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Commit c987301f authored by Matteo Barcella's avatar Matteo Barcella
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remoing methylation script - wrong place

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WES/MethylAnalysis_bulk_v3.R deleted 100644 → 0
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MethylAnalysis_bulk_v3 <- function(methcall.folder = NULL, # input folder with .cov files (bismark)
outfolder = NULL, # output folder (if not exist it will be created)
proj.id = "myproject", # project id to prefix in putput files
samplesheet = NULL,
sheetnum = 1,
design.var = "Disease", # variable to subset samplesheet
case.var = "Case", # 1 in treatment vector
control.var = "Control", # 0 in treatment vector
idcol = "SampleID",
mincoverage = 10,
lowperc = 5,
lowcount = 10,
assem="hg38",
do.subset = T,
chr.subset = "chr9",
start.subset = 136668000,
end.subset = 136671000,
pipeline.meth = "bismarkCoverage",
plot.covariates = c("Condition","Batch","MRD",
"Torelapse","Chemorefractory",
"Sex","CytoA","Tissue"),
idstoremove = NULL,
delta.meth = 10,
plot.categorical.vars = c("Condition","Batch","MRD","Torelapse","Chemorefractory","Sex","CytoA"),
plot.continuous.vars = c("miR-126","egfl7"),
plot.height = 9,
plot.width = 12,
cellw = 15,
cellh = 15,
fontnumsize = 5,
fontsize = 8
){
# load libraries
require(methylKit)
require(ggplot2)
require(reshape2)
require(plyr)
require(ggrepel)
library(GenomicRanges)
library(openxlsx)
library(pheatmap)
# check vars
if(is.null(methcall.folder)){
stop("Please set methcall.folder")
}
if(is.null(outfolder)){
stop("Please set outfolder")
}
if(is.null(samplesheet)){
stop("Please set samplesheet")
}
# Setup variables
infolder = paste0(methcall.folder, "/")
dir.create(infolder, showWarnings = F)
outfolder = paste0(outfolder, "/")
dir.create(outfolder, showWarnings = F)
qcfolder <- paste0(outfolder, "QC/")
dir.create(qcfolder, showWarnings = F)
# reading sample sheet with metadata
ssheet <- read.xlsx(samplesheet, sheet = sheetnum)
if(!is.null(idstoremove)){
ssheet <- subset.data.frame(x = ssheet, subset = !ssheet[[idcol]] %in% idstoremove)
}
# defining design vector according to variable
ssheet <- subset.data.frame(x = ssheet, subset = ssheet[[design.var]] %in% c(case.var, control.var))
d.vec <- ssheet[[design.var]]
d.vector <- ifelse(d.vec == case.var, 1, 0)
# initialzing coverage .cov files list
covs <- list()
sampleids <- as.list(ssheet[[idcol]])
names(sampleids) <- ssheet[[idcol]]
for (i in ssheet[[idcol]]) {
covs[[i]] <- paste0(infolder, "/", i, ".bismark.cov")
}
saveRDS(object = covs, file = "covs_object.rds")
# creating object
myobj <- methRead(location = covs,
sample.id = sampleids,
assembly = assem,
pipeline = pipeline.meth,
treatment = d.vector,
context="CpG",
mincov = mincoverage)
saveRDS(myobj, file = "Myobject.rds")
names(myobj) <- ssheet[[idcol]]
saveRDS(myobj, file = "Myobject_with_names.rds")
# subsetting if declared
if(isTRUE(do.subset)){
my.win = GRanges(seqnames = chr.subset, ranges = IRanges(start = start.subset, end = end.subset))
myobj <- selectByOverlap(myobj,my.win)
}
saveRDS(myobj, file = "Myobject_with_names_aftersubset.rds")
# filtering on minimum coverage
myobj <- filterByCoverage(methylObj = myobj, lo.count=lowcount, lo.perc = lowperc)
# Normalization
myobj <- normalizeCoverage(obj = myobj)
# saveRDS(object = myobj, file = "Initial_object.rds")
# Calculate basic stats and PCs
metricsfolder <- paste0(qcfolder, "Metrics/")
dir.create(path = metricsfolder, showWarnings = F)
for (id in ssheet[[idcol]]) {
png(filename = paste0(metricsfolder,proj.id,"_CpG_pct_methylation_sample_", id, ".png"),
width = 9, height = 6, units = "in", res = 96)
print(getMethylationStats(myobj[[id]],plot=TRUE,both.strands=FALSE))
dev.off()
png(filename = paste0(metricsfolder, proj.id,"_Coverage_stats_sample_", id, ".png"),
width = 9, height = 6, units = "in", res = 96)
print(getCoverageStats(myobj[[id]],plot=TRUE,both.strands=FALSE))
dev.off()
}
# create meth obj
#meth <- unite(object = myobj, destrand=FALSE)
meth <- unite(object = myobj, destrand=FALSE) # for debugging
saveRDS(meth, "savemeth.tmp.rds")
# Perform correlation
sink(paste0(qcfolder, proj.id, "_Correlations.txt"))
getCorrelation(meth,plot=FALSE)
sink()
if(length(ssheet[[idcol]]) < 15){
png(filename = paste0(qcfolder,proj.id, "_Correlations_pearson_pairwise.png"),
width = 9, height = 6, units = "in", res = 96)
print(getCorrelation(meth,plot=TRUE))
dev.off()
}
png(filename = paste0(qcfolder, proj.id, "_Clustering.png"),
width = 9, height = 6, units = "in", res = 96)
clusterSamples(meth, dist="euclidean", plot=TRUE, method = "ward.D2")
dev.off()
# Re-plotting PCs (custom chart)
# compute PCs and store in object
pca_compt <- PCASamples(meth, obj.return = T, screeplot = F)
# extract PCs components
pcafolder <- paste0(qcfolder, "PCA/")
dir.create(path = pcafolder, showWarnings = F)
pca_pc1_2 <- as.data.frame(x = pca_compt$x[,1:2])
for(myvars in plot.covariates){
pca_pc1_2$condition <- as.factor(ssheet[[myvars]])
png(filename = paste0(pcafolder, proj.id, "_PCA_",myvars,".png"),
width = 9, height = 6, units = "in",res=96)
print(ggplot(data = pca_pc1_2,
mapping = aes(x = PC1, y=PC2, col=condition, label=rownames(pca_pc1_2))) +
geom_point(size=3) + geom_text_repel(size=3) + ggtitle(label = "Principal component analysis", subtitle = myvars) +
theme(plot.title = element_text(size = 16, face = "bold", hjust = 0.5)) +
theme(plot.subtitle=element_text(size=12, hjust=0.5, face="italic", color="black")) +
theme(axis.title = element_text(size=12, hjust=0.5, face="bold", color="black")) +
theme(legend.text = element_text(size=8, hjust=0.5)) +
theme(legend.title = element_blank()) +
theme(axis.text = element_text(size=12, hjust=0.5, color="black")))
dev.off()
}
# retrieve and store % of methylation
perc.meth <- percMethylation(meth)
saveRDS(perc.meth,"pctmethly.rds")
base::rownames(perc.meth) <- paste0(meth$chr, "_", meth$start)
# Perform diff methylation
myDiff=calculateDiffMeth(meth)
write.table(myDiff,paste0(outfolder,proj.id,"_DiffMeth_single_CpG.txt"), row.names = F)
difftest <- read.table(paste0(outfolder,proj.id,"_DiffMeth_single_CpG.txt"), header = T)
difftest$comparison <- proj.id
difftest$qvalue_r <- as.character(cut(x = difftest$qvalue,
breaks = c(-1, 1e-100, 1e-10, 1e-02, 1),
labels = c("***","**","*","ns")))
myindex <- abs(difftest$meth.diff) < delta.meth
difftest$meth.diff <- abs(difftest$meth.diff)
difftest$qvalue_r[myindex] <- "ns"
write.table(difftest,paste0(outfolder,proj.id,"_DiffMeth_single_CpG.txt"), row.names = F)
# Adding color list and annotations
library(RColorBrewer)
color_list <- list()
annrows <- NULL
anncols <- subset.data.frame(difftest, select = c("qvalue_r","meth.diff"))
base::rownames(anncols) <- difftest$start
rows.annot.vars.cat = plot.categorical.vars
rows.annot.vars.con = plot.continuous.vars
# if(!is.null(rows.annot.vars.cat) | !is.null(rows.annot.vars.con)){
# rownames(ssheet) <- ssheet$SampleID
# annrows <- subset.data.frame(x = ssheet, select = c(rows.annot.vars.cat, rows.annot.vars.con))
# concolors <- RColorBrewer::brewer.pal(n = 9, name = "Set1")
# catcolors <- NULL
# for (varcon in 1:length(rows.annot.vars.con)) {
# color_list[[rows.annot.vars.con[varcon]]] <- colorRampPalette(c("lightgrey", concolors[varcon]))(10)
# }
# for (varcat in 1:length(rows.annot.vars.cat)) {
# ncolor <- 1
# for (val in unique(ssheet[[rows.annot.vars.cat[varcat]]])[order(unique(ssheet[[rows.annot.vars.cat[varcat]]]))]){
# if(length(unique(ssheet[[rows.annot.vars.cat[varcat]]])) > 9){
# catcolors <- colorRampPalette(RColorBrewer::brewer.pal(n = 9, name = "Set3"))(length(unique(ssheet[[rows.annot.vars.cat[varcat]]])))
# }
# else{
# catcolors <- RColorBrewer::brewer.pal(n = 9, name = "Set3")
# }
# color_list[[rows.annot.vars.cat[varcat]]][[val]] <- catcolors[ncolor]
# ncolor <- ncolor + 1
# }
# }
# }
color_list[["qvalue_r"]] = c("*" = "#6497b1", "**" = "#03396c", "***" = "#011f4b", ns= "#c4cacf")
color_list[["meth.diff"]] = colorRampPalette(brewer.pal(n = 11, "Reds"))(100)
color_list[["miR-126"]] <- brewer.pal(n = 9, "PuRd")
# Heatmap
pctmeth_matrix <- t(perc.meth)
base::colnames(pctmeth_matrix) <- gsub(x = base::colnames(pctmeth_matrix), pattern = "chr[0-9]+_", replacement = "")
pheatmap(mat = pctmeth_matrix, main = gsub(x = proj.id, pattern = "_", replacement = " "),
filename = paste0(outfolder, proj.id,"_CpG_percent_methylation_matrix_pheatmap.pdf"), width = plot.width, height = plot.height,
na_col = "black",
cluster_cols = FALSE,
cluster_rows = TRUE,
annotation_row = annrows,
cellwidth = cellw,
cellheight = cellh,
display_numbers = T,
fontsize = fontsize,
fontsize_number = fontnumsize,
number_format = "%.0f",
#border_color = "#CBBEB5",
annotation_col = anncols,
#labels_row = lrow,
#labels_col = lcol,
#gaps_row = gaps.row,
gaps_col = c(8),
annotation_colors = color_list,
color = c("#F5F5F5","#EEEEEE","#CCCCCC","#999999", "#666666","#333333","#000000"), breaks = c(0,10,20,30,50,70,90,100)
)
saveRDS(object = pctmeth_matrix, paste0(outfolder, proj.id,"_CpG_percent_methylation_matrix_pheatmap.rds"))
saveRDS(object = list(anncol = anncols, annrow = annrows, anncolors = color_list), paste0(outfolder, proj.id,"_annotations_matrix_pheatmap.rds"))
saveRDS(object = difftest, paste0(outfolder, proj.id,"_CpG_differential_methylation.rds"))
saveRDS(object = perc.meth, paste0(outfolder, proj.id,"_CpG_percent_methylation.rds"))
write.table(x = perc.meth, file = paste0(outfolder, proj.id,"_CpG_percent_methylation.txt"))
write.table(x = difftest, file = paste0(outfolder, proj.id,"_CpG_differential_methylation.txt"))
saveRDS(myobj, file = paste0(outfolder,proj.id,"_methylkit.rds"))
saveRDS(myobj, file = paste0(outfolder,proj.id,"_methylkit_meth.rds"))
}
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