Differential Used Intronic
Briana Mittleman
2/23/2020
Last updated: 2020-05-15
Checks: 7 0
Knit directory: Comparative_APA/analysis/
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library(workflowr)This is workflowr version 1.6.0
Run ?workflowr for help getting startedlibrary(ggpubr)Loading required package: ggplot2Loading required package: magrittrlibrary(limma)
library(qvalue)
library(tidyverse)── Attaching packages ───────────────────────────────────────────────── tidyverse 1.2.1 ──✔ tibble 2.1.1 ✔ purrr 0.3.2
✔ tidyr 0.8.3 ✔ dplyr 0.8.0.1
✔ readr 1.3.1 ✔ stringr 1.3.1
✔ tibble 2.1.1 ✔ forcats 0.3.0 ── Conflicts ──────────────────────────────────────────────────── tidyverse_conflicts() ──
✖ tidyr::extract() masks magrittr::extract()
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Attaching package: 'cowplot'The following object is masked from 'package:ggpubr':
get_legendThe following object is masked from 'package:ggplot2':
ggsaveFor this analysis I will look at the differentially used PAS in introns and ask if I can used information from DE and dribosome to better understand these. I subset intornic because I believe the intronic and utr mechanisms are different.
Meta=read.table("../data/PAS_doubleFilter/PAS_5perc_either_HumanCoord_BothUsage_meta_doubleFilter.txt", header = T, stringsAsFactors = F) %>% dplyr::select(PAS, chr, start,end, loc)
DiffIso= read.table("../data/DiffIso_Nuclear_DF/AllPAS_withGeneSig.txt", header = T,stringsAsFactors = F) %>% inner_join(Meta, by=c("chr", 'start','end')) %>% filter(loc %in% c("intron","utr3"))
DiffIsoSig= DiffIso %>% filter(SigPAU2=="Yes")I can compare the effect sizes with these genes in the DE.
Compare with expression
nameID=read.table("../../genome_anotation_data/ensemble_to_genename.txt",sep="\t", header = T, stringsAsFactors = F) %>% dplyr::select(Gene_stable_ID, Gene.name)
DE=read.table("../data/DiffExpression/DEtested_allres.txt",stringsAsFactors = F,header = F, col.names = c("Gene_stable_ID" ,"logFC" ,"AveExpr" , "t" , "P.Value" , "adj.P.Val", "B" )) %>% inner_join(nameID,by="Gene_stable_ID") %>% dplyr::rename('gene'=Gene.name) %>% dplyr::select(-Gene_stable_ID)First do all of the genes:
DeandAPA= DiffIso %>% inner_join(DE, by="gene")This pas I will include each PAS
ggplot(DeandAPA,aes(y=deltaPAU, x=logFC,col=loc)) + geom_point(alpha=.3) + geom_smooth(aes(col=loc), method="lm") + labs(title="Intronic and 3' UTR APA v DE") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(DeandAPA,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="Intronic and 3' UTR APA v DE") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 3)
Just the genes with significant differences in PAS
DeandAPA_sigAPA= DeandAPA %>% filter(SigPAU2=="Yes")ggplot(DeandAPA_sigAPA,aes(y=deltaPAU, x=logFC, col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant Intronic and 3' UTR APA v DE")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(DeandAPA_sigAPA,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant Intronic and 3' UTR APA v DE")+ scale_color_brewer(palette = "Set1")+ stat_cor(label.x = 3)
Sig both:
DeandAPA_sigAPAandE= DeandAPA %>% filter(SigPAU2=="Yes", adj.P.Val<.05)ggplot(DeandAPA_sigAPAandE,aes(y=deltaPAU, x=logFC,col=loc)) + geom_point() + geom_smooth(method="lm")+ labs(title="Significant Intronic and 3' UTR APA v Significant DE") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(DeandAPA_sigAPAandE,aes(y=deltaPAU, x=logFC)) + geom_point() + geom_smooth(method="lm")+ labs(title="Significant Intronic and 3' UTR APA v Significant DE") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 3)
Choose most Sig PAS
To break ties I will use the top average usage. I will not worry about location when chosing top PAS.
DeandAPA_topPAS= DeandAPA %>% mutate(AvgUsageBoth=(Human+Chimp)/2) %>% group_by(gene) %>% arrange(p.adjust,desc(AvgUsageBoth)) %>% slice(1) %>% ungroup()
#intron
nrow(DeandAPA_topPAS %>% filter(loc=="intron"))[1] 1313nrow(DeandAPA_topPAS %>% filter(loc=="intron", SigPAU2=="Yes"))[1] 161#3 utr
nrow(DeandAPA_topPAS %>% filter(loc=="utr3"))[1] 6075nrow(DeandAPA_topPAS %>% filter(loc=="utr3", SigPAU2=="Yes"))[1] 826Plot the correlation in effect size
ggplot(DeandAPA_topPAS,aes(y=deltaPAU, x=logFC, col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="Intronic and 3' UTR APA top PAS v DE") + scale_color_brewer(palette = "Set1") + stat_cor(aes(color = loc), label.x = 3)
ggplot(DeandAPA_topPAS,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.3) + geom_smooth(method="lm") + stat_cor(label.x = 3)
DeandAPA_topPASsigAPA= DeandAPA_topPAS %>% filter(SigPAU2=="Yes")ggplot(DeandAPA_topPASsigAPA,aes(y=deltaPAU, x=logFC,col=loc)) + geom_point(alpha=.4) + geom_smooth(method="lm") + labs(title="Significant APA, Top PAS v DE ") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(DeandAPA_topPASsigAPA,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.4) + geom_smooth(method="lm") + labs(title="Significant APA, Top PAS v DE ") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 3)
Sig both:
DeandAPA_topPASsigAPAandE= DeandAPA_topPASsigAPA %>% filter(SigPAU2=="Yes", adj.P.Val<.05)ggplot(DeandAPA_topPASsigAPAandE,aes(y=deltaPAU, x=logFC,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, Top PAS v Significant DE") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(DeandAPA_topPASsigAPAandE,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, Top PAS v Significant DE") + scale_color_brewer(palette = "Set1")+ stat_cor(label.x = 3)
Plot together:
allboth=ggplot(DeandAPA,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.3) + geom_smooth( method="lm") + labs(title="All APA and all expression") + scale_color_brewer(palette = "Set1")+ stat_cor(label.x = c(-6), label.y=-1) +theme_classic(base_size = 12)
allSep= ggplot(DeandAPA_topPAS,aes(y=deltaPAU, x=logFC, col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="All APA and all expression") + scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR")) + stat_cor(aes(color = loc), label.x = c(-8,1),label.y=-1)+theme_classic(base_size = 12)
sigAPAboth=ggplot(DeandAPA_sigAPA,aes(y=deltaPAU, x=logFC)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA and all Expression")+ scale_color_brewer(palette = "Set1")+ stat_cor(label.x = -8,label.y=-1)+theme_classic(base_size = 12)
sigAPSep=ggplot(DeandAPA_topPASsigAPA,aes(y=deltaPAU, x=logFC,col=loc)) + geom_point(alpha=.4) + geom_smooth(method="lm") + labs(title="Significant APA and all Expression") + scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR"))+ stat_cor(aes(color = loc), label.x = c(-8,1),label.y=-1)+theme_classic(base_size = 12)
SigBoth= ggplot(DeandAPA_sigAPAandE,aes(y=deltaPAU, x=logFC)) + geom_point() + geom_smooth(method="lm")+ labs(title="Significant APA and Significant DE") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = -8,label.y=-1)+theme_classic(base_size = 12)
SigSep=ggplot(DeandAPA_topPASsigAPAandE,aes(y=deltaPAU, x=logFC,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA and Significant DE") + scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR"))+ stat_cor(aes(color = loc), label.x = c(-8,1),label.y=-1)+theme_classic(base_size = 12) plot_grid(allboth,allSep,sigAPAboth,sigAPSep,SigBoth,SigSep, ncol=2)
Ribosome occupancy
Flip direction to match the expression results
Ribo=read.table("../data/Wang_ribo/Additionaltable5_translationComparisons.txt",header = T, stringsAsFactors = F) %>% rename("Gene_stable_ID"= ENSG) %>% inner_join(nameID,by="Gene_stable_ID") %>% dplyr::select(Gene.name, HvC.beta, HvC.pvalue, HvC.FDR) %>% rename("gene"=Gene.name) %>% mutate(HvC.beta2=-1*HvC.beta)Join with APA
RiboandAPA=DiffIso %>% inner_join(Ribo, by="gene")
RiboandAPA %>% group_by(gene) %>% n_distinct()[1] 23590ggplot(RiboandAPA,aes(y=deltaPAU, x=HvC.beta2, col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="APA v Ribosome Occupany", x="-HvC.beta")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(RiboandAPA,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="APA v Ribosome Occupany",x="-HvC.beta")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 3)
Just the genes with significant differences in PAS
RiboandAPA_sigAPA= RiboandAPA %>% filter(SigPAU2=="Yes")ggplot(RiboandAPA_sigAPA,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Ribosome Occupany")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(RiboandAPA_sigAPA,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Ribosome Occupany")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 3)
Sig both:
RiboandAPA_sigAPAandR= RiboandAPA_sigAPA %>% filter(SigPAU2=="Yes", HvC.FDR<.05)ggplot(RiboandAPA_sigAPAandR,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Significant Ribosome Occupany") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(RiboandAPA_sigAPAandR,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Significant Ribosome Occupany") + scale_color_brewer(palette = "Set1")+ stat_cor(label.x = 3)
The correlation in expression with intronic is not there in ribosome occupancy.
Choose most Sig PAS
To break ties I will use the top average usage. I will not worry about location at first.
RiboandAPA_topPAS= RiboandAPA %>% mutate(AvgUsageBoth=(Human+Chimp)/2) %>% group_by(gene) %>% arrange(p.adjust,desc(AvgUsageBoth)) %>% slice(1) %>% ungroup()
nrow(RiboandAPA %>% filter(loc=="intron"))[1] 9994nrow(RiboandAPA_topPAS %>% filter(loc=="intron"))[1] 1138nrow(RiboandAPA %>% filter(loc=="utr3"))[1] 13596nrow(RiboandAPA_topPAS %>% filter(loc=="utr3"))[1] 5269Plot the correlation in effect size
ggplot(RiboandAPA_topPAS,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="APA, top PAS v Ribosome Occupany")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(RiboandAPA_topPAS,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="APA, top PAS v Ribosome Occupany")+ scale_color_brewer(palette = "Set1")+ stat_cor(label.x = 3)
Sig APA
RiboandAPA_topPASsigAPA= RiboandAPA_topPAS %>% filter(SigPAU2=="Yes")
nrow(RiboandAPA_topPASsigAPA %>% filter(loc=="intron"))[1] 136nrow(RiboandAPA_topPASsigAPA %>% filter(loc=="utr3"))[1] 675199 intronic significant, 910 significant 3’ utr
ggplot(RiboandAPA_topPASsigAPA,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, top PAS v Ribosome Occupany") +scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(RiboandAPA_topPASsigAPA,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, top PAS v Ribosome Occupany") +scale_color_brewer(palette = "Set1")+ stat_cor(label.x = 3)
Sig both:
RiboandAPA_topPASsigAPAandR= RiboandAPA_topPASsigAPA %>% filter(SigPAU2=="Yes", HvC.FDR<.05)
nrow(RiboandAPA_topPASsigAPAandR %>% filter(loc=="intron"))[1] 37nrow(RiboandAPA_topPASsigAPAandR %>% filter(loc=="utr3"))[1] 17747 PAS for intrnic 229 for 3’ UTR
ggplot(RiboandAPA_topPASsigAPAandR,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, top PAS v Significant Ribosome Occupany") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 3)
ggplot(RiboandAPA_topPASsigAPAandR,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, top PAS v Significant Ribosome Occupany") + scale_color_brewer(palette = "Set1")+ stat_cor(label.x = 3)
Correlation in UTR but not intronic. Not sure if this is due to the number of PAS.
Plot all together
riboBoth=ggplot(RiboandAPA,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="All APA and translation values",x="-HvC.beta")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = c( -8, 5), label.y = -1)+theme_classic(base_size = 12)
riboTop=ggplot(RiboandAPA_topPAS,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="All APA and translation values",x="-HvC.beta")+ scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR"))+ stat_cor(aes(color = loc), label.x =c( -8, 1),label.y = -1)+theme_classic(base_size = 12)
ribosigapaboth=ggplot(RiboandAPA_sigAPA,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA and all Translation",x="-HvC.beta")+ scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR"))+ stat_cor( label.x = -8,label.y = -1)+theme_classic(base_size = 12)
ribosigapasep=ggplot(RiboandAPA_topPASsigAPA,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA and Significant Translation",x="-HvC.beta") +scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR"))+ stat_cor(aes(color = loc), label.x =c( -8, 1),label.y = -1)+theme_classic(base_size = 12)
sigapasigriboboth=ggplot(RiboandAPA_sigAPAandR,aes(y=deltaPAU, x=HvC.beta2)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA and all Translation",x="-HvC.beta") + scale_color_brewer(palette = "Set1", name="")+ stat_cor(label.x = -8,label.y = -1)+theme_classic(base_size = 12)
sigapasigribosep=ggplot(RiboandAPA_topPASsigAPAandR,aes(y=deltaPAU, x=HvC.beta2,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA and Significant Translation",x="-HvC.beta") + scale_color_brewer(palette = "Set1",name="", labels=c("Intronic", "3' UTR"))+ stat_cor(aes(color = loc), label.x = c( -8, 1),label.y = -1)+theme_classic(base_size = 12) plot_grid(riboBoth,riboTop,ribosigapaboth, ribosigapasep,sigapasigriboboth,sigapasigribosep, ncol=2)
plot_grid(riboTop, ribosigapasep,sigapasigribosep)
Plot top ribo and expression next to eachother:
allboth,allSep,sigAPAboth,sigAPSep,SigBoth,SigSep
plot_grid(allSep,riboTop,sigAPSep, ribosigapasep,SigSep,sigapasigribosep, ncol=2)
Protein
I will use code from https://github.com/siddisis/project_primate_ribo to fit the linear model again and get effect sizes.
load(“../tables/fileS4.RData”)
load(“../rdas/HCR.protein.TMM.RData”)
Put both of these in ../data/Khan_prot
load("../data/Khan_prot/fileS4.RData")
load("../data/Khan_prot/HCR.protein.TMM.RData")expressed.gene.names <- as.character(HCR.protein.TMM.norm.ESNGlabeled[rownames(HCR.protein.TMM.norm.ESNGlabeled) %in% rownames(protein.expressed.data),16])
names(expressed.gene.names) <- rownames(protein.expressed.data)Use to make design matrix
# HvC
RNA.expressed.data.HC<-RNA.expressed.data[,1:10]
species.label <- substring(colnames(RNA.expressed.data.HC),1,1)
design <- model.matrix(~species.label)
colnames(design)<-c("Chimp","Human")Protien
protein.expressed.data.HC<-protein.expressed.data[,1:10]
protein.fit<-lmFit(protein.expressed.data.HC ,design = design)
HvC.prot<- eBayes(protein.fit)
top.table <- topTable(HvC.prot, n = Inf)
volcanoplot(HvC.prot,coef=2,highlight=2)
effectsizeDF= as.data.frame(cbind(Gene_stable_ID=rownames(protein.expressed.data.HC),logEf=HvC.prot$coefficients[,2], pval=top.table$adj.P.Val)) %>% inner_join(nameID,by="Gene_stable_ID") %>% dplyr::rename('gene'=Gene.name) %>% dplyr::select(-Gene_stable_ID)Warning: Column `Gene_stable_ID` joining factor and character vector,
coercing into character vectorwrite.table(effectsizeDF, "../data/Khan_prot/ProtData_effectSize.txt",col.names = T, row.names = F, quote = F)DPandAPA= DiffIso %>% inner_join(effectsizeDF, by="gene")
DPandAPA %>% group_by(gene) %>% summarise(n()) %>% nrow()[1] 2611DPandAPA$logEf= as.numeric(as.character(DPandAPA$logEf))
DPandAPA$pval= as.numeric(as.character(DPandAPA$pval))Looking at 2557 common genes.
ggplot(DPandAPA,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(aes(col=loc), method="lm") + labs(title="Intronic and 3' UTR APA v DP") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)
ggplot(DPandAPA,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="Intronic and 3' UTR APA v DP") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)
Just the genes with significant differences in PAS
PandAPA_sigAPA= DPandAPA %>% filter(SigPAU2=="Yes")ggplot(PandAPA_sigAPA,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)
ggplot(PandAPA_sigAPA,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)
Sig both:
PandAPA_sigAPAandP= PandAPA_sigAPA %>% filter(SigPAU2=="Yes", pval <.05)ggplot(PandAPA_sigAPAandP,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Signficant Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)
ggplot(PandAPA_sigAPAandP,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Signficant Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)
Choose most Sig PAS
To break ties I will use the top average usage. I will not worry about location at first.
PandAPA_topPAS= DPandAPA %>% mutate(AvgUsageBoth=(Human+Chimp)/2) %>% group_by(gene) %>% arrange(p.adjust,desc(AvgUsageBoth)) %>% slice(1) %>% ungroup()ggplot(PandAPA_topPAS,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(aes(col=loc), method="lm") + labs(title="Intronic and 3' UTR APA, top PAS v DP") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)
ggplot(PandAPA_topPAS,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="Intronic and 3' UTR APA v DP") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)
PandAPA_topPAS_sigAPA= PandAPA_topPAS %>% filter(SigPAU2=="Yes")ggplot(PandAPA_topPAS_sigAPA,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, top PAS v Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)
ggplot(PandAPA_topPAS_sigAPA,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA top PAS v Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)
Sig both:
PandAPA_topPAS_sigAPAandP= PandAPA_topPAS_sigAPA %>% filter(SigPAU2=="Yes", pval <.05)ggplot(PandAPA_topPAS_sigAPAandP,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA top PAS v Signficant Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)
ggplot(PandAPA_topPAS_sigAPAandP,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA top PAS v Signficant Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)
Check pvalues:
protKhan=read.csv("../data/Khan_prot/Khan_TableS4.csv",header = T) %>% rename("Gene_stable_ID"= ENSG) %>% inner_join(nameID,by="Gene_stable_ID") %>% rename("gene"=Gene.name) Warning: Column `Gene_stable_ID` joining factor and character vector,
coercing into character vectorprotKhanwmine= protKhan %>% inner_join(effectsizeDF, by="gene")
protKhanwmine$logEf=as.numeric(as.character(protKhanwmine$logEf))
protKhanwmine$pval=as.numeric(as.character(protKhanwmine$pval))
cor.test(protKhanwmine$pval,protKhanwmine$HC.pvalues.protein )
Pearson's product-moment correlation
data: protKhanwmine$pval and protKhanwmine$HC.pvalues.protein
t = -0.71198, df = 3248, p-value = 0.4765
alternative hypothesis: true correlation is not equal to 0
95 percent confidence interval:
-0.04685410 0.02189991
sample estimates:
cor
-0.01249186 This is not good. Try the difference in means approach:
Chimp-human
protKhanSmall= protKhan %>% select(gene,mean.H.protein,mean.C.protein, HC.qvalues.rna) %>% mutate(Effect=mean.C.protein-mean.H.protein)deltaPandAPA= DiffIso %>% inner_join(protKhanSmall, by="gene")
deltaPandAPA %>% group_by(gene) %>% summarise(n()) %>% nrow()[1] 26662607 genes
ggplot(deltaPandAPA,aes(y=deltaPAU, x=Effect,col=loc)) + geom_point(alpha=.3) + geom_smooth(aes(col=loc), method="lm") + labs(title="Intronic and 3' UTR APA v DP") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 2)
ggplot(deltaPandAPA,aes(y=deltaPAU, x=Effect)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="Intronic and 3' UTR APA v DP") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 2)
Correlation effect sizes:
protKhanSmall_withmone= protKhanSmall %>% inner_join(effectsizeDF, by="gene")
protKhanSmall_withmone$logEf=as.numeric(as.character(protKhanSmall_withmone$logEf))
cor.test(protKhanSmall_withmone$logEf, protKhanSmall_withmone$Effect)
Pearson's product-moment correlation
data: protKhanSmall_withmone$logEf and protKhanSmall_withmone$Effect
t = -11459, df = 3248, p-value < 2.2e-16
alternative hypothesis: true correlation is not equal to 0
95 percent confidence interval:
-0.9999885 -0.9999868
sample estimates:
cor
-0.9999876 Ok this is equal but opposite. So this is correct.
I need to check the direction of the effects.
Plot together:
protboth=ggplot(DPandAPA,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm") + labs(title="APA v Protein") + scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 2)+theme_classic(base_size = 12)
protsep=ggplot(PandAPA_topPAS,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(aes(col=loc), method="lm") + labs(title="APA, top PAS v Protein") + scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)+theme_classic(base_size = 12)
protsigapa=ggplot(PandAPA_sigAPA,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)+theme_classic(base_size = 12)
protsigapasep=ggplot(PandAPA_topPAS_sigAPA,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA, top PAS v Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)+theme_classic(base_size = 12)
protsigall=ggplot(PandAPA_sigAPAandP,aes(y=deltaPAU, x=logEf)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA v Signficant Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor( label.x = 1)+theme_classic(base_size = 12)
protsigallsep=ggplot(PandAPA_topPAS_sigAPAandP,aes(y=deltaPAU, x=logEf,col=loc)) + geom_point(alpha=.3) + geom_smooth(method="lm")+ labs(title="Significant APA top PAS v Signficant Protein")+ scale_color_brewer(palette = "Set1")+ stat_cor(aes(color = loc), label.x = 1)+theme_classic(base_size = 12) plot_grid(protboth,protsep,protsigapa,protsigapasep,protsigall,protsigallsep, ncol=2)
sessionInfo()R version 3.5.1 (2018-07-02)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)
Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so
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] stats graphics grDevices utils datasets methods base
other attached packages:
[1] cowplot_0.9.4 forcats_0.3.0 stringr_1.3.1 dplyr_0.8.0.1
[5] purrr_0.3.2 readr_1.3.1 tidyr_0.8.3 tibble_2.1.1
[9] tidyverse_1.2.1 qvalue_2.14.0 limma_3.38.2 ggpubr_0.2
[13] magrittr_1.5 ggplot2_3.1.1 workflowr_1.6.0
loaded via a namespace (and not attached):
[1] tidyselect_0.2.5 reshape2_1.4.3 splines_3.5.1
[4] haven_1.1.2 lattice_0.20-38 colorspace_1.3-2
[7] generics_0.0.2 htmltools_0.3.6 yaml_2.2.0
[10] rlang_0.4.0 later_0.7.5 pillar_1.3.1
[13] glue_1.3.0 withr_2.1.2 RColorBrewer_1.1-2
[16] modelr_0.1.2 readxl_1.1.0 plyr_1.8.4
[19] munsell_0.5.0 gtable_0.2.0 cellranger_1.1.0
[22] rvest_0.3.2 evaluate_0.12 labeling_0.3
[25] knitr_1.20 httpuv_1.4.5 broom_0.5.1
[28] Rcpp_1.0.4.6 promises_1.0.1 scales_1.0.0
[31] backports_1.1.2 jsonlite_1.6 fs_1.3.1
[34] hms_0.4.2 digest_0.6.18 stringi_1.2.4
[37] grid_3.5.1 rprojroot_1.3-2 cli_1.1.0
[40] tools_3.5.1 lazyeval_0.2.1 crayon_1.3.4
[43] whisker_0.3-2 pkgconfig_2.0.2 xml2_1.2.0
[46] lubridate_1.7.4 rstudioapi_0.10 assertthat_0.2.0
[49] rmarkdown_1.10 httr_1.3.1 R6_2.3.0
[52] nlme_3.1-137 git2r_0.26.1 compiler_3.5.1