Last updated: 2018-07-26

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    File Version Author Date Message
    html 94153b3 kevinlkx 2018-07-26 Build site.
    Rmd 4d492e6 kevinlkx 2018-07-26 compare centipede predictions for CTCF in DiffAC regions
    html ec5a6ef kevinlkx 2018-07-25 Build site.
    Rmd c628625 kevinlkx 2018-07-25 compare centipede predictions for CTCF in DiffAC regions 

library(ggplot2)
library(grid)
library(gridExtra)
suppressPackageStartupMessages(library(GenomicRanges))
library(limma)

Attaching package: 'limma'
The following object is masked from 'package:BiocGenerics':

    plotMA
library(edgeR)
library(VennDiagram)
Warning: package 'VennDiagram' was built under R version 3.4.4
Loading required package: futile.logger
message <- futile.logger::flog.threshold(futile.logger::ERROR, name = "VennDiagramLogger")

## venn diagram
plot_venn_overlaps <- function(overlaps.m, title = "", col_fill = NULL, category.names = NULL){
  grid.newpage()
  overlaps_venn.l <- lapply(as.data.frame(overlaps.m), function(x) which(x == 1))
  if(is.null(col_fill)){
    col_fill <-  1:length(overlaps_venn.l)
  }
  if(is.null(category.names)){
    category.names <- names(x)
  }
  
  venn.plot <- venn.diagram( 
    x = overlaps_venn.l,
    category.names = category.names, 
    filename = NULL,
    fill = col_fill,
    alpha=rep(0.5, length(overlaps_venn.l)), 
    cex = 1.5, 
    cat.fontface=4, 
    main=title) 
  grid.draw(venn.plot)
}

parameters

tf_name <- "CTCF"
pwm_name <- "CTCF_MA0139.1_1e-4"

thresh_PostPr_bound <- 0.99

flank <- 100

cat("PWM name: ", pwm_name, "\n")
PWM name:  CTCF_MA0139.1_1e-4

load diff accessibility test results, comparing hypoxia vs. normoxia.

  • log fold change > 0 indicates differentially open in hypoxia.
  • log fold change < 0 indicates differentially open in normoxia.
diffAC_regions.df <- read.csv("~/Dropbox/research/ATAC_DNase/ATAC-seq_Olivia_Gray/results/DiffAC_regions/ordered_results_withcoords.csv")

cat(nrow(diffAC_regions.df), "regions in total \n")
138927 regions in total 
diffAC_regions.df <- diffAC_regions.df[, c("chr", "Start", "End","GeneID", "baseMean", "Strand",  "log2FoldChange", "lfcSE", "stat", "pvalue", "padj")]

diffAC_sig_regions.df <- diffAC_regions.df[diffAC_regions.df$padj < 0.1, ]
cat(nrow(diffAC_sig_regions.df), "significant regions \n")
2390 significant regions 
hist(diffAC_regions.df$log2FoldChange, xlab = "log2FoldChange", main = "Differentially open regions (FDR < 10%)")

Expand here to see past versions of unnamed-chunk-3-1.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25 

diffAC_sigH_regions.df <- diffAC_sig_regions.df[diffAC_sig_regions.df$log2FoldChange > 0, ]
cat(nrow(diffAC_sigH_regions.df), "regions differentially open in hypoxia. \n")
201 regions differentially open in hypoxia. 
diffAC_sigN_regions.df <- diffAC_sig_regions.df[diffAC_sig_regions.df$log2FoldChange < 0, ]
cat(nrow(diffAC_sigN_regions.df), "regions differentially open in normoxia. \n")
2189 regions differentially open in normoxia. 
diffAC_sig_regions.gr <- makeGRangesFromDataFrame(diffAC_sig_regions.df, start.field = "Start", end.field = "End", keep.extra.columns = T)

diffAC_sigH_regions.gr <- makeGRangesFromDataFrame(diffAC_sigH_regions.df, start.field = "Start", end.field = "End", keep.extra.columns = T)

diffAC_sigN_regions.gr <- makeGRangesFromDataFrame(diffAC_sigN_regions.df, start.field = "Start", end.field = "End", keep.extra.columns = T)

load CENTIPEDE predictions

dir_predictions <- paste0("~/Dropbox/research/ATAC_DNase/ATAC-seq_Olivia_Gray/results/centipede_predictions/", pwm_name)

## condition: N
bam_namelist_N <- c("N1_nomito_rdup.bam", "N2_nomito_rdup.bam", "N3_nomito_rdup.bam")

site_predictions_N.l <- vector("list", 3)
names(site_predictions_N.l) <- bam_namelist_N

for(i in 1:length(bam_namelist_N)){
  bam_basename <- tools::file_path_sans_ext(basename(bam_namelist_N[[i]]))
  site_predictions_N.l[[i]] <- read.table(paste0(dir_predictions, "/", pwm_name, "_", bam_basename, "_predictions.txt"), header = T, stringsAsFactors = F)
}

CentPostPr_N.df <- data.frame(N1 = site_predictions_N.l[[1]]$CentPostPr, 
                              N2 = site_predictions_N.l[[2]]$CentPostPr, 
                              N3 = site_predictions_N.l[[3]]$CentPostPr)

CentLogRatios_N.df <- data.frame(N1 = site_predictions_N.l[[1]]$CentLogRatios, 
                                 N2 = site_predictions_N.l[[2]]$CentLogRatios, 
                                 N3 = site_predictions_N.l[[3]]$CentLogRatios)

## condition: H
bam_namelist_H <- c("H1_nomito_rdup.bam", "H2_nomito_rdup.bam", "H3_nomito_rdup.bam")

site_predictions_H.l <- vector("list", 3)
names(site_predictions_H.l) <- bam_namelist_H

for(i in 1:length(bam_namelist_H)){
  bam_basename <- tools::file_path_sans_ext(basename(bam_namelist_H[[i]]))
  site_predictions_H.l[[i]] <- read.table(paste0(dir_predictions, "/", pwm_name, "_", bam_basename, "_predictions.txt"), header = T, stringsAsFactors = F)
}

CentPostPr_H.df <- data.frame(H1 = site_predictions_H.l[[1]]$CentPostPr, 
                              H2 = site_predictions_H.l[[2]]$CentPostPr, 
                              H3 = site_predictions_H.l[[3]]$CentPostPr)

CentLogRatios_H.df <- data.frame(H1 = site_predictions_H.l[[1]]$CentLogRatios, 
                                 H2 = site_predictions_H.l[[2]]$CentLogRatios, 
                                 H3 = site_predictions_H.l[[3]]$CentLogRatios)

if(any(site_predictions_N.l[[1]]$name != site_predictions_H.l[[1]]$name)){
  stop("sites not match!")
}

sites.df <- site_predictions_N.l[[1]][,1:7]

## get motif coordinates
if(sites.df[1, "end"] - sites.df[1, "start"] > flank){
  sites.df$start <- sites.df$start + flank
  sites.df$end <- sites.df$end - flank
}


sites.gr <- makeGRangesFromDataFrame(sites.df, start.field = "start", end.field = "end", keep.extra.columns = F)


CentPostPr.df <- cbind(CentPostPr_N.df, CentPostPr_H.df)
CentLogRatios.df <- cbind(CentLogRatios_N.df, CentLogRatios_H.df)

sites_CentPostPr.df <- cbind(sites.df, CentPostPr_N.df, CentPostPr_H.df)
sites_CentLogRatios.df <- cbind(sites.df, CentLogRatios_N.df, CentLogRatios_H.df)

intersect CENTIPEDE sites with diffAC regions

overlaps_diffAC.df <- as.data.frame(findOverlaps(query = sites.gr, subject = diffAC_sig_regions.gr, type = "within", ignore.strand = T))
idx_sites_diffAC <- unique(overlaps_diffAC.df$queryHits)
cat(length(idx_sites_diffAC), "candidate motif sites differentially open in hypoxia or normoxia. \n")
412 candidate motif sites differentially open in hypoxia or normoxia. 
overlaps_sigH.df <- as.data.frame(findOverlaps(query = sites.gr, subject = diffAC_sigH_regions.gr, type = "within", ignore.strand = T))
idx_sites_sigH <- unique(overlaps_sigH.df$queryHits)

cat(length(idx_sites_sigH), "candidate motif sites differentially open in hypoxia. \n")
22 candidate motif sites differentially open in hypoxia. 
overlaps_sigN.df <- as.data.frame(findOverlaps(query = sites.gr, subject = diffAC_sigN_regions.gr, type = "within", ignore.strand = T))
idx_sites_sigN <- unique(overlaps_sigN.df$queryHits)

cat(length(idx_sites_sigN), "candidate motif sites differentially open in normoxia. \n")
390 candidate motif sites differentially open in normoxia.

binarize to bound and unbound

cat("Number of bound sites that are differentially open in hypoxia: \n")
Number of bound sites that are differentially open in hypoxia: 
colSums(CentPostPr.df[idx_sites_sigH, ] > thresh_PostPr_bound)
N1 N2 N3 H1 H2 H3 
14 16 13 18 15 17 
cat("Number of bound sites that are differentially open in normoxia: \n")
Number of bound sites that are differentially open in normoxia: 
colSums(CentPostPr.df[idx_sites_sigN, ] > thresh_PostPr_bound)
 N1  N2  N3  H1  H2  H3 
385 372 362 176 148 227

Average binding probablity and average logRatios

all motif sites

# binding probablity
par(pty="s")
plot(rowMeans(CentPostPr_N.df), rowMeans(CentPostPr_H.df), 
     xlab = "N average P(Bound)", ylab = "H average P(Bound)", main = tf_name,
     pch = ".", col = rgb(0,0,1,0.7))
abline(a=0, b=1, col = "darkgray")

Expand here to see past versions of unnamed-chunk-7-1.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25 

# logRatios
par(mfrow = c(1,2))
par(pty="s")
plot(rowMeans(CentLogRatios_N.df), rowMeans(CentLogRatios_H.df), 
     xlab = "N average logRatios", ylab = "H average logRatios", main = tf_name, 
     pch = ".", col = rgb(0,0,1,0.7))
abline(a=0,b=1,col = "darkgray")

plot(x = (rowMeans(CentLogRatios_H.df)+rowMeans(CentLogRatios_N.df))/2, 
     y = rowMeans(CentLogRatios_H.df) - rowMeans(CentLogRatios_N.df),
     xlab = "average logRatios", ylab = "Difference in logRatios (H - N)", main = tf_name,
     pch = ".", col = rgb(0,0,1,0.7))
abline(v=0, h=0, col = "darkgray")

Expand here to see past versions of unnamed-chunk-7-2.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25

sites that are differentially open in hypoxia

cat(length(idx_sites_sigH), "candidate motif sites differentially open in hypoxia. \n")
22 candidate motif sites differentially open in hypoxia. 
# binding probablity
par(pty="s")
plot(rowMeans(CentPostPr_N.df[idx_sites_sigH,]), rowMeans(CentPostPr_H.df[idx_sites_sigH,]), 
     xlab = "N average P(Bound)", ylab = "H average P(Bound)", main = paste(tf_name, "bound sites"),
     pch = 20, col = rgb(0,0,1,0.7))
abline(a=0, b=1, col = "darkgray")

Expand here to see past versions of unnamed-chunk-8-1.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25 

# logRatios
par(mfrow = c(1,2))
par(pty="s")
plot(rowMeans(CentLogRatios_N.df[idx_sites_sigH,]), rowMeans(CentLogRatios_H.df[idx_sites_sigH,]), 
     xlab = "N average logRatios", ylab = "H average logRatios", main = tf_name, 
     pch = 20, col = rgb(0,0,1,0.7))
abline(a=0,b=1,col = "darkgray")

plot(x = (rowMeans(CentLogRatios_H.df[idx_sites_sigH,])+rowMeans(CentLogRatios_N.df[idx_sites_sigH,]))/2, 
     y = rowMeans(CentLogRatios_H.df[idx_sites_sigH,]) - rowMeans(CentLogRatios_N.df[idx_sites_sigH,]),
     xlab = "average logRatios", ylab = "Difference in logRatios (H - N)", main = tf_name,
     pch = 20, col = rgb(0,0,1,0.7))
abline(v=0, h=0, col = "darkgray")

Expand here to see past versions of unnamed-chunk-8-2.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25

sites that are differentially open in normoxia

cat(length(idx_sites_sigN), "candidate motif sites differentially open in normoxia \n")
390 candidate motif sites differentially open in normoxia 
# binding probablity
par(pty="s")
plot(rowMeans(CentPostPr_N.df[idx_sites_sigN,]), rowMeans(CentPostPr_H.df[idx_sites_sigN,]), 
     xlab = "N average P(Bound)", ylab = "H average P(Bound)", main = paste(tf_name, "bound sites"),
     pch = 20, col = rgb(0,0,1,0.7))
abline(a=0, b=1, col = "darkgray")

Expand here to see past versions of unnamed-chunk-9-1.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25 

# logRatios
par(mfrow = c(1,2))
par(pty="s")
plot(rowMeans(CentLogRatios_N.df[idx_sites_sigN,]), rowMeans(CentLogRatios_H.df[idx_sites_sigN,]), 
     xlab = "N average logRatios", ylab = "H average logRatios", main = tf_name, 
     pch = 20, col = rgb(0,0,1,0.7))
abline(a=0,b=1,col = "darkgray")

plot(x = (rowMeans(CentLogRatios_H.df[idx_sites_sigN,])+rowMeans(CentLogRatios_N.df[idx_sites_sigN,]))/2, 
     y = rowMeans(CentLogRatios_H.df[idx_sites_sigN,]) - rowMeans(CentLogRatios_N.df[idx_sites_sigN,]),
     xlab = "average logRatios", ylab = "Difference in logRatios (H - N)", main = tf_name,
     pch = 20, col = rgb(0,0,1,0.7))
abline(v=0, h=0, col = "darkgray")

Expand here to see past versions of unnamed-chunk-9-2.png:
Version Author Date
ec5a6ef kevinlkx 2018-07-25

Compare logRatios for differentially accessible sites using limma

targets <- data.frame(bam = c(bam_namelist_N, bam_namelist_H), 
                      label = colnames(CentLogRatios.df), 
                      condition = rep(c("N", "H"), each = 3))

print(targets)
                 bam label condition
1 N1_nomito_rdup.bam    N1         N
2 N2_nomito_rdup.bam    N2         N
3 N3_nomito_rdup.bam    N3         N
4 H1_nomito_rdup.bam    H1         H
5 H2_nomito_rdup.bam    H2         H
6 H3_nomito_rdup.bam    H3         H
condition <- factor(targets$condition, levels = c("N", "H"))
design <- model.matrix(~0+condition)
colnames(design) <- levels(condition)
print(design)
  N H
1 1 0
2 1 0
3 1 0
4 0 1
5 0 1
6 0 1
attr(,"assign")
[1] 1 1
attr(,"contrasts")
attr(,"contrasts")$condition
[1] "contr.treatment"
CentLogRatios_diffAC.df <- CentLogRatios.df[idx_sites_diffAC, ]

fit <- lmFit(CentLogRatios_diffAC.df, design)
contrasts <- makeContrasts(H-N, levels=design)
fit2 <- contrasts.fit(fit, contrasts)
fit2 <- eBayes(fit2, trend=TRUE)
num_diffbind <- summary(decideTests(fit2))

percent_diffbind <- round(num_diffbind / sum(num_diffbind) * 100, 2)

cat(num_diffbind[1], "sites differentially open in normoxia (", percent_diffbind[1], "%) \n", 
    num_diffbind[3], "sites differentially open in hypoxia (", percent_diffbind[3], "%) \n",
    num_diffbind[2], "sites not significantly different (", percent_diffbind[2], "%) \n")
388 sites differentially open in normoxia ( 94.17 %) 
 3 sites differentially open in hypoxia ( 0.73 %) 
 21 sites not significantly different ( 5.1 %) 
# volcanoplot(fit2, main="H vs. N", xlab = "Difference in logRatios (H - N)")

plot(x = fit2$coef, y = -log10(fit2$p.value),
     xlab = "Difference in logRatios (H - N)", ylab = "-log10(P-value)", main= paste(tf_name, "H vs. N"),
     pch = 16, cex = 0.35)

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Version Author Date
94153b3 kevinlkx 2018-07-26
ec5a6ef kevinlkx 2018-07-25

Session information

sessionInfo()
R version 3.4.3 (2017-11-30)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS High Sierra 10.13.6

Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
 [1] parallel  stats4    grid      stats     graphics  grDevices utils    
 [8] datasets  methods   base     

other attached packages:
 [1] VennDiagram_1.6.20   futile.logger_1.4.3  edgeR_3.20.9        
 [4] limma_3.34.9         GenomicRanges_1.30.3 GenomeInfoDb_1.14.0 
 [7] IRanges_2.12.0       S4Vectors_0.16.0     BiocGenerics_0.24.0 
[10] gridExtra_2.3        ggplot2_2.2.1       

loaded via a namespace (and not attached):
 [1] Rcpp_0.12.16           formatR_1.5            XVector_0.18.0        
 [4] compiler_3.4.3         pillar_1.2.2           git2r_0.21.0          
 [7] plyr_1.8.4             workflowr_1.1.1        futile.options_1.0.1  
[10] zlibbioc_1.24.0        R.methodsS3_1.7.1      R.utils_2.6.0         
[13] bitops_1.0-6           tools_3.4.3            digest_0.6.15         
[16] lattice_0.20-35        evaluate_0.10.1        tibble_1.4.2          
[19] gtable_0.2.0           rlang_0.2.0            yaml_2.1.18           
[22] GenomeInfoDbData_1.0.0 stringr_1.3.0          knitr_1.20            
[25] locfit_1.5-9.1         rprojroot_1.3-2        rmarkdown_1.9         
[28] lambda.r_1.2.2         magrittr_1.5           whisker_0.3-2         
[31] splines_3.4.3          backports_1.1.2        scales_0.5.0          
[34] htmltools_0.3.6        colorspace_1.3-2       stringi_1.1.7         
[37] RCurl_1.95-4.10        lazyeval_0.2.1         munsell_0.4.3         
[40] R.oo_1.22.0

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