiviral mechanism"
## [13] "M Phase"
## [14] "RHO GTPase Effectors"
## [15] "SUMOylation"
## [16] "Mitotic Metaphase and Anaphase"
## [17] "Mitotic Prometaphase"
## [18] "Mitotic Anaphase"
## [19] "Resolution of Sister Chromatid Cohesion"
## [20] "SUMO E3 ligases SUMOylate target proteins"
## [21] "Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs)"
## [22] "Signaling by Interleukins"
## [23] "Mitotic G1 phase and G1/S transition"
## [24] "Post-translational protein phosphorylation"
## [25] "Post-translational protein modification"
## [26] "SUMOylation of DNA replication proteins"
## [27] "EML4 and NUDC in mitotic spindle formation"
## [28] "Interactions of Rev with host cellular proteins"
## [29] "Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal"
## [30] "Amplification of signal from the kinetochores"
## [31] "RHO GTPases Activate Formins"
## [32] "SUMOylation of DNA damage response and repair proteins"
## [33] "DNA Repair"
## [34] "Interleukin-4 and Interleukin-13 signaling"
## [35] "Postmitotic nuclear pore complex (NPC) reformation"
## [36] "NS1 Mediated Effects on Host Pathways"
## [37] "G1/S Transition"
## [38] "Nuclear import of Rev protein"
## [39] "Separation of Sister Chromatids"
## [40] "S Phase"
## [41] "Rev-mediated nuclear export of HIV RNA"
## [42] "Mitotic Spindle Checkpoint"
## [43] "Extracellular matrix organization"
## [44] "Transport of the SLBP Dependant Mature mRNA"
## [45] "Interleukin-20 family signaling"
## [46] "Transport of the SLBP independent Mature mRNA"
## [47] "Hemostasis"
## [48] "Nuclear Pore Complex (NPC) Disassembly"
## [49] "Metabolism of proteins"
## [50] "Transport of Ribonucleoproteins into the Host Nucleus"
## [51] "DNA Replication"
## [52] "Mitotic Prophase"
## [53] "Export of Viral Ribonucleoproteins from Nucleus"
## [54] "NEP/NS2 Interacts with the Cellular Export Machinery"
## [55] "SUMOylation of ubiquitinylation proteins"
## [56] "Estrogen-dependent gene expression"
## [57] "G1/S-Specific Transcription"
## [58] "DNA Double-Strand Break Repair"
## [59] "Nuclear Envelope Breakdown"
## [60] "Transport of Mature mRNAs Derived from Intronless Transcripts"
## [61] "SUMOylation of chromatin organization proteins"
## [62] "G2/M Checkpoints"
## [63] "Nuclear Envelope (NE) Reassembly"
## [64] "Transport of Mature mRNA Derived from an Intronless Transcript"
## [65] "Gene Silencing by RNA"
## [66] "G0 and Early G1"
## [67] "Cellular response to heat stress"
## [68] "Transcriptional Regulation by TP53"
## [69] "Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC)"
## [70] "Regulation of Glucokinase by Glucokinase Regulatory Protein"
## [71] "SUMOylation of SUMOylation proteins"
## [72] "Glycolysis"
## [73] "Synthesis of DNA"
## [74] "DNA strand elongation"
## [75] "Signaling by GPCR"
## [76] "Host Interactions of HIV factors"
## [77] "Vpr-mediated nuclear import of PICs"
## [78] "HCMV Early Events"
## [79] "Interactions of Vpr with host cellular proteins"
## [80] "Growth hormone receptor signaling"
## [81] "G alpha (i) signalling events"
## [82] "Chromosome Maintenance"
## [83] "Binding and Uptake of Ligands by Scavenger Receptors"
## [84] "Glucose metabolism"
## [85] "Condensation of Prometaphase Chromosomes"
## [86] "GPCR downstream signalling"
## [87] "Interleukin-10 signaling"
## [88] "DNA Damage Bypass"
## [89] "TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest"
## [90] "Mitotic G2-G2/M phases"
## [91] "RHO GTPases Activate NADPH Oxidases"
## [92] "Gene expression (Transcription)"
## [93] "G2/M Transition"
## [94] "Mitotic Telophase/Cytokinesis"
## [95] "SUMOylation of RNA binding proteins"
## [96] "Condensation of Prophase Chromosomes"
## [97] "Transport of Mature mRNA derived from an Intron-Containing Transcript"
## [98] "Homology Directed Repair"
## [99] "Formation of tubulin folding intermediates by CCT/TriC"
## [100] "Transport of Mature Transcript to Cytoplasm"
## [101] "Metabolism of non-coding RNA"
## [102] "snRNP Assembly"
## [103] "Leishmania infection"
## [104] "TP53 Regulates Transcription of Cell Death Genes"
## [105] "G2/M DNA damage checkpoint"
## [106] "Metabolism of fat-soluble vitamins"
## [107] "tRNA processing in the nucleus"
## [108] "Regulation of mRNA stability by proteins that bind AU-rich elements"
## [109] "Polo-like kinase mediated events"
## [110] "HDR through Homologous Recombination (HRR) or Single Strand Annealing (SSA)"
## [111] "RHOA GTPase cycle"
## [112] "Regulation of HSF1-mediated heat shock response"
## [113] "Extension of Telomeres"
## [114] "Transcriptional regulation by small RNAs"
## [115] "Nucleotide biosynthesis"
## [116] "Inflammasomes"
## [117] "Folding of actin by CCT/TriC"
## [118] "FGFR1 mutant receptor activation"
## [119] "Retinoid metabolism and transport"
## [120] "Association of TriC/CCT with target proteins during biosynthesis"
## [121] "RAC1 GTPase cycle"
## [122] "SLC transporter disorders"
## [123] "Processing of Capped Intron-Containing Pre-mRNA"
## [124] "Cyclin A/B1/B2 associated events during G2/M transition"
## [125] "Activation of the pre-replicative complex"
## [126] "DNA Replication Pre-Initiation"
## [127] "Viral Messenger RNA Synthesis"
## [128] "Signaling by cytosolic FGFR1 fusion mutants"
## [129] "Unwinding of DNA"
## [130] "HDR through Homologous Recombination (HRR)"
## [131] "Deposition of new CENPA-containing nucleosomes at the centromere"
## [132] "Nucleosome assembly"
## [133] "Platelet activation, signaling and aggregation"
## [134] "Cohesin Loading onto Chromatin"
## [135] "Activation of ATR in response to replication stress"
## [136] "Diseases associated with the TLR signaling cascade"
## [137] "Diseases of Immune System"
## [138] "Response to elevated platelet cytosolic Ca2+"
## [139] "Class A/1 (Rhodopsin-like receptors)"
## [140] "Cyclin E associated events during G1/S transition"
## [141] "RMTs methylate histone arginines"
## [142] "Telomere Maintenance"
## [143] "Amyloid fiber formation"
## [144] "Metabolism of RNA"
## [145] "Signal regulatory protein family interactions"
## [146] "Endosomal/Vacuolar pathway"
## [147] "Platelet degranulation"
## [148] "Cell surface interactions at the vascular wall"
## [149] "Other interleukin signaling"
## [150] "Lagging Strand Synthesis"
## [151] "Fcgamma receptor (FCGR) dependent phagocytosis"
## [152] "AURKA Activation by TPX2"Euler diagrams comparing FCS and ORA methods
par(cex.main=0.5)
par(mar=c(2,2,2,2))
l3 <- list("ORA up"=o_up,"ORA dn"=o_dn,"ORA comb"=o_com,
"FCS up"=ms_up,"FCS dn"=ms_dn,"FCS comb"=mc_up)
plot(euler(l3),quantities = TRUE, edges = "gray", main="FCS compared to ORA")
Save data
dat <- list( "FCS_up"=ms_up,
"FCS_dn"=ms_dn,
"FCS_com"=mc_up,
"ORA_up"= o_up,
"ORA_dn"=o_dn,
"ORA_com"=o_com)
str(dat)## List of 6
## $ FCS_up : chr [1:192] "Innate Immune System" "Immune System" "Interferon alpha/beta signaling" "Neutrophil degranulation" ...
## $ FCS_dn : chr [1:317] "Cell Cycle" "Metabolism of RNA" "Cell Cycle, Mitotic" "Cell Cycle Checkpoints" ...
## $ FCS_com: chr [1:209] "Immune System" "Interferon Signaling" "Cytokine Signaling in Immune system" "Neutrophil degranulation" ...
## $ ORA_up : chr [1:68] "Immune System" "Innate Immune System" "Neutrophil degranulation" "Cytokine Signaling in Immune system" ...
## $ ORA_dn : chr [1:191] "Cell Cycle" "Cell Cycle, Mitotic" "Cell Cycle Checkpoints" "M Phase" ...
## $ ORA_com: chr [1:83] "Interferon Signaling" "Immune System" "Cytokine Signaling in Immune system" "Interferon alpha/beta signaling" ...saveRDS(dat,file = "ex2dat.rds")Conclusion
For mitch, it would appear that performing direction informed (DI) analysis clearly yields more differentially regulated pathways (413) as compared to the direction agnostic (DA) method (55).
That being said, there were 18 pathways identified only in the DA method that appeared to be related to the physiology of the model. These gene sets are likely to contain a mix of genes affected by the stimulus in different ways - for example a mix of up and downregulated genes. Are these really real? Not sure.
This pattern was consistent with ORA, where 80 sets were identified with separate analysis and only 23 with the combined analysis.
When comparing ORA to FCS, we found that FCS identified many more sets than ORA. In fact all gene sets that were identified by ORA were also identified by FCS, except for 3 that were specific to the ORA up set.
Let’s look at those now.
myfcs <- c(ms_up, mc_up)
setdiff(o_up,myfcs)## [1] "Signaling by SCF-KIT"Session information
sessionInfo()## R version 4.1.2 (2021-11-01)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.3 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_AU.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_AU.UTF-8 LC_COLLATE=en_AU.UTF-8
## [5] LC_MONETARY=en_AU.UTF-8 LC_MESSAGES=en_AU.UTF-8
## [7] LC_PAPER=en_AU.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] parallel stats4 stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] gplots_3.1.1 eulerr_6.1.1
## [3] kableExtra_1.3.4 mitch_1.4.1
## [5] clusterProfiler_4.0.5 DESeq2_1.32.0
## [7] SummarizedExperiment_1.22.0 Biobase_2.52.0
## [9] MatrixGenerics_1.4.3 matrixStats_0.61.0
## [11] GenomicRanges_1.44.0 GenomeInfoDb_1.28.4
## [13] IRanges_2.26.0 S4Vectors_0.30.2
## [15] BiocGenerics_0.38.0 getDEE2_1.2.0
##
## loaded via a namespace (and not attached):
## [1] shadowtext_0.1.1 fastmatch_1.1-3 systemfonts_1.0.3
## [4] plyr_1.8.6 igraph_1.2.11 lazyeval_0.2.2
## [7] polylabelr_0.2.0 splines_4.1.2 BiocParallel_1.26.2
## [10] ggplot2_3.3.5 digest_0.6.29 yulab.utils_0.0.4
## [13] htmltools_0.5.2 GOSemSim_2.18.1 viridis_0.6.2
## [16] GO.db_3.13.0 fansi_1.0.0 magrittr_2.0.1
## [19] memoise_2.0.1 Biostrings_2.60.2 annotate_1.70.0
## [22] graphlayouts_0.8.0 svglite_2.0.0 enrichplot_1.12.3
## [25] colorspace_2.0-2 rvest_1.0.2 blob_1.2.2
## [28] ggrepel_0.9.1 xfun_0.29 dplyr_1.0.7
## [31] crayon_1.4.2 RCurl_1.98-1.5 jsonlite_1.7.2
## [34] scatterpie_0.1.7 genefilter_1.74.1 survival_3.2-13
## [37] ape_5.6-1 glue_1.6.0 polyclip_1.10-0
## [40] gtable_0.3.0 zlibbioc_1.38.0 XVector_0.32.0
## [43] webshot_0.5.2 htm2txt_2.1.1 DelayedArray_0.18.0
## [46] scales_1.1.1 DOSE_3.18.3 DBI_1.1.2
## [49] GGally_2.1.2 Rcpp_1.0.7 viridisLite_0.4.0
## [52] xtable_1.8-4 gridGraphics_0.5-1 tidytree_0.3.7
## [55] bit_4.0.4 htmlwidgets_1.5.4 httr_1.4.2
## [58] fgsea_1.18.0 RColorBrewer_1.1-2 ellipsis_0.3.2
## [61] pkgconfig_2.0.3 reshape_0.8.8 XML_3.99-0.8
## [64] farver_2.1.0 sass_0.4.0 locfit_1.5-9.4
## [67] utf8_1.2.2 ggplotify_0.1.0 tidyselect_1.1.1
## [70] rlang_0.4.12 reshape2_1.4.4 later_1.3.0
## [73] AnnotationDbi_1.54.1 munsell_0.5.0 tools_4.1.2
## [76] cachem_1.0.6 downloader_0.4 generics_0.1.1
## [79] RSQLite_2.2.9 evaluate_0.14 stringr_1.4.0
## [82] fastmap_1.1.0 yaml_2.2.1 ggtree_3.0.4
## [85] knitr_1.37 bit64_4.0.5 tidygraph_1.2.0
## [88] caTools_1.18.2 purrr_0.3.4 KEGGREST_1.32.0
## [91] ggraph_2.0.5 nlme_3.1-155 mime_0.12
## [94] aplot_0.1.2 xml2_1.3.3 DO.db_2.9
## [97] rstudioapi_0.13 compiler_4.1.2 beeswarm_0.4.0
## [100] png_0.1-7 treeio_1.16.2 tibble_3.1.6
## [103] tweenr_1.0.2 geneplotter_1.70.0 bslib_0.3.1
## [106] stringi_1.7.6 highr_0.9 lattice_0.20-45
## [109] Matrix_1.4-0 vctrs_0.3.8 pillar_1.6.4
## [112] lifecycle_1.0.1 jquerylib_0.1.4 data.table_1.14.2
## [115] cowplot_1.1.1 bitops_1.0-7 httpuv_1.6.5
## [118] patchwork_1.1.1 qvalue_2.24.0 R6_2.5.1
## [121] promises_1.2.0.1 KernSmooth_2.23-20 echarts4r_0.4.3
## [124] gridExtra_2.3 gtools_3.9.2 MASS_7.3-55
## [127] assertthat_0.2.1 GenomeInfoDbData_1.2.6 grid_4.1.2
## [130] ggfun_0.0.4 tidyr_1.1.4 rmarkdown_2.11
## [133] ggforce_0.3.3 shiny_1.7.1