Gene set testing for Illumina HumanMethylation Arrays
Jovana Maksimovic, Alicia Oshlack and Belinda Phipson
April 24, 2020
Last updated: 2020-04-24
Checks: 7 0
Knit directory: methyl-geneset-testing/
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| File | Version | Author | Date | Message |
|---|---|---|---|---|
| Rmd | 9373b7c | Jovana Maksimovic | 2020-04-24 | wflow_publish(“analysis/exploreData.Rmd”) |
| Rmd | 3ae5062 | Jovana Maksimovic | 2020-04-21 | Updated code to reverse bubble plot y-axis |
| html | e82bc51 | Jovana Maksimovic | 2020-04-03 | Build site. |
| Rmd | f7696d6 | Jovana Maksimovic | 2020-04-03 | wflow_publish(“analysis/exploreData.Rmd”) |
| html | 76dacfc | Jovana Maksimovic | 2020-03-31 | Build site. |
| Rmd | 43ca6b6 | Jovana Maksimovic | 2020-03-31 | wflow_publish(“analysis/exploreData.Rmd”) |
| Rmd | 2bcb4c9 | JovMaksimovic | 2020-03-23 | New script and modifications for methylGSA analysis. |
| Rmd | 250d8ae | JovMaksimovic | 2020-03-16 | Comitting local changes. |
| Rmd | 24fb166 | Jovana Maksimovic | 2020-03-16 | Adding Rmd file changes. |
| Rmd | d7cd66e | Jovana Maksimovic | 2020-03-02 | Initial Commit |
library(here)
library(minfi)
library(paletteer)
library(limma)
library(BiocParallel)
library(reshape2)
library(gridExtra)
library(missMethyl)
library(ggplot2)
library(glue)
library(tidyverse)
library(rbin)
library(patchwork)
library(ChAMPdata)
source(here("code/utility.R"))Load data
We are using publicly available EPIC data GSE110554 generated from flow sorted blood cells. The data is normalised and filtered (bad probes, multi-mapping probes, SNP probes, sex chromosomes).
# load data
dataFile <- here("data/GSE110554-data.RData")
if(file.exists(dataFile)){
# load processed data and sample information
load(dataFile)
} else {
# get data from experiment hub, normalise, filter and save objects
readData(dataFile)
# load processed data and sample information
load(dataFile)
}QC plots
# plot mean detection p-values across all samples
dat <- tibble::tibble(mean = colMeans(detP), cellType = targets$CellType)
ggplot(dat, aes(y = mean, x = cellType, fill = cellType)) +
geom_bar(stat = "identity") +
labs(fill = "Cell Type")
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
# plot normalised beta value distribution
bVals <- getBeta(normGr)
dat <- data.frame(reshape2::melt(bVals))
colnames(dat) <- c("cpg", "sample", "bVal")
dat <- dplyr::bind_cols(dat, cellType = rep(targets$CellType,
each = nrow(bVals)))
ggplot(dat, aes(x = bVal, colour = cellType)) +
geom_density() +
labs(colour = "Cell Type")
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
# MDS plots to look at largest sources of variation
p <- plotMDS(getM(fltGr), top=1000, gene.selection="common", plot = FALSE)
dat <- tibble::tibble(x = p$x, y = p$y, cellType = targets$CellType)
ggplot(dat, aes(x = x, y = y, colour = cellType)) +
geom_point() +
labs(colour = "Cell Type")
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Statistical analysis
Compare several sets of sorted immune cells. Consider results significant at FDR < 0.05 and delta beta ~ 10% (~ lfc = 0.5).
mVals <- getM(fltGr)
bVals <- getBeta(fltGr)design <- model.matrix(~0+targets$CellType)
colnames(design) <- levels(factor(targets$CellType))
fit <- lmFit(mVals, design)
cont.matrix <- makeContrasts(CD4vCD8=CD4T-CD8T,
#BcellvMono=Bcell-Mono,
MonovNeu=Mono-Neu,
BcellvNK=Bcell-NK,
#NeuvNK=Neu-NK,
#MonovNK=Mono-NK,
levels=design)
fit2 <- contrasts.fit(fit, cont.matrix)
tfit <- eBayes(fit2, robust=TRUE, trend=TRUE)
tfit <- treat(tfit, lfc = 0.5)
pval <- 0.05
fitSum <- summary(decideTests(tfit, p.value = pval))
fitSum CD4vCD8 MonovNeu BcellvNK
Down 5072 9324 34803
NotSig 725611 712480 667559
Up 3202 12081 31523Examine only the independent contrasts.
dat <- melt(fitSum[rownames(fitSum) != "NotSig", ])
colnames(dat) <- c("dir","comp","num")
ggplot(dat, aes(x = comp, y = num, fill = dir)) +
geom_bar(stat = "identity", position = "dodge") +
labs(x = "Comparison", y = "No. DM CpGs (FDR < 0.05)", fill = "Direction")
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Compare performance of different gene set testing methods
Compare the gene set testing methods available for methylation arrays; hypergeometric test (HGT), gometh (best), methylRRA (GLM), methylRRA (ORA) and methylRRA (GSEA). As methylRRA does not work well with sets that only contain very few genes or very large sets, we will only test sets with at least 5 genes and maximum of 5000 genes.
Save analysis results
minsize <- 5
maxsize <- 5000
outFile <- here("data/blood.contrasts.rds")
if(!file.exists(outFile)){
obj <- NULL
obj$tfit <- tfit
obj$maxsize <- maxsize
obj$minsize <- minsize
obj$mVals <- mVals
obj$targets <- targets
saveRDS(obj, file = outFile)
} Load output for all methods
inFiles <- list.files(here("output/compare-methods"), pattern = "rds",
full.names = TRUE)
res <- lapply(inFiles, function(file){
readRDS(file)
})
dat <- as_tibble(dplyr::bind_rows(res))sub <- dat %>% filter(method %in% c("mmethyl.hgt", "mmethyl.gometh"))
ggplot(sub, aes(x = pvalue, colour = sub)) +
geom_density() +
facet_grid(cols = vars(contrast), rows = vars(method)) +
labs(colour = "Sig. CpG Selection", x = "P-value", y = "Density") +
scale_color_hue(labels = c("Top 5000", "Top 10000", "FDR < 0.01",
"FDR < 0.05")) +
theme(legend.position = "bottom", legend.text = element_text(size=6))
Test GO categories
ann <- loadAnnotation("EPIC")
flatAnn <- missMethyl:::.getFlatAnnotation("EPIC", anno = ann)
cpgEgGo <- cpgsEgGoFreqs(flatAnn)cpgEgGo %>%
group_by(GO) %>%
summarise(med = median(Freq)) -> medCpgEgGo
dat %>% filter(set == "GO") %>%
filter(sub %in% c("n","c1")) %>%
inner_join(medCpgEgGo, by = c("ID" = "GO")) -> sub
bins <- rbin_quantiles(sub, ID, med, bins = 12)
sub$bin <- as.factor(findInterval(sub$med, bins$upper_cut))
binLabs <- paste0("<", bins$upper_cut)
names(binLabs) <- levels(sub$bin)
binLabs[length(binLabs)] <- gsub("<", "\u2265", binLabs[length(binLabs) - 1])
sub %>% group_by(contrast, method, bin) %>%
summarise(prop = sum(pvalue < 0.05)/n()) -> pdat
p <- ggplot(pdat, aes(x = as.numeric(bin), y = prop, color = method)) +
geom_line() +
facet_wrap(vars(contrast), ncol = 3) +
scale_x_continuous(breaks = as.numeric(levels(pdat$bin)), labels = binLabs) +
theme(axis.text.x = element_text(angle=45, hjust = 1, vjust = 1,
size = 7),
legend.position = "bottom") +
labs(x = "Med. No. CpGs per Gene per GO Cat. (binned)",
y = "Prop. GO Cat. with p-value < 0.05")
p
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
As we are comparing immune cells, we expect GO categories related to the immune system and its processes to be highly ranked. To evalue this, we downloaded all of the child terms for the GO category “immune system process” (GO:002376) from AminGO 2; http://amigo.geneontology.org/amigo/term/GO:0002376.
immuneGO <- unique(read.csv(here("data/GO-immune-system-process.txt"),
stringsAsFactors = FALSE, header = FALSE,
col.names = "GOID"))
# immuneGO <- NULL
#
# for(i in 1:ncol(cont.matrix)){
# tmp <- read.csv(here(glue("code/{colnames(cont.matrix)[i]}.GO.csv")),
# stringsAsFactors = FALSE)
# immuneGO <- bind_rows(immuneGO, data.frame("GOID" = tmp$X[1:100],
# contrast = colnames(cont.matrix)[i]))
# }
dat %>% filter(set == "GO") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(csum = cumsum(ID %in% immuneGO$GOID)) %>% #[immuneGO$contrast == contrast])) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 100) -> sub
p <- ggplot(sub, aes(x = rank, y = csum, colour = method)) +
geom_line() +
facet_wrap(vars(contrast), ncol=3) +
geom_vline(xintercept = 10, linetype = "dotted") +
labs(colour = "Method", x = "Rank", y = "Cumulative no. immune sets") +
theme(legend.position = "bottom")
p
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Examine results when top ranked 100 GO terms from RNA-seq analysis of the same cell type comparisons is used as “truth”.
immuneGO <- NULL
for(i in 1:ncol(cont.matrix)){
tmp <- read.csv(here(glue("code/{colnames(cont.matrix)[i]}.GO.csv")),
stringsAsFactors = FALSE)
immuneGO <- bind_rows(immuneGO, data.frame("GOID" = tmp$X[1:100],
contrast = colnames(cont.matrix)[i]))
}Warning in bind_rows_(x, .id): Unequal factor levels: coercing to characterWarning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vectorWarning in bind_rows_(x, .id): Unequal factor levels: coercing to characterWarning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vectordat %>% filter(set == "GO") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(csum = cumsum(ID %in% immuneGO$GOID[immuneGO$contrast == contrast])) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 100) -> subWarning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object lengthWarning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object length
Warning in immuneGO$contrast == contrast: longer object length is not a multiple
of shorter object lengthp <- ggplot(sub, aes(x = rank, y = csum, colour = method)) +
geom_line() +
facet_wrap(vars(contrast), ncol=3) +
geom_vline(xintercept = 10, linetype = "dotted") +
labs(colour = "Method", x = "Rank", y = "Cumulative no. immune sets") +
theme(legend.position = "bottom")
p
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Examine what the top 10 ranked gene sets are and how many genes they contain, for each method and comparison.
terms <- missMethyl:::.getGO()$idTable
nGenes <- rownames_to_column(data.frame(n = sapply(missMethyl:::.getGO()$idList,
length)),
var = "ID")
dat %>% filter(set == "GO") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(FDR = p.adjust(pvalue)) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 10) %>%
inner_join(terms, by = c("ID" = "GOID")) %>%
inner_join(nGenes) -> subJoining, by = "ID"p <- vector("list", length(unique(sub$contrast)) * length(unique(sub$method)))
i = 1
for(cont in unique(sub$contrast)){
c = 1
for(meth in unique(sub$method)){
tmp <- sub %>% filter(contrast == cont & method == meth) %>%
mutate(rank = factor(rank),
rank = factor(rank, levels = rev(levels(rank))))
p[[i]] <- ggplot(tmp, aes(x = -log10(FDR), y = rank)) +
geom_point(aes(size = n), alpha = 0.5,
colour = scales::hue_pal()(length(unique(sub$method)))[c]) +
scale_y_discrete(labels = rev(tmp$TERM)) +
labs(y = "", size = "No. genes", title = meth) +
theme(axis.text.y = element_text(size = 6),
plot.title = element_text(size = 8),
legend.position = "right",
legend.key.size = unit(0.25, "cm"),
legend.text = element_text(size = 6),
legend.title = element_text(size = 8),
axis.text.x = element_text(size = 6),
axis.title.x = element_text(size = 8)) +
coord_cartesian(xlim = c(-log10(0.99), -log10(10^-100))) +
geom_vline(xintercept = -log10(0.05), linetype = "dashed")
i = i + 1
c = c + 1
}
}
(p[[1]] / p[[2]] / p[[3]] / p[[4]] / p[[5]]) +
plot_annotation(title = unique(sub$contrast)[1],
theme = theme(plot.title = element_text(size = 10))) 
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
(p[[6]] / p[[7]] / p[[8]] / p[[9]] / p[[10]]) +
plot_annotation(title = unique(sub$contrast)[2],
theme = theme(plot.title = element_text(size = 10))) 
(p[[11]] / p[[12]] / p[[13]] / p[[14]] / p[[15]]) +
plot_annotation(title = unique(sub$contrast)[3],
theme = theme(plot.title = element_text(size = 10))) 
P-value histograms for the different methods for all contrasts on GO categories.
dat %>% filter(set == "GO") %>%
filter(sub %in% c("n","c1")) -> sub
ggplot(sub, aes(pvalue, fill = method)) +
geom_histogram(binwidth = 0.025) +
facet_grid(cols = vars(contrast), rows = vars(method)) +
theme(legend.position = "bottom") +
labs(x = "P-value", y = "Frequency", fill = "Method")
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Test KEGG pathways
Now test KEGG pathways with at least 5 genes and 5000 at most.
Again, as we are comparing immune cells we expecte pathways from the following categories to be highly ranked: Immune system, Immune disease, Signal transduction, Signaling molecules and interaction; https://www.genome.jp/kegg/pathway.html.
immuneKEGG <- read.csv(here("data/kegg-immune-related-pathways.csv"),
stringsAsFactors = FALSE, header = FALSE,
col.names = c("ID","pathway"))
immuneKEGG$PID <- paste0("path:hsa0",immuneKEGG$ID)
dat %>% filter(set == "KEGG") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(csum = cumsum(ID %in% immuneKEGG$PID)) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 100) -> sub
p <- ggplot(sub, aes(x = rank, y = csum, colour = method)) +
geom_line() +
facet_wrap(vars(contrast), ncol=3) +
geom_vline(xintercept = 10, linetype = "dotted") +
labs(colour = "Method", x = "Rank", y = "Cumulative no. immune sets") +
theme(legend.position = "bottom")
p
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Examine results when top ranked 100 KEGG pathways from RNA-seq analysis of the same cell type comparisons is used as “truth”.
immuneKEGG <- NULL
for(i in 1:ncol(cont.matrix)){
tmp <- read.csv(here(glue("code/{colnames(cont.matrix)[i]}.KEGG.csv")),
stringsAsFactors = FALSE)
immuneKEGG <- bind_rows(immuneKEGG, data.frame("PID" = tmp$X[1:100],
contrast = colnames(cont.matrix)[i]))
}Warning in bind_rows_(x, .id): Unequal factor levels: coercing to characterWarning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vectorWarning in bind_rows_(x, .id): Unequal factor levels: coercing to characterWarning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vectordat %>% filter(set == "KEGG") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(csum = cumsum(ID %in% immuneKEGG$PID[immuneKEGG$contrast == contrast])) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 50) -> subWarning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object lengthWarning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneKEGG$contrast == contrast: longer object length is not a
multiple of shorter object lengthp <- ggplot(sub, aes(x = rank, y = csum, colour = method)) +
geom_line() +
facet_wrap(vars(contrast), ncol=3, scales = "free_y") +
geom_vline(xintercept = 10, linetype = "dotted") +
labs(colour = "Method", x = "Rank", y = "Cumulative no. immune sets") +
theme(legend.position = "bottom")
p
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Examine what the top 10 ranked gene sets are and how many genes they contain, for each method and comparison.
terms <- missMethyl:::.getKEGG()$idTable
nGenes <- rownames_to_column(data.frame(n = sapply(missMethyl:::.getKEGG()$idList,
length)),
var = "ID")
dat %>% filter(set == "KEGG") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(FDR = p.adjust(pvalue)) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 10) %>%
inner_join(terms, by = c("ID" = "PathwayID")) %>%
inner_join(nGenes) -> subJoining, by = "ID"p <- vector("list", length(unique(sub$contrast)) * length(unique(sub$method)))
i = 1
for(cont in unique(sub$contrast)){
c = 1
for(meth in unique(sub$method)){
tmp <- sub %>% filter(contrast == cont & method == meth) %>%
mutate(rank = factor(rank),
rank = factor(rank, levels = rev(levels(rank))))
p[[i]] <- ggplot(tmp, aes(x = -log10(FDR), y = rank)) +
geom_point(aes(size = n), alpha = 0.5,
colour = scales::hue_pal()(length(unique(sub$method)))[c]) +
scale_y_discrete(labels = rev(tmp$Description)) +
labs(y = "", size = "No. genes", title = meth) +
theme(axis.text.y = element_text(size = 6),
plot.title = element_text(size = 8),
legend.position = "right",
legend.key.size = unit(0.25, "cm"),
legend.text = element_text(size = 6),
legend.title = element_text(size = 8),
axis.text.x = element_text(size = 6),
axis.title.x = element_text(size = 8)) +
coord_cartesian(xlim = c(-log10(0.99), -log10(10^-15))) +
geom_vline(xintercept = -log10(0.05), linetype = "dashed")
i = i + 1
c = c + 1
}
}
(p[[1]] / p[[2]] / p[[3]] / p[[4]] / p[[5]]) +
plot_annotation(title = unique(sub$contrast)[1],
theme = theme(plot.title = element_text(size = 10))) 
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
(p[[6]] / p[[7]] / p[[8]] / p[[9]] / p[[10]]) +
plot_annotation(title = unique(sub$contrast)[2],
theme = theme(plot.title = element_text(size = 10))) 
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
(p[[11]] / p[[12]] / p[[13]] / p[[14]] / p[[15]]) +
plot_annotation(title = unique(sub$contrast)[3],
theme = theme(plot.title = element_text(size = 10))) 
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
P-value histograms for the different methods for all contrasts on KEGG pathways.
dat %>% filter(set == "KEGG") %>%
filter(sub %in% c("n","c1")) -> sub
ggplot(sub, aes(pvalue, fill = method)) +
geom_histogram(binwidth = 0.025) +
facet_grid(cols = vars(contrast), rows = vars(method)) +
theme(legend.position = "bottom") +
labs(x = "P-value", y = "Frequency", fill = "Method")
| Version | Author | Date |
|---|---|---|
| 76dacfc | Jovana Maksimovic | 2020-03-31 |
Test BROAD gene sets
As the ebGSEA method from the ChAMP package only allows testing of their in-built database of Broad Institute gene sets, we will compare to other methods by testing these same gene sets.
immuneBROAD <- NULL
for(i in 1:ncol(cont.matrix)){
tmp <- read.csv(here(glue("code/{colnames(cont.matrix)[i]}.BROAD.csv")),
stringsAsFactors = FALSE)
tmp <- tmp[order(tmp$PValue),]
immuneBROAD <- bind_rows(immuneBROAD, data.frame("ID" = tmp$X[1:100],
contrast = colnames(cont.matrix)[i]))
}Warning in bind_rows_(x, .id): Unequal factor levels: coercing to characterWarning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vectorWarning in bind_rows_(x, .id): Unequal factor levels: coercing to characterWarning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vector
Warning in bind_rows_(x, .id): binding character and factor vector, coercing
into character vectordat %>% filter(set == "BROAD") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(csum = cumsum(ID %in% immuneBROAD$ID[immuneBROAD$contrast == contrast])) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 100) -> subWarning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object lengthWarning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object length
Warning in immuneBROAD$contrast == contrast: longer object length is not a
multiple of shorter object lengthp <- ggplot(sub, aes(x = rank, y = csum, colour = method)) +
geom_line() +
facet_wrap(vars(contrast), ncol=3, scales = "free_y") +
geom_vline(xintercept = 10, linetype = "dotted") +
labs(colour = "Method", x = "Rank", y = "Cumulative no. immune sets") +
theme(legend.position = "bottom")
p
data(PathwayList)
nGenes <- rownames_to_column(data.frame(n = sapply(PathwayList,
length)),
var = "ID")
dat %>% filter(set == "BROAD") %>%
filter(sub %in% c("n","c1")) %>%
arrange(contrast, method, pvalue) %>%
group_by(contrast, method) %>%
mutate(FDR = p.adjust(pvalue)) %>%
mutate(rank = 1:n()) %>%
filter(rank <= 10) %>%
inner_join(nGenes) -> subJoining, by = "ID"p <- vector("list", length(unique(sub$contrast)) * length(unique(sub$method)))
i = 1
for(cont in unique(sub$contrast)){
c = 1
for(meth in unique(sub$method)){
tmp <- sub %>% filter(contrast == cont & method == meth) %>%
mutate(rank = factor(rank),
rank = factor(rank, levels = rev(levels(rank))))
p[[i]] <- ggplot(tmp, aes(x = -log10(FDR), y = rank)) +
geom_point(aes(size = n), alpha = 0.5,
colour = scales::hue_pal()(length(unique(sub$method)))[c]) +
scale_y_discrete(labels = rev(tmp$ID)) +
labs(y = "", size = "No. genes", title = meth) +
theme(axis.text.y = element_text(size = 6),
plot.title = element_text(size = 8),
legend.position = "right",
legend.key.size = unit(0.25, "cm"),
legend.text = element_text(size = 6),
legend.title = element_text(size = 8),
axis.text.x = element_text(size = 6),
axis.title.x = element_text(size = 8)) +
coord_cartesian(xlim = c(-log10(0.99), -log10(10^-100))) +
geom_vline(xintercept = -log10(0.05), linetype = "dashed")
i = i + 1
c = c + 1
}
}
(p[[1]] / p[[2]] / p[[3]] / p[[4]] / p[[5]] / p[[6]] / p[[7]]) +
plot_annotation(title = unique(sub$contrast)[1],
theme = theme(plot.title = element_text(size = 10))) 
(p[[8]] / p[[9]] / p[[10]] / p[[11]] / p[[12]] / p[[13]] / p[[14]]) +
plot_annotation(title = unique(sub$contrast)[2],
theme = theme(plot.title = element_text(size = 10))) 
(p[[15]] / p[[16]] / p[[17]] / p[[18]] / p[[19]] / p[[20]] / p[[21]]) +
plot_annotation(title = unique(sub$contrast)[3],
theme = theme(plot.title = element_text(size = 10))) 
P-value histograms for the different methods for all contrasts on BROAD gene sets.
dat %>% filter(set == "BROAD") %>%
filter(sub %in% c("n","c1")) -> sub
ggplot(sub, aes(pvalue, fill = method)) +
geom_histogram(binwidth = 0.025) +
facet_grid(cols = vars(contrast), rows = vars(method)) +
theme(legend.position = "bottom") +
labs(x = "P-value", y = "Frequency", fill = "Method")
sessionInfo()R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)
Matrix products: default
BLAS: /config/RStudio/R/3.6.1/lib64/R/lib/libRblas.so
LAPACK: /config/RStudio/R/3.6.1/lib64/R/lib/libRlapack.so
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] stats4 parallel stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] ChAMPdata_2.18.0 patchwork_1.0.0
[3] rbin_0.1.2 forcats_0.4.0
[5] stringr_1.4.0 dplyr_0.8.3
[7] purrr_0.3.4 readr_1.3.1
[9] tidyr_1.0.2 tibble_2.1.3
[11] tidyverse_1.3.0 glue_1.4.0
[13] ggplot2_3.3.0 missMethyl_1.20.4
[15] gridExtra_2.3 reshape2_1.4.3
[17] limma_3.42.2 paletteer_1.1.0
[19] minfi_1.32.0 bumphunter_1.26.0
[21] locfit_1.5-9.1 iterators_1.0.12
[23] foreach_1.5.0 Biostrings_2.54.0
[25] XVector_0.24.0 SummarizedExperiment_1.16.1
[27] DelayedArray_0.12.3 BiocParallel_1.20.1
[29] matrixStats_0.56.0 Biobase_2.46.0
[31] GenomicRanges_1.38.0 GenomeInfoDb_1.22.1
[33] IRanges_2.20.2 S4Vectors_0.24.4
[35] BiocGenerics_0.32.0 here_0.1
[37] workflowr_1.6.1
loaded via a namespace (and not attached):
[1] tidyselect_0.2.5
[2] RSQLite_2.1.2
[3] AnnotationDbi_1.46.1
[4] grid_3.6.1
[5] munsell_0.5.0
[6] codetools_0.2-16
[7] preprocessCore_1.48.0
[8] statmod_1.4.32
[9] withr_2.1.2
[10] colorspace_1.4-1
[11] knitr_1.28
[12] rstudioapi_0.11
[13] labeling_0.3
[14] git2r_0.26.1
[15] GenomeInfoDbData_1.2.1
[16] farver_2.0.3
[17] bit64_0.9-7
[18] rhdf5_2.30.1
[19] rprojroot_1.3-2
[20] vctrs_0.2.4
[21] generics_0.0.2
[22] xfun_0.13
[23] BiocFileCache_1.10.2
[24] R6_2.4.1
[25] illuminaio_0.28.0
[26] palr_0.2.0
[27] pals_1.6
[28] bitops_1.0-6
[29] reshape_0.8.8
[30] assertthat_0.2.1
[31] promises_1.1.0
[32] IlluminaHumanMethylation450kanno.ilmn12.hg19_0.6.0
[33] scales_1.1.0
[34] gtable_0.3.0
[35] methylumi_2.30.0
[36] rlang_0.4.5
[37] genefilter_1.68.0
[38] splines_3.6.1
[39] rtracklayer_1.44.4
[40] GEOquery_2.54.1
[41] dichromat_2.0-0
[42] broom_0.5.2
[43] scico_1.1.0
[44] yaml_2.2.1
[45] modelr_0.1.6
[46] GenomicFeatures_1.36.4
[47] backports_1.1.6
[48] httpuv_1.5.2
[49] tools_3.6.1
[50] nor1mix_1.3-0
[51] RColorBrewer_1.1-2
[52] siggenes_1.60.0
[53] Rcpp_1.0.4.6
[54] plyr_1.8.6
[55] progress_1.2.2
[56] zlibbioc_1.30.0
[57] RCurl_1.95-4.12
[58] BiasedUrn_1.07
[59] prettyunits_1.0.2
[60] openssl_1.4.1
[61] IlluminaHumanMethylationEPICmanifest_0.3.0
[62] haven_2.2.0
[63] cluster_2.1.0
[64] fs_1.4.1
[65] magrittr_1.5
[66] data.table_1.12.8
[67] reprex_0.3.0
[68] whisker_0.4
[69] IlluminaHumanMethylationEPICanno.ilm10b4.hg19_0.6.0
[70] hms_0.5.3
[71] evaluate_0.14
[72] xtable_1.8-4
[73] XML_3.98-1.20
[74] mclust_5.4.6
[75] readxl_1.3.1
[76] compiler_3.6.1
[77] biomaRt_2.42.1
[78] maps_3.3.0
[79] crayon_1.3.4
[80] htmltools_0.4.0
[81] later_1.0.0
[82] jcolors_0.0.4
[83] lubridate_1.7.4
[84] DBI_1.0.0
[85] dbplyr_1.4.2
[86] MASS_7.3-51.5
[87] rappdirs_0.3.1
[88] Matrix_1.2-18
[89] cli_2.0.2
[90] quadprog_1.5-8
[91] pkgconfig_2.0.3
[92] GenomicAlignments_1.20.1
[93] registry_0.5-1
[94] IlluminaHumanMethylation450kmanifest_0.4.0
[95] oompaBase_3.2.9
[96] xml2_1.3.1
[97] annotate_1.62.0
[98] rngtools_1.4
[99] pkgmaker_0.27
[100] multtest_2.40.0
[101] beanplot_1.2
[102] ruv_0.9.7.1
[103] rvest_0.3.5
[104] bibtex_0.4.2
[105] doRNG_1.7.1
[106] scrime_1.3.5
[107] digest_0.6.25
[108] rmarkdown_2.1
[109] base64_2.0
[110] cellranger_1.1.0
[111] DelayedMatrixStats_1.8.0
[112] curl_4.3
[113] Rsamtools_2.0.1
[114] lifecycle_0.2.0
[115] nlme_3.1-147
[116] jsonlite_1.6.1
[117] Rhdf5lib_1.6.1
[118] mapproj_1.2.6
[119] fansi_0.4.1
[120] viridisLite_0.3.0
[121] askpass_1.1
[122] pillar_1.4.3
[123] lattice_0.20-41
[124] httr_1.4.1
[125] survival_2.44-1.1
[126] GO.db_3.8.2
[127] bit_1.1-14
[128] stringi_1.4.6
[129] HDF5Array_1.14.4
[130] rematch2_2.1.0
[131] blob_1.2.0
[132] org.Hs.eg.db_3.8.2
[133] memoise_1.1.0