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CG_DMR_analysis.Rmd
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CG_DMR_analysis.Rmd
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---
title: "CG-DMR analyses"
author: "Sam Buckberry"
date: '2022-08-03'
output: github_document
---
**This part of the analysis is run on external HPC**
```{r, eval=FALSE}
source("R/server_libraries_and_functions.R")
```
Sync required files (too large for git)
```{bash, eval=FALSE}
rsync -av --progress --partial-dir=/tmp --files-from=wgbs/dmrs/dmr_bsseq_CG_obj_list.txt hg19ips_samb@dug:/d/home/hg19ips/hg19ips_samb/mcc_hg19ips/sam/hs-reprogram/wgbs/CG_bsobj/ wgbs/CG_bsobj/
```
```{bash, eval=FALSE}
rsync -av --progress --partial-dir=/tmp hg19ips_samb@dug:/d/home/hg19ips/hg19ips_samb/mcc_hg19ips/sam/hs-reprogram/wgbs/CG_bsobj/ wgbs/CG_bsobj/
```
Read in the sample data
```{r, eval=FALSE}
mdat <- read.csv("wgbs/metadata/wgbs_metadata_local.csv")
```
### Merge ESC replicates for DMR calling
Merge and collapse H9 ESC KSR replicates from Smith lab
```{r, eval=FALSE}
smith_primed_files <- mdat$BSseq_CG[mdat$Background == "H9" &
mdat$Media == "KSR" &
mdat$Lab == "Smith"]
stopifnot(all(file.exists(smith_primed_files)))
smith_primed_list <- lapply(X = smith_primed_files, FUN = readRDS)
smith_primed_obj <- bsseq::combineList(smith_primed_list)
merge_vec <- c("H9_KSR", "H9_KSR", "H9_KSR")
names(merge_vec) <- sampleNames(smith_primed_obj)
smith_collapsed_obj <- bsseq::collapseBSseq(BSseq = smith_primed_obj,
group = merge_vec)
saveRDS(object = smith_collapsed_obj, file = "wgbs/CG_bsobj/Smith_H9_Primed_KSR_combined_CG_bsseq.Rds")
```
Merge MEL1 ESC E8 replicates
```{r, eval=FALSE}
mel1_esc_files <- mdat$BSseq_CG[mdat$Background == "MEL1" &
mdat$Media == "E8" &
mdat$State == "ESC"]
stopifnot(all(file.exists(mel1_esc_files)))
mel1_esc_list <- lapply(X = mel1_esc_files, FUN = readRDS)
mel1_esc_obj <- bsseq::combineList(mel1_esc_list)
merge_vec <- c("MEL1_ESC_E8", "MEL1_ESC_E8")
names(merge_vec) <- sampleNames(mel1_esc_obj)
mel1_collapsed_obj <- bsseq::collapseBSseq(BSseq = mel1_esc_obj,
group = merge_vec)
saveRDS(object = mel1_collapsed_obj,
file = "wgbs/CG_bsobj/MEL1_ESC_E8_combined_CG_bsseq.Rds")
```
Merge H9 ESC E8 replicates
```{r, eval=FALSE}
h9_esc_files <- mdat$BSseq_CG[mdat$Background == "H9" &
mdat$Media == "E8" &
mdat$State == "ESC"]
stopifnot(all(file.exists(h9_esc_files)))
h9_esc_list <- lapply(X = h9_esc_files, FUN = readRDS)
h9_esc_obj <- bsseq::combineList(h9_esc_list)
merge_vec <- c("H9_ESC_E8", "H9_ESC_E8")
names(merge_vec) <- sampleNames(h9_esc_obj)
mel1_collapsed_obj <- bsseq::collapseBSseq(BSseq = h9_esc_obj,
group = merge_vec)
saveRDS(object = mel1_collapsed_obj,
file = "wgbs/CG_bsobj/H9_ESC_E8_combined_CG_bsseq.Rds")
```
### DMR calling
DMRs called using the script ```"wgbs/dmrs/run_dmrseq_dug.R"```
### DMR assessment
```{r, echo=FALSE}
source("R/project_functions.R")
```
Load the metadata
```{r, cache=TRUE}
mdat <- read.csv("wgbs/metadata/wgbs_metadata_local.csv")
```
#### Primed vs ESC original submission data
```{r, cache=TRUE}
process_dmr <- function(dmr_obj, p = 0.05, delta = 0.2){
# make mCG matrix
bsseq_fls <- basename(dmr_obj$manifest_data$rds_path)
bsseq_fls <- str_c("wgbs/CG_bsobj/", bsseq_fls)
mC_dat <- make_mC_matrix(obj_fls = bsseq_fls,
gr = dmr_obj$dmr_granges,
cores = 3)
ind <- match(colnames(mC_dat), basename(dmr_obj$manifest_data$rds_path))
colnames(mC_dat) <- dmr_obj$manifest_data$id[ind]
## Calculate delta for groups
groups <- unique(dmr_obj$manifest_data$group)
g1_mean <- rowMeans(mC_dat[ ,dmr_obj$manifest_data$group == groups[1]])
g2_mean <- rowMeans(mC_dat[ ,dmr_obj$manifest_data$group == groups[2]])
group_delta <- g1_mean - g2_mean
## Setup mcols for output
df <- data.frame(g1_mean = g1_mean, g2_mean = g2_mean, delta = group_delta)
mcols(dmr_obj$dmr_granges) <- cbind(mcols(dmr_obj$dmr_granges), df, mC_dat)
# Set if DMR passes significance threshold
dmr_obj$dmr_granges$significant <- (dmr_obj$dmr_granges$pval < p) &
(abs(dmr_obj$dmr_granges$delta) >= delta)
# remove sex chromosomes
dmr_obj$dmr_granges <- dmr_obj$dmr_granges[seqnames(dmr_obj$dmr_granges) %in%
str_c("chr", 1:22)]
return(dmr_obj)
}
```
```{r, cache=TRUE}
dmr_a <- readRDS("wgbs/dmrs/dmrseq_primed_vs_esc_original/dmrseq_primed_vs_esc_original_ESC_vs_Primed_dmrseq_dmrs.Rds")
```
```{r, cache=TRUE}
dmr_a <- process_dmr(dmr_a)
```
Heatmap
```{r, cache=TRUE}
pheatmap(mcols(dmr_a$dmr_granges)[dmr_a$dmr_granges$significant,
dmr_a$manifest_data$id])
```
#### Primed vs TNT matched replicates
```{r, cache=TRUE}
dmr_matched <- readRDS("wgbs/dmrs/dmrseq_primed_vs_tnt_matched/primed_vs_tnt_matched_TNT_vs_Primed_dmrseq_dmrs.Rds")
```
Process and filter DMRs
```{r, cache=TRUE}
dmr_matched <- process_dmr(dmr_obj = dmr_matched)
esc_lines <- c("SRR1561745_merge", "RL2351_merge", "RL2352_merge",
"SRS004213", "SRS114877", "SRS606777", "SRS606778")
fib_ind <- match(c("RL415", esc_lines, dmr_matched$manifest_data$id),
mdat$Library_id)
fib_dmr_mCG <- make_mC_matrix(obj_fls = mdat$BSseq_CG[fib_ind],
gr = dmr_matched$dmr_granges[dmr_matched$dmr_granges$significant],
cores = 3)
dmr_matched_table <- as.data.frame(dmr_matched$dmr_granges)
write.csv(dmr_matched_table, "wgbs/processed_data/32F-matched-primed-tnt-cg-dmrs.csv")
colnames(fib_dmr_mCG) <- mdat$Manuscript.Name[fib_ind]
fib_coldat <- data.frame(row.names = colnames(fib_dmr_mCG),
Group = mdat$Group[fib_ind])
pdf("wgbs/plots/cg-dmr-32F-repeat-endpoint-heatmap.pdf", width = 4, height = 4)
pheatmap(fib_dmr_mCG[complete.cases(fib_dmr_mCG), ],
show_rownames = FALSE, fontsize = 6,
annotation_col = fib_coldat,
clustering_distance_cols = "correlation",
clustering_method = "average")
dev.off()
```
```{r, eval=FALSE}
wb_ed_fig9f <- openxlsx::createWorkbook()
openxlsx::addWorksheet(wb_ed_fig9f, sheetName = "ED_Fig_9f_hdf")
openxlsx::writeData(wb = wb_ed_fig9f, sheet = "ED_Fig_9f_hdf",
x = fib_dmr_mCG[complete.cases(fib_dmr_mCG), ])
```
### MSCs
```{r, cache=TRUE}
dmr_msc <- readRDS("wgbs/dmrs/dmrseq_msc_primed_vs_tnt/dmrseq_msc_primed_vs_tnt_MSC_TNT_vs_MSC_Primed_dmrseq_dmrs.Rds")
dmr_msc <- process_dmr(dmr_msc)
msc_ind <- match(c("RL3161", "RL3162",
esc_lines, dmr_msc$manifest_data$id), mdat$Library_id)
msc_dmr_mCG <- make_mC_matrix(obj_fls = mdat$BSseq_CG[msc_ind],
gr = dmr_msc$dmr_granges[dmr_msc$dmr_granges$significant],
cores = 3)
colnames(msc_dmr_mCG) <- mdat$Manuscript.Name[msc_ind]
pheatmap(msc_dmr_mCG[complete.cases(msc_dmr_mCG), ], show_rownames = FALSE)
pdf("wgbs/plots/cg-dmr-msc-tnt-vs-primed-heatmap.pdf", width = 3, height = 4)
pheatmap(msc_dmr_mCG[complete.cases(msc_dmr_mCG), ],
show_rownames = FALSE, fontsize = 6,
clustering_distance_cols = "correlation")
dev.off()
```
```{r, eval=FALSE}
openxlsx::addWorksheet(wb_ed_fig9f, sheetName = "ED_Fig_9f_msc")
openxlsx::writeData(wb = wb_ed_fig9f, sheet = "ED_Fig_9f_msc",
x = msc_dmr_mCG[complete.cases(msc_dmr_mCG), ])
```
#### Keratinocytes
```{r, cache=TRUE}
dmr_nhek <- readRDS("wgbs/dmrs/dmrseq_nhek_primed_vs_tnt/dmrseq_nhek_primed_vs_tnt_NHEK_Primed_vs_NHEK_TNT_dmrseq_dmrs.Rds")
dmr_nhek <- process_dmr(dmr_nhek)
nhek_ind <- match(c("RL3245", "RL3246", "RL3247",
esc_lines, dmr_nhek$manifest_data$id), mdat$Library_id)
nhek_dmr_mCG <- make_mC_matrix(obj_fls = mdat$BSseq_CG[nhek_ind],
gr = dmr_nhek$dmr_granges[dmr_nhek$dmr_granges$significant],
cores = 3)
colnames(nhek_dmr_mCG) <- mdat$Manuscript.Name[nhek_ind]
pdf("wgbs/plots/cg-dmr-nhek-tnt-vs-primed-heatmap.pdf", width = 3, height = 4)
pheatmap(nhek_dmr_mCG[complete.cases(nhek_dmr_mCG), ],
show_rownames = FALSE, fontsize = 6,
clustering_distance_cols = "correlation")
dev.off()
```
```{r, eval=FALSE}
openxlsx::addWorksheet(wb_ed_fig9f, sheetName = "ED_Fig_9f_nhek")
openxlsx::writeData(wb = wb_ed_fig9f, sheet = "ED_Fig_9f_nhek",
x = nhek_dmr_mCG[complete.cases(nhek_dmr_mCG), ])
openxlsx::saveWorkbook(wb = wb_ed_fig9f,
file = "ED_Figure_9f_source_data.xlsx", overwrite = TRUE)
```
Compare DMRs in context of ESCs and Fibroblasts
```{r, cache=TRUE}
esc_lines <- c("SRR1561745_merge", "RL2351_merge", "RL2352_merge",
"SRS004213", "SRS114877", "SRS606777", "SRS606778")
ips_lines <- c(dmr_matched$manifest_data$id,
dmr_msc$manifest_data$id,
dmr_nhek$manifest_data$id)
origin_lines <- c("RL415", "RL3245", "RL3161")
lib_ids_a <- c(esc_lines, ips_lines, origin_lines)
ind_a <- match(lib_ids_a, mdat$Library_id)
# Make union DMRs
union_dmr_matched <- GRangesList(
dmr_matched$dmr_granges[dmr_matched$dmr_granges$significant],
dmr_nhek$dmr_granges[dmr_nhek$dmr_granges$significant],
dmr_msc$dmr_granges[dmr_msc$dmr_granges$significant]
) %>% unlist() %>% reduce()
dmr_union_mCG <- make_mC_matrix(obj_fls = mdat$BSseq_CG[ind_a],
gr = union_dmr_matched, cores = 3)
colnames(dmr_union_mCG) <- mdat$ID[ind_a]
pheatmap(dmr_union_mCG[complete.cases(dmr_union_mCG), c(1:7, 26:31)],
show_rownames = FALSE,
clustering_distance_cols = 'correlation')
pheatmap(dmr_union_mCG[complete.cases(dmr_union_mCG), c(1:7, 20:25)],
show_rownames = FALSE,
clustering_distance_cols = 'correlation')
pheatmap(dmr_union_mCG[complete.cases(dmr_union_mCG), c(1:19)],
show_rownames = FALSE,
clustering_distance_cols = 'correlation')
plot_pca(dmr_union_mCG, dim1 = 2, dim2 = 3)
plot_dendrogram(dmr_union_mCG[complete.cases(dmr_union_mCG), c(1:7, 26:31)],
method = "pearson")
```
Heatmap the DMRs
```{r}
#
# dmr_mdat <- mdat[ind_a, c(1, 10)]
# rownames(dmr_mdat) <- dmr_mdat$Library_id
# dmr_mdat$Library_id <- NULL
#
# hm1_labs <- mdat$Manuscript.Name[ind_a]
# hm1_labs[grepl(pattern = "(32F)", hm1_labs)] <- "Fibroblast-32F"
# hm1_labs[grepl(pattern = "(38F)", hm1_labs)] <- "Fibroblast-38F"
#
# dmr_hm1 <- pheatmap(dmr_match_mCG[complete.cases(dmr_match_mCG) &
# dmr_matched$dmr_granges$pval < 0.05, ],
# annotation_col = dmr_mdat,
# labels_col = hm1_labs,
# fontsize = 6,
# show_rownames = FALSE,
# border_color = NA,
# clustering_distance_cols = "correlation")
#
# ## Export plot for manuscript layout
# pdf("manuscript_figure_plots/revision/cg-dmr-batch-2-heatmap.pdf",
# height = 4, width = 4)
# dmr_hm1
#
# dev.off()
#
# dmr_hm1
```
DMR delta plots
```{r}
# sig <- dmr_matched$dmr_granges$pval < 0.05
#
# primed_mean <- rowMeans(dmr_match_mCG[sig, dmr_mdat$Group == "Primed-hiPSC"], na.rm = TRUE)
# tnt_mean <- rowMeans(dmr_match_mCG[sig, dmr_mdat$Group == "TNT-hiPSC"], na.rm = TRUE)
# esc_mean <- rowMeans(dmr_match_mCG[sig, dmr_mdat$Group == "hESC"], na.rm = TRUE)
# fblast_mean <- dmr_match_mCG[sig ,"RL415"]
#
# primed_esc_delta <- primed_mean - esc_mean
# tnt_esc_delta <- tnt_mean - esc_mean
# primed_fblast_delta <- primed_mean - fblast_mean
# tnt_fblast_delta <- tnt_mean - fblast_mean
#
# df <- data.frame(iPSC = c(primed_esc_delta, tnt_esc_delta),
# HDF = c(primed_fblast_delta, tnt_fblast_delta),
# group = c(rep("Primed-hiPSC", times=length(primed_esc_delta)),
# rep("TNT-hiPSC", times=length(tnt_esc_delta))))
#
# ggMarg_primed <- ggplot(df[df$group == "Primed-hiPSC", ],
# aes(x = iPSC, y = HDF)) +
# #scale_colour_manual(values = cols[1]) +
# geom_vline(xintercept = c(-.2, .2), alpha=0.5, linetype = "dashed") +
# geom_hline(yintercept = c(-.2, .2), alpha=0.5, linetype = "dashed") +
# geom_point(alpha=0.5, inherit.aes = TRUE, size=1) +
# scale_x_continuous(limits = c(-1, 1), expand = c(0,0)) +
# scale_y_continuous(limits = c(-1, 1), expand = c(0,0)) +
# ylab("Fibroblast mCG - hiPSC mCG") +
# xlab("hESC mCG - hiPSC mCG") +
# sams_pub_theme(x.text.angle = 0, hjust = 0.5)
#
# ggMarg_primed <- ggExtra::ggMarginal(ggMarg_primed)
#
# ggMarg_tnt <- ggplot(df[df$group == "TNT-hiPSC", ],
# aes(x = iPSC, y = HDF)) +
# geom_vline(xintercept = c(-.2, .2), alpha=0.5, linetype = "dashed") +
# geom_hline(yintercept = c(-.2, .2), alpha=0.5, linetype = "dashed") +
# geom_point(alpha=0.5, inherit.aes = TRUE, size=1) +
# scale_x_continuous(limits = c(-1, 1), expand = c(0,0)) +
# scale_y_continuous(limits = c(-1, 1), expand = c(0,0)) +
# ylab("Fibroblast mCG - hiPSC mCG") +
# xlab("hESC mCG - hiPSC mCG") +
# scale_colour_manual(values = vitC[c(6,1)]) +
# sams_pub_theme(x.text.angle = 0, hjust = 0.5)
#
# ggMarg_tnt <- ggExtra::ggMarginal(ggMarg_tnt)
#
# cols <- reprog_pal2[c(2,1)] %>% str_sub(end = -3)
#
# gg_dmr_hist <- ggplot(df, aes(x = iPSC, fill=group)) +
# geom_histogram(bins = 40) +
# scale_fill_manual(values = cols, drop = FALSE) +
# facet_grid(group~.) +
# geom_vline(xintercept = 0, linetype="dashed") +
# scale_x_continuous(limits = c(-1, 1), expand = c(0,0)) +
# scale_y_continuous(expand = c(0,0)) +
# #scale_colour_manual(values = c("grey")) +
# xlab("mCG DMR difference \n (hiPSC mCG - hESC mCG)") +
# ylab("CG-DMR count") +
# sams_pub_theme(x.text.angle = 0, hjust = 0.5)
#
# pdf("manuscript_figure_plots/revision/cg-dmr-batch-2-group-histogram.pdf",
# width = 2, height = 2.5)
# gg_dmr_hist
# dev.off()
```
#### MEL1 DMRs
```{r, cache=TRUE}
dmr_mel1 <- readRDS("wgbs/dmrs/MEL1_dmrs/dmr_out/esc_vs_primed_dmrseq_dmrs.Rds")
dmr_mel1 <- process_dmr(dmr_mel1, p = 0.05, delta = 0)
```
#### Re-analysis Lister 2011
```{r, cache=TRUE}
dmr_lister <- readRDS("wgbs/dmrs/dmrseq_lister_reanalysis/lister_primed_vs_esc_Primed_vs_ESC_dmrseq_dmrs.Rds")
dmr_lister <- process_dmr(dmr_lister, p = 0.05, delta = 0)
pheatmap(mcols(dmr_lister$dmr_granges)[dmr_lister$dmr_granges$significant,
dmr_lister$manifest_data$id])
## Add our data and plot
primary_dat <- mdat$BSseq_CG[mdat$Background %in% c("32F", "38F") &
mdat$Group %in% c("TNT-hiPSC", "Primed-hiPSC") &
mdat$In_manuscript == "YES"]
dmr_lister_mCG <- make_mC_matrix(obj_fls = primary_dat,
gr = dmr_lister$dmr_granges[dmr_lister$dmr_granges$significant],
cores = 3)
dmr_lister_mCG <- cbind(mcols(dmr_lister$dmr_granges)
[dmr_lister$dmr_granges$significant,
dmr_lister$manifest_data$id],
dmr_lister_mCG)
pheatmap(dmr_lister_mCG[complete.cases(dmr_lister_mCG), ],
cluster_cols = FALSE, gaps_col = c(4, 6, 18, 22),
show_rownames = FALSE)
```
#### Re-analysis Ma 2015
```{r, cache=TRUE}
dmr_ma <- readRDS("wgbs/dmrs/dmrseq_mitalipov_reanalysis/mitalipov_primed_sendai_vs_esc_Primed_vs_ESC_dmrseq_dmrs.Rds")
dmr_ma <- process_dmr(dmr_ma)
pheatmap(mcols(dmr_ma$dmr_granges)[dmr_ma$dmr_granges$significant,
dmr_ma$manifest_data$id])
```
Delta ESC plots
```{r, cache=TRUE}
plot_esc_delta_histograms <- function(dmr_obj, title=""){
# Get the GRanges DMR object
dmr_gr <- dmr_obj$dmr_granges[dmr_obj$dmr_granges$significant]
# Get the library IDs for plot
esc_lines <- c("SRR1561745_merge", "RL2351_merge", "RL2352_merge",
"SRS004213", "SRS114877", "SRS606777", "SRS606778")
ips_lines <- dmr_obj$manifest_data$id
# Get the files matching IDs
ind <- match(c(esc_lines, ips_lines), mdat$Library_id)
fls <- mdat$BSseq_CG[ind]
# Make mC matrix
mat <- make_mC_matrix(obj_fls = fls, gr = dmr_gr, cores = 3)
colnames(mat) <- mdat$Library_id[ind]
# Calculate ESC delta
esc_mean <- rowMeans(mat[ ,esc_lines], na.rm = TRUE)
delta_mat <- mat[ ,ips_lines] - esc_mean
# Format for plotting
delta_mat <- reshape2::melt(delta_mat)
ind2 <- match(delta_mat$Var2, mdat$Library_id)
delta_mat$Group <- mdat$Group[ind2]
ggplot(delta_mat, aes(x = value)) +
geom_histogram(bins = 50) +
facet_grid(Group+Var2~.) +
geom_vline(xintercept = 0) +
scale_x_continuous(limits = c(-1, 1)) +
ylab("DMR count") + xlab("hiPSC mCG - hESC mCG") +
ggtitle(title) +
sams_pub_theme(x.text.angle = 0, hjust = 0.5)
}
gg_delta_nhek <- plot_esc_delta_histograms(dmr_obj = dmr_nhek,
title = "Keratinocytes")
gg_delta_msc <- plot_esc_delta_histograms(dmr_obj = dmr_msc,
title = "MSCs")
gg_delta_fib <- plot_esc_delta_histograms(dmr_obj = dmr_matched,
title = "Fibroblasts")
```
### Plot combined DMRs
```{r, cache=TRUE}
all_dmrs <- c(dmr_a$dmr_granges[dmr_a$dmr_granges$significant],
dmr_matched$dmr_granges[dmr_matched$dmr_granges$significant],
dmr_lister$dmr_granges[dmr_lister$dmr_granges$significant],
dmr_ma$dmr_granges[dmr_ma$dmr_granges$significant])
all_fls <- c(dmr_a$manifest_data$rds_path,
dmr_matched$manifest_data$rds_path,
dmr_lister$manifest_data$rds_path,
dmr_ma$manifest_data$rds_path,
mdat$BSseq_CG[mdat$Group == "TNT-hiPSC" &
mdat$Batch == "A"])
all_fls <- str_remove(all_fls, "/d/home/hg19ips/hg19ips_samb/mcc_hg19ips/sam/hs-reprogram/")
all_fls <- unique(all_fls)
all_ind <- match(all_fls, mdat$BSseq_CG)
all_groups <- mdat$Group[all_ind]
all_labs <- mdat$Lab[all_ind]
all_batch <- mdat$Batch[all_ind]
all_link <- str_c(all_groups, all_labs, all_batch, sep = "_")
all_link <- factor(all_link, levels = c("hESC_Ecker_Ecker",
"hESC_Mitalipov_Mitalipov",
"hESC_Smith_Smith",
"hESC_Lister_A",
"Primed-hiPSC_Lister_A",
"Primed-hiPSC_Lister_B",
"TNT-hiPSC_Lister_A",
"Primed-hiPSC_Lister_C",
"TNT-hiPSC_Lister_C",
"Primed-hiPSC_Ecker_Ecker",
"Primed-hiPSC_Mitalipov_Mitalipov"))
all_link[order(all_link)]
plot_ind <- order(all_link)
all_dmr_mCG <- make_mC_matrix(obj_fls = all_fls, all_dmrs, cores = 3)
colnames(all_dmr_mCG) <- mdat$Library_id[match(colnames(all_dmr_mCG),
basename(mdat$BSseq_CG))]
gaps_row <- cumsum(c(length(dmr_a$dmr_granges[dmr_a$dmr_granges$significant]),
length(dmr_matched$dmr_granges[dmr_matched$dmr_granges$significant]),
length(dmr_lister$dmr_granges[dmr_lister$dmr_granges$significant]),
length(dmr_ma$dmr_granges[dmr_ma$dmr_granges$significant])))
# pheatmap(all_dmr_mCG[ ,plot_ind],
# gaps_col = c(7, 11, 13, 19, 25, 29),
# gaps_row = gaps_row,
# cluster_rows = FALSE,
# cluster_cols = FALSE,
# show_rownames = FALSE)
```
### Plot DMR overlaps
```{r, cache=TRUE}
# Just use P < 0.05 to make a permissive comparison (no delta filter)
# h3k9 <-
# fib_lad <-
# esc_lad <-
# constit_lad <-
# pmd <-
# ch_dmr <-
#
dmr_list <- list(dmr_a$dmr_granges[dmr_a$dmr_granges$pval < 0.05],
dmr_matched$dmr_granges[dmr_matched$dmr_granges$pval < 0.05],
dmr_lister$dmr_granges[dmr_lister$dmr_granges$pval < 0.05],
dmr_ma$dmr_granges[dmr_ma$dmr_granges$pval < 0.05])
names(dmr_list) <- c("Batch_A", "Matched", "Lister", "Ma")
saveRDS(dmr_list, "wgbs/dmrs/dmr_list_filtered.Rds")
dmr_list <- readRDS("wgbs/dmrs/dmr_list_filtered.Rds")
gr_to_bed(dmr_list$Batch_A,
out_path = "wgbs/processed_data/primary_fibroblast_dmrs_filt.bed")
gr_to_bed(dmr_list$Lister,
out_path = "wgbs/processed_data/lister_2011_recall_dmrs_filt.bed")
gr_to_bed(dmr_list$Ma,
out_path = "wgbs/processed_data/ma_2014_recall_dmrs_filt.bed")
# Make union DMR set
union_dmrs <- GRangesList(dmr_list) %>% unlist %>% reduce()
dmr_olaps <- lapply(dmr_list, overlapsAny, query = union_dmrs)
dmr_olaps <- do.call(cbind, dmr_olaps)
dmr_olaps <- dmr_olaps + 0 #binarise the data
colnames(dmr_olaps) <- c("Primed vs ESC",
"Primed vs TNT (B)",
"Lister Primed vs TNT",
"Ma Primed vs ESC")
dmr_olaps <- data.frame(dmr_olaps)
rownames(dmr_olaps) <- gr_to_loci(union_dmrs)
pdf("wgbs/plots/cg-dmr-overlap-upset.pdf",
width = 4, height = 2.5)
UpSetR::upset(dmr_olaps[ ,-c(2)], order.by = "freq")
dev.off()
```
```{r, eval=FALSE}
wb_ed_fig5 <- openxlsx::createWorkbook()
openxlsx::addWorksheet(wb_ed_fig5, sheetName = "ED_Fig_5a")
openxlsx::writeData(wb = wb_ed_fig5, sheet = "ED_Fig_5a",
x = dmr_olaps[ ,-c(2)])
```
```{r, eval=FALSE}
source("R/server_libraries_and_functions.R")
source("R/project_functions.R")
all_elements_gr <- readRDS("resources/all_genomic_elements_granges_final.Rds")
dmr_list <- readRDS("wgbs/dmrs/dmr_list_filtered.Rds")
CH_dmr <- bed_to_gr("resources/nature13551-s3.bed")
mcols(CH_dmr)$class <- "CH_DMR"
all_elements_gr <- c(all_elements_gr, CH_dmr)
#### Peak enrichment
test_dmr_enrichment <- function(target_element="Fibroblast_H3K9me3", test_gr,
permutations=200, cores=20, alt="auto"){
#stopifnot(file.exists(test_bed))
gc()
message(Sys.time())+
message(str_c("Running ", target_element, "..."))
target_gr <- all_elements_gr[all_elements_gr$class == target_element]
message(str_c("n = "), length(target_gr))
#test_gr <- bed_to_gr(test_bed)
mcols(test_gr) <- NULL
test_gr <- resize(test_gr, width = 100, fix = "center")
set.seed(123)
perm_test_results <- regioneR::permTest(A=test_gr, B=target_gr,
#alternative = "auto",
alternative = alt,
randomize.function=regioneR::randomizeRegions,
ntimes = permutations,
evaluate.function=regioneR::numOverlaps,
count.once = TRUE,
genome="hg19",
#mask = unmappable_gr,
allow.overlaps=FALSE,
per.chromosome=TRUE,
mc.cores=cores,
mc.set.seed=FALSE,
force.parallel=FALSE)
lz <- regioneR::localZScore(pt=perm_test_results, A=test_gr, B=target_gr,
step = mean(width(test_gr)/2),
window = 100000)
results <- list(pt=perm_test_results, lz=lz, element=target_element)
message("Done!")
return(results)
}
regions <- c("Fibroblast_LAD", "Fibroblast_PMD", "Fibroblast_H3K9me3",
"ESC_H3K9me3", "ESC_LAD",
"Constitutive_LAD", "Constitutive_H3K9me3")
lister_enrich <- lapply(X = regions, FUN = test_dmr_enrichment,
test_gr = dmr_list$Lister)
saveRDS(lister_enrich, "wgbs/processed_data/lister-2011-cg-dmr-enrichment-list.Rds")
ma_enrich <- lapply(X = regions, FUN = test_dmr_enrichment,
test_gr = dmr_list$Ma)
saveRDS(ma_enrich, "wgbs/processed_data/ma-2014-cg-dmr-enrichment-list.Rds")
## Ch-DMR enrichments
primary_ch_dmr_enrich <- test_dmr_enrichment(target_element = "CH_DMR",
cores = 3,
test_gr = dmr_list$Batch_A)
saveRDS(primary_ch_dmr_enrich, "wgbs/processed_data/primary_ch_dmr_enrich.Rds")
lister_ch_dmr_enrich <- test_dmr_enrichment(target_element = "CH_DMR",
cores = 3,
test_gr = dmr_list$Lister)
saveRDS(lister_ch_dmr_enrich, "wgbs/processed_data/lister_ch_dmr_enrich.Rds")
ma_ch_dmr_enrich <- test_dmr_enrichment(target_element = "CH_DMR",
cores = 3,
test_gr = dmr_list$Ma)
saveRDS(ma_ch_dmr_enrich, "wgbs/processed_data/ma_ch_dmr_enrich.Rds")
## Get data on promoter and enhancer enrichment in CH-DMRs
promoter_ch_dmr_enrich <- test_dmr_enrichment(target_element = "CH_DMR",
cores = 3,
test_gr = all_elements_gr[all_elements_gr$class == "Promoter"])
saveRDS(promoter_ch_dmr_enrich, "wgbs/processed_data/promoter_ch_dmr_enrich.Rds")
enh_ch_dmr_enrich <- test_dmr_enrichment(target_element = "CH_DMR",
cores = 3,
test_gr = all_elements_gr[all_elements_gr$class == "Enhancer"])
saveRDS(enh_ch_dmr_enrich, "wgbs/processed_data/enh_ch_dmr_enrich.Rds")
grps <- c("Primary_fibroblasts", "Lister_2011", "Ma 2014", "Promoters", "Enhancers")
enrich_ch_df <- data.frame(Group=factor(grps, levels=rev(grps)), Zscore=c(primary_ch_dmr_enrich$pt$`regioneR::numOverlaps`$zscore,
lister_ch_dmr_enrich$pt$`regioneR::numOverlaps`$zscore,
ma_ch_dmr_enrich$pt$`regioneR::numOverlaps`$zscore,
promoter_ch_dmr_enrich$pt$`regioneR::numOverlaps`$zscore,
enh_ch_dmr_enrich$pt$`regioneR::numOverlaps`$zscore))
gg_enricg_cg <- ggplot(data = enrich_ch_df, aes(x = Group, y = Zscore)) +
geom_col() + coord_flip() + sams_pub_theme()
pdf("wgbs/plots/lister-ma-cg-dmr-in-ch-dmr-enrichment-plots.pdf",
height = 1.3, width = 3)
gg_enricg_cg
dev.off()
```
```{r, eval=FALSE}
wb_ed_fig5d <- openxlsx::createWorkbook()
openxlsx::addWorksheet(wb_ed_fig5d, sheetName = "ED_Fig_5d")
openxlsx::writeData(wb = wb_ed_fig5d, sheet = "ED_Fig_5d",
x = gg_enricg_cg$data)
openxlsx::saveWorkbook(wb = wb_ed_fig5d,
file = "ED_Figure_5d_source_data.xlsx", overwrite = TRUE)
```
```{r, eval=FALSE}
## Percentage of overlapping CG-DMRs with CH-DMRs
lapply(X = dmr_list,
FUN = function(x){sum(overlapsAny(x, subject = CH_dmr))/length(x)})
lister_enrich <- readRDS("wgbs/processed_data/lister-2011-cg-dmr-enrichment-list.Rds")
ma_enrich <- readRDS("wgbs/processed_data/ma-2014-cg-dmr-enrichment-list.Rds")
process_enrichments <- function(enrich_list){
get_stats <- function(x){
results <- enrich_list[[x]]
df <- data.frame(#file=results$test_bed,
element=results$element,
z_score=results[[1]]$`regioneR::numOverlaps`$zscore,
p_value=results[[1]]$`regioneR::numOverlaps`$pval,
permutations=results[[1]]$`regioneR::numOverlaps`$ntimes,
alternative=results[[1]]$`regioneR::numOverlaps`$alternative,
observed=results[[1]]$`regioneR::numOverlaps`$observed)
return(df)
}
stats_df <- lapply(1:length(enrich_list), get_stats) %>% do.call(rbind, .)
return(stats_df)
}
ma_dat <- process_enrichments(enrich_list = ma_enrich)
ma_dat$dataset <- "Ma et al. 2014"
lister_dat <- process_enrichments(enrich_list = lister_enrich)
lister_dat$dataset <- "Lister et al. 2011"
enrich_dat <- rbind(ma_dat, lister_dat)
## Remove constitutive to simplify comparison
enrich_dat <- enrich_dat[!grepl(pattern = "Constitutive", enrich_dat$element), ]
enrich_dat <- enrich_dat[!grepl(pattern = "PMD", enrich_dat$element), ]
enrich_dat$element <- factor(enrich_dat$element,
levels=c("ESC_LAD", "ESC_H3K9me3",
"Fibroblast_LAD", "Fibroblast_H3K9me3"))
gg_enrich_bar <- ggplot(enrich_dat, aes(x = element, y=z_score)) +
geom_col() +
facet_grid(.~dataset, space = "free", drop = TRUE, scales = "free") +
sams_pub_theme(legend_pos = "right", x.text.angle = 0, hjust = 0.5) +
coord_flip()
gg_enrich_bar
pdf("wgbs/plots/lister-ma-cg-dmr-enrichment-plots.pdf", height = 1.3, width = 4)
gg_enrich_bar
dev.off()
```
```{r, eval=FALSE}
openxlsx::addWorksheet(wb_ed_fig5, sheetName = "ED_Fig_5c")
openxlsx::writeData(wb = wb_ed_fig5, sheet = "ED_Fig_5c",
x = gg_enrich_bar$data)
```
Plot DMR delta histograms
```{r, cache=TRUE}
hist(dmr_list$Lister$delta)
hist(dmr_list$Ma$delta)
hist(dmr_list$Batch_A$delta)
hist_df <- data.frame(Group=c(
rep("This study", times=length(dmr_list$Batch_A)),
rep("Lister_2011", times=length(dmr_list$Lister)),
rep("Ma 2014", times=length(dmr_list$Ma))),
Delta=c(
dmr_list$Batch_A$delta,
dmr_list$Lister$delta,
dmr_list$Ma$delta))
gg_comparitive_hist <- ggplot(hist_df, aes(x = -Delta)) +
geom_histogram() +
facet_grid(.~Group) +
sams_pub_theme(x.text.angle = 0, hjust = 0.5)
pdf("wgbs/plots/lister-ma-cg-dmr-histograms.pdf", width = 3.5,
height = 1.5)
gg_comparitive_hist
dev.off()
```
```{r, eval=FALSE}
openxlsx::addWorksheet(wb_ed_fig5, sheetName = "ED_Fig_5b")
openxlsx::writeData(wb = wb_ed_fig5, sheet = "ED_Fig_5b",
x = gg_comparitive_hist$data)
```
Plot heatmap of union DMRs
```{r, cache=TRUE}
fl_list <- c(dmr_a$manifest_data$rds_path,
dmr_matched$manifest_data$rds_path,
dmr_lister$manifest_data$rds_path,
dmr_ma$manifest_data$rds_path) %>%
str_remove("/d/home/hg19ips/hg19ips_samb/mcc_hg19ips/sam/hs-reprogram/") %>%
unique()
fl_list <- c(fl_list, mdat$BSseq_CG[mdat$Group == "TNT-hiPSC" &
mdat$Batch == "A"])
union_dmr_mCG <- make_mC_matrix(obj_fls = fl_list,
gr = union_dmrs, cores = 3)
ind_b <- match(colnames(union_dmr_mCG), basename(mdat$BSseq_CG))
colnames(union_dmr_mCG) <- mdat$Library_id[ind_b]
coldat <- data.frame(group=mdat$Group[ind_b],
lab=mdat$Lab[ind_b])
rownames(coldat) <- mdat$Library_id[ind_b]
pheatmap(union_dmr_mCG[complete.cases(union_dmr_mCG), ], scale = 'row',
annotation_col = coldat,
show_rownames = FALSE)
## PCA plot
# Remove incomplete cases
mat <- union_dmr_mCG[complete.cases(union_dmr_mCG), ]
# Transpose
mat <- t(mat)
# Calculate PC's
pr <- prcomp(x = mat, scale.=TRUE)
## Scree plot
gg_scree <- ggplot2::qplot(x = factor(1:10),
y = ((summary(pr)$importance[2, ] * 100)[1:10])) +
sams_pub_theme(x.text.angle = 0, hjust = 0.5) +
ylab("Varaince explained (%)") + xlab("Principal component")
pc1 <- (summary(pr)$importance[2, 1] * 100) %>% round(digits = 1)
pc2 <- (summary(pr)$importance[2, 2] * 100) %>% round(digits = 1)
pc3 <- (summary(pr)$importance[2, 3] * 100) %>% round(digits = 1)
pc4 <- (summary(pr)$importance[2, 4] * 100) %>% round(digits = 1)
pc1_dat <- pr$x[ ,1]
pc2_dat <- pr$x[ ,2]
pc3_dat <- pr$x[ ,3]
pc4_dat <- pr$x[ ,4]
samples <- rownames(pr$x)
pca_df <- data.frame(Sample=samples, PC1=pc1_dat,
PC2=pc2_dat, PC3=pc3_dat, PC4=pc4_dat)
ind_pca <- match(pca_df$Sample, mdat$Library_id)
pca_df$group <- mdat$Group[ind_pca]
pca_df$id <- mdat$Manuscript.Name[ind_pca]
pca_df$lab <- mdat$Lab[ind_pca]
pca_df$background <- mdat$Background[ind_pca]
gg_pc12 <- ggplot(data = pca_df,
mapping = aes(x = PC1, y = PC2, label=id,
fill=group, colour=group)) +
geom_point(alpha=0.8, size=2) +
theme_linedraw() +
stat_ellipse(level=0.9,
geom = "polygon",
aes(fill = group, colour=group),
alpha = 0.1) +
theme(panel.grid = element_line(colour = 'grey')) +
scale_color_manual(values = reprog_pal) +
scale_fill_manual(values = reprog_pal) +
#geom_text_repel(data = subset(pca_df,
# samples %in% samples),
# point.padding = unit(1, "lines"), size=3) +
xlab(str_c("PC1 (", pc1, "%)")) +
ylab(str_c("PC2 (", pc2, "%)")) +
theme(legend.position = "none")
gg_pc23 <- ggplot(data = pca_df,
mapping = aes(x = PC2, y = PC3, label=id,
fill=group, colour=group)) +
geom_point(alpha=0.8, size=2) +
theme_linedraw() +
stat_ellipse(level=0.9,
geom = "polygon",
aes(fill = group, colour=group),
alpha = 0.1) +
theme(panel.grid = element_line(colour = 'grey')) +
scale_color_manual(values = reprog_pal) +
scale_fill_manual(values = reprog_pal) +
#geom_text_repel(data = subset(pca_df,
# samples %in% samples),
# point.padding = unit(1, "lines"), size=3) +
xlab(str_c("PC2 (", pc2, "%)")) +
ylab(str_c("PC3 (", pc3, "%)")) +
theme(legend.position = "none")
gg_pc34 <- ggplot(data = pca_df,
mapping = aes(x = PC3, y = PC4, label=id,
fill=group, colour=group)) +
geom_point(alpha=0.8, size=2) +
theme_linedraw() +
stat_ellipse(level=0.9,
geom = "polygon",
aes(fill = group, colour=group),
alpha = 0.1) +
theme(panel.grid = element_line(colour = 'grey')) +
scale_color_manual(values = reprog_pal) +
scale_fill_manual(values = reprog_pal) +
#geom_text_repel(data = subset(pca_df,
# samples %in% samples),
# point.padding = unit(1, "lines"), size=3) +
xlab(str_c("PC3 (", pc3, "%)")) +
ylab(str_c("PC4 (", pc4, "%)")) +
theme(legend.position = "none")
pdf("wgbs/plots/cg-dmr-all-union-pca-plots.pdf",
width = 8.5, height = 2)
plot_grid(gg_scree, gg_pc12, gg_pc23, gg_pc34,
nrow = 1, ncol = 4)
dev.off()
```
```{r, eval=FALSE}
openxlsx::addWorksheet(wb_ed_fig5, sheetName = "ED_Fig_5g_upper_left")
openxlsx::writeData(wb = wb_ed_fig5, sheet = "ED_Fig_5g_upper_left",
x = ((summary(pr)$importance[2, ] * 100)[1:10]))
openxlsx::addWorksheet(wb_ed_fig5, sheetName = "ED_Fig_5g_upper_right")
openxlsx::writeData(wb = wb_ed_fig5, sheet = "ED_Fig_5g_upper_right",
x = gg_pc12$data)
openxlsx::addWorksheet(wb_ed_fig5, sheetName = "ED_Fig_5g_lower_left")