Last updated: 2024-09-27

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Knit directory: ATAC_learning/

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Untracked files:
    Untracked:  DOX_DAR_assess.Rmd
    Untracked:  EAR_2_plot.pdf
    Untracked:  ESR_1_plot.pdf
    Untracked:  Firstcorr plotATAC.pdf
    Untracked:  IND1_2_3_6_corrplot.pdf
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    Untracked:  trimmed_Ind1_75DA3h_S1.nodup.fragment.size.distribution.pdf

Unstaged changes:
    Modified:   analysis/CorMotif_data_n45.Rmd
    Modified:   analysis/Enrichment_motif.Rmd
    Modified:   analysis/GO_KEGG_analysis.Rmd
    Modified:   analysis/Jaspar_motif_ff.Rmd
    Modified:   analysis/Peak_calling.Rmd
    Modified:   analysis/Raodah.Rmd
    Modified:   analysis/Raodah_mycount.Rmd
    Modified:   analysis/Smaller_set_DAR.Rmd
    Modified:   analysis/TE_analysis.Rmd

Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.


These are the previous versions of the repository in which changes were made to the R Markdown (analysis/final_four_analysis.Rmd) and HTML (docs/final_four_analysis.html) files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view the files as they were in that past version.

File Version Author Date Message
Rmd c0456fb reneeisnowhere 2024-09-27 updates using C and D separately
html 8acb1e8 reneeisnowhere 2024-09-23 Build site.
Rmd 1777f5a reneeisnowhere 2024-09-23 updated group of eight based on 4 individuals
Rmd 82f2027 reneeisnowhere 2024-09-16 updates to plots
Rmd 7778182 reneeisnowhere 2024-09-10 updates
Rmd 437a792 reneeisnowhere 2024-09-05 new updates
Rmd a540a01 reneeisnowhere 2024-09-04 adding in high conf peak set based on final files

library(tidyverse)
library(kableExtra)
library(broom)
library(RColorBrewer)
library(ChIPseeker)
library("TxDb.Hsapiens.UCSC.hg38.knownGene")
library("org.Hs.eg.db")
library(rtracklayer)
library(edgeR)
library(ggfortify)
library(limma)
library(readr)
library(BiocGenerics)
library(gridExtra)
library(VennDiagram)
library(scales)
library(Cormotif)
library(BiocParallel)
library(ggpubr)
library(devtools)
library(eulerr)
library(genomation)
library(ggsignif)
library(plyranges)
library(ggrepel)
library(ComplexHeatmap)
library(cowplot)
## Fit limma model using code as it is found in the original cormotif code. It has
## only been modified to add names to the matrix of t values, as well as the
## limma fits

limmafit.default <- function(exprs,groupid,compid) {
  limmafits  <- list()
  compnum    <- nrow(compid)
  genenum    <- nrow(exprs)
  limmat     <- matrix(0,genenum,compnum)
  limmas2    <- rep(0,compnum)
  limmadf    <- rep(0,compnum)
  limmav0    <- rep(0,compnum)
  limmag1num <- rep(0,compnum)
  limmag2num <- rep(0,compnum)

  rownames(limmat)  <- rownames(exprs)
  colnames(limmat)  <- rownames(compid)
  names(limmas2)    <- rownames(compid)
  names(limmadf)    <- rownames(compid)
  names(limmav0)    <- rownames(compid)
  names(limmag1num) <- rownames(compid)
  names(limmag2num) <- rownames(compid)

  for(i in 1:compnum) {
    selid1 <- which(groupid == compid[i,1])
    selid2 <- which(groupid == compid[i,2])
    eset   <- new("ExpressionSet", exprs=cbind(exprs[,selid1],exprs[,selid2]))
    g1num  <- length(selid1)
    g2num  <- length(selid2)
    designmat <- cbind(base=rep(1,(g1num+g2num)), delta=c(rep(0,g1num),rep(1,g2num)))
    fit <- lmFit(eset,designmat)
    fit <- eBayes(fit)
    limmat[,i] <- fit$t[,2]
    limmas2[i] <- fit$s2.prior
    limmadf[i] <- fit$df.prior
    limmav0[i] <- fit$var.prior[2]
    limmag1num[i] <- g1num
    limmag2num[i] <- g2num
    limmafits[[i]] <- fit

    # log odds
    # w<-sqrt(1+fit$var.prior[2]/(1/g1num+1/g2num))
    # log(0.99)+dt(fit$t[1,2],g1num+g2num-2+fit$df.prior,log=TRUE)-log(0.01)-dt(fit$t[1,2]/w, g1num+g2num-2+fit$df.prior, log=TRUE)+log(w)
  }
  names(limmafits) <- rownames(compid)
  limmacompnum<-nrow(compid)
  result<-list(t       = limmat,
               v0      = limmav0,
               df0     = limmadf,
               s20     = limmas2,
               g1num   = limmag1num,
               g2num   = limmag2num,
               compnum = limmacompnum,
               fits    = limmafits)
}

limmafit.counts <-
  function (exprs, groupid, compid, norm.factor.method = "TMM", voom.normalize.method = "none")
  {
    limmafits  <- list()
    compnum    <- nrow(compid)
    genenum    <- nrow(exprs)
    limmat     <- matrix(NA,genenum,compnum)
    limmas2    <- rep(0,compnum)
    limmadf    <- rep(0,compnum)
    limmav0    <- rep(0,compnum)
    limmag1num <- rep(0,compnum)
    limmag2num <- rep(0,compnum)

    rownames(limmat)  <- rownames(exprs)
    colnames(limmat)  <- rownames(compid)
    names(limmas2)    <- rownames(compid)
    names(limmadf)    <- rownames(compid)
    names(limmav0)    <- rownames(compid)
    names(limmag1num) <- rownames(compid)
    names(limmag2num) <- rownames(compid)

    for (i in 1:compnum) {
      message(paste("Running limma for comparision",i,"/",compnum))
      selid1 <- which(groupid == compid[i, 1])
      selid2 <- which(groupid == compid[i, 2])
      # make a new count data frame
      counts <- cbind(exprs[, selid1], exprs[, selid2])

      # remove NAs
      not.nas <- which(apply(counts, 1, function(x) !any(is.na(x))) == TRUE)

      # runn voom/limma
      d <- DGEList(counts[not.nas,])
      d <- calcNormFactors(d, method = norm.factor.method)
      g1num <- length(selid1)
      g2num <- length(selid2)
      designmat <- cbind(base = rep(1, (g1num + g2num)), delta = c(rep(0,
                                                                       g1num), rep(1, g2num)))

      y <- voom(d, designmat, normalize.method = voom.normalize.method)
      fit <- lmFit(y, designmat)
      fit <- eBayes(fit)

      limmafits[[i]] <- fit
      limmat[not.nas, i] <- fit$t[, 2]
      limmas2[i] <- fit$s2.prior
      limmadf[i] <- fit$df.prior
      limmav0[i] <- fit$var.prior[2]
      limmag1num[i] <- g1num
      limmag2num[i] <- g2num
    }
    limmacompnum <- nrow(compid)
    names(limmafits) <- rownames(compid)
    result <- list(t       = limmat,
                   v0      = limmav0,
                   df0     = limmadf,
                   s20     = limmas2,
                   g1num   = limmag1num,
                   g2num   = limmag2num,
                   compnum = limmacompnum,
                   fits    = limmafits)
  }

limmafit.list <-
  function (fitlist, cmp.idx=2)
  {
    compnum    <- length(fitlist)

    genes <- c()
    for (i in 1:compnum) genes <- unique(c(genes, rownames(fitlist[[i]])))

    genenum    <- length(genes)
    limmat     <- matrix(NA,genenum,compnum)
    limmas2    <- rep(0,compnum)
    limmadf    <- rep(0,compnum)
    limmav0    <- rep(0,compnum)
    limmag1num <- rep(0,compnum)
    limmag2num <- rep(0,compnum)

    rownames(limmat)  <- genes
    colnames(limmat)  <- names(fitlist)
    names(limmas2)    <- names(fitlist)
    names(limmadf)    <- names(fitlist)
    names(limmav0)    <- names(fitlist)
    names(limmag1num) <- names(fitlist)
    names(limmag2num) <- names(fitlist)

    for (i in 1:compnum) {
      this.t <- fitlist[[i]]$t[,cmp.idx]
      limmat[names(this.t),i] <- this.t

      limmas2[i]    <- fitlist[[i]]$s2.prior
      limmadf[i]    <- fitlist[[i]]$df.prior
      limmav0[i]    <- fitlist[[i]]$var.prior[cmp.idx]
      limmag1num[i] <- sum(fitlist[[i]]$design[,cmp.idx]==0)
      limmag2num[i] <- sum(fitlist[[i]]$design[,cmp.idx]==1)
    }

    limmacompnum <- compnum
    result <- list(t       = limmat,
                   v0      = limmav0,
                   df0     = limmadf,
                   s20     = limmas2,
                   g1num   = limmag1num,
                   g2num   = limmag2num,
                   compnum = limmacompnum,
                   fits    = limmafits)

  }

## Rank genes based on statistics
generank<-function(x) {
  xcol<-ncol(x)
  xrow<-nrow(x)
  result<-matrix(0,xrow,xcol)
  z<-(1:1:xrow)
  for(i in 1:xcol) {
    y<-sort(x[,i],decreasing=TRUE,na.last=TRUE)
    result[,i]<-match(x[,i],y)
    result[,i]<-order(result[,i])
  }
  result
}

## Log-likelihood for moderated t under H0
modt.f0.loglike<-function(x,df) {
  a<-dt(x, df, log=TRUE)
  result<-as.vector(a)
  flag<-which(is.na(result)==TRUE)
  result[flag]<-0
  result
}

## Log-likelihood for moderated t under H1
## param=c(df,g1num,g2num,v0)
modt.f1.loglike<-function(x,param) {
  df<-param[1]
  g1num<-param[2]
  g2num<-param[3]
  v0<-param[4]
  w<-sqrt(1+v0/(1/g1num+1/g2num))
  dt(x/w, df, log=TRUE)-log(w)
  a<-dt(x/w, df, log=TRUE)-log(w)
  result<-as.vector(a)
  flag<-which(is.na(result)==TRUE)
  result[flag]<-0
  result
}

## Correlation Motif Fit
cmfit.X<-function(x, type, K=1, tol=1e-3, max.iter=100) {
  ## initialize
  xrow <- nrow(x)
  xcol <- ncol(x)
  loglike0 <- list()
  loglike1 <- list()
  p <- rep(1, K)/K
  q <- matrix(runif(K * xcol), K, xcol)
  q[1, ] <- rep(0.01, xcol)
  for (i in 1:xcol) {
    f0 <- type[[i]][[1]]
    f0param <- type[[i]][[2]]
    f1 <- type[[i]][[3]]
    f1param <- type[[i]][[4]]
    loglike0[[i]] <- f0(x[, i], f0param)
    loglike1[[i]] <- f1(x[, i], f1param)
  }
  condlike <- list()
  for (i in 1:xcol) {
    condlike[[i]] <- matrix(0, xrow, K)
  }
  loglike.old <- -1e+10
  for (i.iter in 1:max.iter) {
    if ((i.iter%%50) == 0) {
      print(paste("We have run the first ", i.iter, " iterations for K=",
                  K, sep = ""))
    }
    err <- tol + 1
    clustlike <- matrix(0, xrow, K)
    #templike <- matrix(0, xrow, 2)
    templike1 <- rep(0, xrow)
    templike2 <- rep(0, xrow)
    for (j in 1:K) {
      for (i in 1:xcol) {
        templike1 <- log(q[j, i]) + loglike1[[i]]
        templike2 <- log(1 - q[j, i]) + loglike0[[i]]
        tempmax <- Rfast::Pmax(templike1, templike2)

        templike1 <- exp(templike1 - tempmax)
        templike2 <- exp(templike2 - tempmax)

        tempsum <- templike1 + templike2
        clustlike[, j] <- clustlike[, j] + tempmax +
          log(tempsum)
        condlike[[i]][, j] <- templike1/tempsum
      }
      clustlike[, j] <- clustlike[, j] + log(p[j])
    }
    #tempmax <- apply(clustlike, 1, max)
    tempmax <- Rfast::rowMaxs(clustlike, value=TRUE)
    for (j in 1:K) {
      clustlike[, j] <- exp(clustlike[, j] - tempmax)
    }
    #tempsum <- apply(clustlike, 1, sum)
    tempsum <- Rfast::rowsums(clustlike)
    for (j in 1:K) {
      clustlike[, j] <- clustlike[, j]/tempsum
    }
    #p.new <- (apply(clustlike, 2, sum) + 1)/(xrow + K)
    p.new <- (Rfast::colsums(clustlike) + 1)/(xrow + K)
    q.new <- matrix(0, K, xcol)
    for (j in 1:K) {
      clustpsum <- sum(clustlike[, j])
      for (i in 1:xcol) {
        q.new[j, i] <- (sum(clustlike[, j] * condlike[[i]][,
                                                           j]) + 1)/(clustpsum + 2)
      }
    }
    err.p <- max(abs(p.new - p)/p)
    err.q <- max(abs(q.new - q)/q)
    err <- max(err.p, err.q)
    loglike.new <- (sum(tempmax + log(tempsum)) + sum(log(p.new)) +
                      sum(log(q.new) + log(1 - q.new)))/xrow
    p <- p.new
    q <- q.new
    loglike.old <- loglike.new
    if (err < tol) {
      break
    }
  }
  clustlike <- matrix(0, xrow, K)
  for (j in 1:K) {
    for (i in 1:xcol) {
      templike1 <- log(q[j, i]) + loglike1[[i]]
      templike2 <- log(1 - q[j, i]) + loglike0[[i]]
      tempmax <- Rfast::Pmax(templike1, templike2)

      templike1 <- exp(templike1 - tempmax)
      templike2 <- exp(templike2 - tempmax)

      tempsum <- templike1 + templike2
      clustlike[, j] <- clustlike[, j] + tempmax + log(tempsum)
      condlike[[i]][, j] <- templike1/tempsum
    }
    clustlike[, j] <- clustlike[, j] + log(p[j])
  }
  #tempmax <- apply(clustlike, 1, max)
  tempmax <- Rfast::rowMaxs(clustlike, value=TRUE)
  for (j in 1:K) {
    clustlike[, j] <- exp(clustlike[, j] - tempmax)
  }
  #tempsum <- apply(clustlike, 1, sum)
  tempsum <- Rfast::rowsums(clustlike)
  for (j in 1:K) {
    clustlike[, j] <- clustlike[, j]/tempsum
  }
  p.post <- matrix(0, xrow, xcol)
  for (j in 1:K) {
    for (i in 1:xcol) {
      p.post[, i] <- p.post[, i] + clustlike[, j] * condlike[[i]][,
                                                                  j]
    }
  }
  loglike.old <- loglike.old - (sum(log(p)) + sum(log(q) +
                                                    log(1 - q)))/xrow
  loglike.old <- loglike.old * xrow
  result <- list(p.post = p.post, motif.prior = p, motif.q = q,
                 loglike = loglike.old, clustlike=clustlike, condlike=condlike)
}

## Fit using (0,0,...,0) and (1,1,...,1)
cmfitall<-function(x, type, tol=1e-3, max.iter=100) {
  ## initialize
  xrow<-nrow(x)
  xcol<-ncol(x)
  loglike0<-list()
  loglike1<-list()
  p<-0.01

  ## compute loglikelihood
  L0<-matrix(0,xrow,1)
  L1<-matrix(0,xrow,1)
  for(i in 1:xcol) {
    f0<-type[[i]][[1]]
    f0param<-type[[i]][[2]]
    f1<-type[[i]][[3]]
    f1param<-type[[i]][[4]]
    loglike0[[i]]<-f0(x[,i],f0param)
    loglike1[[i]]<-f1(x[,i],f1param)
    L0<-L0+loglike0[[i]]
    L1<-L1+loglike1[[i]]
  }


  ## EM algorithm to get MLE of p and q
  loglike.old <- -1e10
  for(i.iter in 1:max.iter) {
    if((i.iter%%50) == 0) {
      print(paste("We have run the first ", i.iter, " iterations",sep=""))
    }
    err<-tol+1

    ## compute posterior cluster membership
    clustlike<-matrix(0,xrow,2)
    clustlike[,1]<-log(1-p)+L0
    clustlike[,2]<-log(p)+L1

    tempmax<-apply(clustlike,1,max)
    for(j in 1:2) {
      clustlike[,j]<-exp(clustlike[,j]-tempmax)
    }
    tempsum<-apply(clustlike,1,sum)

    ## update motif occurrence rate
    for(j in 1:2) {
      clustlike[,j]<-clustlike[,j]/tempsum
    }

    p.new<-(sum(clustlike[,2])+1)/(xrow+2)

    ## evaluate convergence
    err<-abs(p.new-p)/p

    ## evaluate whether the log.likelihood increases
    loglike.new<-(sum(tempmax+log(tempsum))+log(p.new)+log(1-p.new))/xrow

    loglike.old<-loglike.new
    p<-p.new

    if(err<tol) {
      break;
    }
  }

  ## compute posterior p
  clustlike<-matrix(0,xrow,2)
  clustlike[,1]<-log(1-p)+L0
  clustlike[,2]<-log(p)+L1

  tempmax<-apply(clustlike,1,max)
  for(j in 1:2) {
    clustlike[,j]<-exp(clustlike[,j]-tempmax)
  }
  tempsum<-apply(clustlike,1,sum)

  for(j in 1:2) {
    clustlike[,j]<-clustlike[,j]/tempsum
  }

  p.post<-matrix(0,xrow,xcol)
  for(i in 1:xcol) {
    p.post[,i]<-clustlike[,2]
  }

  ## return

  #calculate back loglikelihood
  loglike.old<-loglike.old-(log(p)+log(1-p))/xrow
  loglike.old<-loglike.old*xrow
  result<-list(p.post=p.post, motif.prior=p, loglike=loglike.old)
}

## Fit each dataset separately
cmfitsep<-function(x, type, tol=1e-3, max.iter=100) {
  ## initialize
  xrow<-nrow(x)
  xcol<-ncol(x)
  loglike0<-list()
  loglike1<-list()
  p<-0.01*rep(1,xcol)
  loglike.final<-rep(0,xcol)

  ## compute loglikelihood
  for(i in 1:xcol) {
    f0<-type[[i]][[1]]
    f0param<-type[[i]][[2]]
    f1<-type[[i]][[3]]
    f1param<-type[[i]][[4]]
    loglike0[[i]]<-f0(x[,i],f0param)
    loglike1[[i]]<-f1(x[,i],f1param)
  }

  p.post<-matrix(0,xrow,xcol)

  ## EM algorithm to get MLE of p
  for(coli in 1:xcol) {
    loglike.old <- -1e10
    for(i.iter in 1:max.iter) {
      if((i.iter%%50) == 0) {
        print(paste("We have run the first ", i.iter, " iterations",sep=""))
      }
      err<-tol+1

      ## compute posterior cluster membership
      clustlike<-matrix(0,xrow,2)
      clustlike[,1]<-log(1-p[coli])+loglike0[[coli]]
      clustlike[,2]<-log(p[coli])+loglike1[[coli]]

      tempmax<-apply(clustlike,1,max)
      for(j in 1:2) {
        clustlike[,j]<-exp(clustlike[,j]-tempmax)
      }
      tempsum<-apply(clustlike,1,sum)

      ## evaluate whether the log.likelihood increases
      loglike.new<-sum(tempmax+log(tempsum))/xrow

      ## update motif occurrence rate
      for(j in 1:2) {
        clustlike[,j]<-clustlike[,j]/tempsum
      }

      p.new<-(sum(clustlike[,2]))/(xrow)

      ## evaluate convergence
      err<-abs(p.new-p[coli])/p[coli]
      loglike.old<-loglike.new
      p[coli]<-p.new

      if(err<tol) {
        break;
      }
    }

    ## compute posterior p
    clustlike<-matrix(0,xrow,2)
    clustlike[,1]<-log(1-p[coli])+loglike0[[coli]]
    clustlike[,2]<-log(p[coli])+loglike1[[coli]]

    tempmax<-apply(clustlike,1,max)
    for(j in 1:2) {
      clustlike[,j]<-exp(clustlike[,j]-tempmax)
    }
    tempsum<-apply(clustlike,1,sum)

    for(j in 1:2) {
      clustlike[,j]<-clustlike[,j]/tempsum
    }

    p.post[,coli]<-clustlike[,2]
    loglike.final[coli]<-loglike.old
  }


  ## return
  loglike.final<-loglike.final*xrow
  result<-list(p.post=p.post, motif.prior=p, loglike=loglike.final)
}

## Fit the full model
cmfitfull<-function(x, type, tol=1e-3, max.iter=100) {
  ## initialize
  xrow<-nrow(x)
  xcol<-ncol(x)
  loglike0<-list()
  loglike1<-list()
  K<-2^xcol
  p<-rep(1,K)/K
  pattern<-rep(0,xcol)
  patid<-matrix(0,K,xcol)

  ## compute loglikelihood
  for(i in 1:xcol) {
    f0<-type[[i]][[1]]
    f0param<-type[[i]][[2]]
    f1<-type[[i]][[3]]
    f1param<-type[[i]][[4]]
    loglike0[[i]]<-f0(x[,i],f0param)
    loglike1[[i]]<-f1(x[,i],f1param)
  }
  L<-matrix(0,xrow,K)
  for(i in 1:K)
  {
    patid[i,]<-pattern
    for(j in 1:xcol) {
      if(pattern[j] < 0.5) {
        L[,i]<-L[,i]+loglike0[[j]]
      } else {
        L[,i]<-L[,i]+loglike1[[j]]
      }
    }

    if(i < K) {
      pattern[xcol]<-pattern[xcol]+1
      j<-xcol
      while(pattern[j] > 1) {
        pattern[j]<-0
        j<-j-1
        pattern[j]<-pattern[j]+1
      }
    }
  }

  ## EM algorithm to get MLE of p and q
  loglike.old <- -1e10
  for(i.iter in 1:max.iter) {
    if((i.iter%%50) == 0) {
      print(paste("We have run the first ", i.iter, " iterations",sep=""))
    }
    err<-tol+1

    ## compute posterior cluster membership
    clustlike<-matrix(0,xrow,K)
    for(j in 1:K) {
      clustlike[,j]<-log(p[j])+L[,j]
    }

    tempmax<-apply(clustlike,1,max)
    for(j in 1:K) {
      clustlike[,j]<-exp(clustlike[,j]-tempmax)
    }
    tempsum<-apply(clustlike,1,sum)

    ## update motif occurrence rate
    for(j in 1:K) {
      clustlike[,j]<-clustlike[,j]/tempsum
    }

    p.new<-(apply(clustlike,2,sum)+1)/(xrow+K)

    ## evaluate convergence
    err<-max(abs(p.new-p)/p)

    ## evaluate whether the log.likelihood increases
    loglike.new<-(sum(tempmax+log(tempsum))+sum(log(p.new)))/xrow

    loglike.old<-loglike.new
    p<-p.new

    if(err<tol) {
      break;
    }
  }

  ## compute posterior p
  clustlike<-matrix(0,xrow,K)
  for(j in 1:K) {
    clustlike[,j]<-log(p[j])+L[,j]
  }

  tempmax<-apply(clustlike,1,max)
  for(j in 1:K) {
    clustlike[,j]<-exp(clustlike[,j]-tempmax)
  }
  tempsum<-apply(clustlike,1,sum)

  for(j in 1:K) {
    clustlike[,j]<-clustlike[,j]/tempsum
  }

  p.post<-matrix(0,xrow,xcol)
  for(j in 1:K) {
    for(i in 1:xcol) {
      if(patid[j,i] > 0.5) {
        p.post[,i]<-p.post[,i]+clustlike[,j]
      }
    }
  }

  ## return
  #calculate back loglikelihood
  loglike.old<-loglike.old-sum(log(p))/xrow
  loglike.old<-loglike.old*xrow
  result<-list(p.post=p.post, motif.prior=p, loglike=loglike.old)
}

generatetype<-function(limfitted)
{
  jtype<-list()
  df<-limfitted$g1num+limfitted$g2num-2+limfitted$df0
  for(j in 1:limfitted$compnum)
  {
    jtype[[j]]<-list(f0=modt.f0.loglike, f0.param=df[j], f1=modt.f1.loglike, f1.param=c(df[j],limfitted$g1num[j],limfitted$g2num[j],limfitted$v0[j]))
  }
  jtype
}

cormotiffit <- function(exprs, groupid=NULL, compid=NULL, K=1, tol=1e-3,
                        max.iter=100, BIC=TRUE, norm.factor.method="TMM",
                        voom.normalize.method = "none", runtype=c("logCPM","counts","limmafits"), each=3)
{
  # first I want to do some typechecking. Input can be either a normalized
  # matrix, a count matrix, or a list of limma fits. Dispatch the correct
  # limmafit accordingly.
  # todo: add some typechecking here
  limfitted <- list()
  if (runtype=="counts") {
    limfitted <- limmafit.counts(exprs,groupid,compid, norm.factor.method, voom.normalize.method)
  } else if (runtype=="logCPM") {
    limfitted <- limmafit.default(exprs,groupid,compid)
  } else if (runtype=="limmafits") {
    limfitted <- limmafit.list(exprs)
  } else {
    stop("runtype must be one of 'logCPM', 'counts', or 'limmafits'")
  }


  jtype<-generatetype(limfitted)
  fitresult<-list()
  ks <- rep(K, each = each)
  fitresult <- bplapply(1:length(ks), function(i, x, type, ks, tol, max.iter) {
    cmfit.X(x, type, K = ks[i], tol = tol, max.iter = max.iter)
  }, x=limfitted$t, type=jtype, ks=ks, tol=tol, max.iter=max.iter)

  best.fitresults <- list()
  for (i in 1:length(K)) {
    w.k <- which(ks==K[i])
    this.bic <- c()
    for (j in w.k) this.bic[j] <- -2 * fitresult[[j]]$loglike + (K[i] - 1 + K[i] * limfitted$compnum) * log(dim(limfitted$t)[1])
    w.min <- which(this.bic == min(this.bic, na.rm = TRUE))[1]
    best.fitresults[[i]] <- fitresult[[w.min]]
  }
  fitresult <- best.fitresults

  bic <- rep(0, length(K))
  aic <- rep(0, length(K))
  loglike <- rep(0, length(K))
  for (i in 1:length(K)) loglike[i] <- fitresult[[i]]$loglike
  for (i in 1:length(K)) bic[i] <- -2 * fitresult[[i]]$loglike + (K[i] - 1 + K[i] * limfitted$compnum) * log(dim(limfitted$t)[1])
  for (i in 1:length(K)) aic[i] <- -2 * fitresult[[i]]$loglike + 2 * (K[i] - 1 + K[i] * limfitted$compnum)
  if(BIC==TRUE) {
    bestflag=which(bic==min(bic))
  }
  else {
    bestflag=which(aic==min(aic))
  }
  result<-list(bestmotif=fitresult[[bestflag]],bic=cbind(K,bic),
               aic=cbind(K,aic),loglike=cbind(K,loglike), allmotifs=fitresult)

}

cormotiffitall<-function(exprs,groupid,compid, tol=1e-3, max.iter=100)
{
  limfitted<-limmafit(exprs,groupid,compid)
  jtype<-generatetype(limfitted)
  fitresult<-cmfitall(limfitted$t,type=jtype,tol=1e-3,max.iter=max.iter)
}

cormotiffitsep<-function(exprs,groupid,compid, tol=1e-3, max.iter=100)
{
  limfitted<-limmafit(exprs,groupid,compid)
  jtype<-generatetype(limfitted)
  fitresult<-cmfitsep(limfitted$t,type=jtype,tol=1e-3,max.iter=max.iter)
}

cormotiffitfull<-function(exprs,groupid,compid, tol=1e-3, max.iter=100)
{
  limfitted<-limmafit(exprs,groupid,compid)
  jtype<-generatetype(limfitted)
  fitresult<-cmfitfull(limfitted$t,type=jtype,tol=1e-3,max.iter=max.iter)
}

plotIC<-function(fitted_cormotif)
{
  oldpar<-par(mfrow=c(1,2))
  plot(fitted_cormotif$bic[,1], fitted_cormotif$bic[,2], type="b",xlab="Motif Number", ylab="BIC", main="BIC")
  plot(fitted_cormotif$aic[,1], fitted_cormotif$aic[,2], type="b",xlab="Motif Number", ylab="AIC", main="AIC")
}

plotMotif<-function(fitted_cormotif,title="")
{
  layout(matrix(1:2,ncol=2))
  u<-1:dim(fitted_cormotif$bestmotif$motif.q)[2]
  v<-1:dim(fitted_cormotif$bestmotif$motif.q)[1]
  image(u,v,t(fitted_cormotif$bestmotif$motif.q),
        col=gray(seq(from=1,to=0,by=-0.1)),xlab="Study",yaxt = "n",
        ylab="Corr. Motifs",main=paste(title,"pattern",sep=" "))
  axis(2,at=1:length(v))
  for(i in 1:(length(u)+1))
  {
    abline(v=(i-0.5))
  }
  for(i in 1:(length(v)+1))
  {
    abline(h=(i-0.5))
  }
  Ng=10000
  if(is.null(fitted_cormotif$bestmotif$p.post)!=TRUE)
    Ng=nrow(fitted_cormotif$bestmotif$p.post)
  genecount=floor(fitted_cormotif$bestmotif$motif.p*Ng)
  NK=nrow(fitted_cormotif$bestmotif$motif.q)
  plot(0,0.7,pch=".",xlim=c(0,1.2),ylim=c(0.75,NK+0.25),
       frame.plot=FALSE,axes=FALSE,xlab="No. of genes",ylab="", main=paste(title,"frequency",sep=" "))
  segments(0,0.7,fitted_cormotif$bestmotif$motif.p[1],0.7)
  rect(0,1:NK-0.3,fitted_cormotif$bestmotif$motif.p,1:NK+0.3,
       col="dark grey")
  mtext(1:NK,at=1:NK,side=2,cex=0.8)
  text(fitted_cormotif$bestmotif$motif.p+0.15,1:NK,
       labels=floor(fitted_cormotif$bestmotif$motif.p*Ng))
}
drug_pal <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")
pca_plot <-
  function(df,
           col_var = NULL,
           shape_var = NULL,
           title = "") {
    ggplot(df) + geom_point(aes_string(
      x = "PC1",
      y = "PC2",
      color = col_var,
      shape = shape_var
    ),
    size = 5) +
      labs(title = title, x = "PC 1", y = "PC 2") +
      scale_color_manual(values = c(
        "#8B006D",
        "#DF707E",
        "#F1B72B",
        "#3386DD",
        "#707031",
        "#41B333"
      ))
  }
pca_var_plot <- function(pca) {
  # x: class == prcomp
  pca.var <- pca$sdev ^ 2
  pca.prop <- pca.var / sum(pca.var)
  var.plot <-
    qplot(PC, prop, data = data.frame(PC = 1:length(pca.prop),
                                      prop = pca.prop)) +
    labs(title = 'Variance contributed by each PC',
         x = 'PC', y = 'Proportion of variance')
  plot(var.plot)
}

calc_pca <- function(x) {
  # Performs principal components analysis with prcomp
  # x: a sample-by-gene numeric matrix
  prcomp(x, scale. = TRUE, retx = TRUE)
}

get_regr_pval <- function(mod) {
  # Returns the p-value for the Fstatistic of a linear model
  # mod: class lm
  stopifnot(class(mod) == "lm")
  fstat <- summary(mod)$fstatistic
  pval <- 1 - pf(fstat[1], fstat[2], fstat[3])
  return(pval)
}

plot_versus_pc <- function(df, pc_num, fac) {
  # df: data.frame
  # pc_num: numeric, specific PC for plotting
  # fac: column name of df for plotting against PC
  pc_char <- paste0("PC", pc_num)
  # Calculate F-statistic p-value for linear model
  pval <- get_regr_pval(lm(df[, pc_char] ~ df[, fac]))
  if (is.numeric(df[, f])) {
    ggplot(df, aes_string(x = f, y = pc_char)) + geom_point() +
      geom_smooth(method = "lm") + labs(title = sprintf("p-val: %.2f", pval))
  } else {
    ggplot(df, aes_string(x = f, y = pc_char)) + geom_boxplot() +
      labs(title = sprintf("p-val: %.2f", pval))
  }
}
x_axis_labels = function(labels, every_nth = 1, ...) {
  axis(side = 1,
       at = seq_along(labels),
       labels = F)
  text(
    x = (seq_along(labels))[seq_len(every_nth) == 1],
    y = par("usr")[3] - 0.075 * (par("usr")[4] - par("usr")[3]),
    labels = labels[seq_len(every_nth) == 1],
    xpd = TRUE,
    ...
  )
}

Counts summary

Adding analysis of first peak counts into one large file

drug_pal <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")
Ind1_summary  <- read.csv("data/Ind1_summary.txt", row.names = 1) %>% 
  dplyr::rename("sample"=X1,"reads"=X2,"mapped"=X3)%>% 
  mutate(sample=gsub(pattern = "flagstat_first/trimmed_","total_",sample)) %>% 
  mutate(sample= gsub(pattern= "flagstat_noM/trimmed_","nuclear_",sample)) %>% 
  mutate(sample=gsub(pattern = "filt_files/trimmed_","other_",sample)) %>% 
  mutate(sample = gsub("24h","_24h",sample), 
       sample = gsub("3h","_3h",sample)) %>%
  separate(sample, into=c("read_type","indv","trt","time",NA,"neat")) %>% 
  mutate(read_type=if_else(read_type=="other",neat,read_type)) %>% 
  mutate(trt= gsub('75DX','DOX',trt),
         trt= gsub('75E','EPI', trt),
         trt=gsub('75DA','DNR',trt),
         trt=gsub('75M','MTX',trt),
         trt=gsub('75T','TRZ',trt),
         trt=gsub('75V','VEH',trt)) %>% 
  separate(reads,into=c("reads",NA),sep= " ") %>% 
   mutate(reads=as.numeric(reads)) %>% 
  separate(mapped, into= c("mapped_reads", NA,NA, "percent_mapped_reads",NA)) %>% 
  mutate(mapped_reads=as.numeric(mapped_reads)) %>% 
  tidyr::unite("sample",indv:time,sep = "_",remove = FALSE) %>% 
  dplyr::select(sample, indv:time, read_type,reads, mapped_reads)

Ind2_summary  <- read.csv("data/Ind2_summary.txt", row.names = 1)%>% 
dplyr::rename("sample"=X1,"reads"=X2,"mapped"=X3)%>% 
  mutate(sample=gsub(pattern = "flagstat_first/trimmed_","total_",sample)) %>% 
  mutate(sample= gsub(pattern= "flagstat_noM/trimmed_","nuclear_",sample)) %>% 
  mutate(sample=gsub(pattern = "filt_files/trimmed_","other_",sample)) %>% 
  mutate(sample = gsub("24h","_24h",sample), 
       sample = gsub("3h","_3h",sample)) %>%
  separate(sample, into=c("read_type","indv","trt","time",NA,"neat")) %>% 
  mutate(read_type=if_else(read_type=="other",neat,read_type)) %>% 
  mutate(trt= gsub('87DX','DOX',trt),
         trt= gsub('87E','EPI', trt),
         trt=gsub('87DA','DNR',trt),
         trt=gsub('87M','MTX',trt),
         trt=gsub('87T','TRZ',trt),
         trt=gsub('87V','VEH',trt)) %>% 
  separate(reads,into=c("reads",NA),sep= " ") %>% 
   mutate(reads=as.numeric(reads)) %>% 
  separate(mapped, into= c("mapped_reads", NA,NA, "percent_mapped_reads",NA)) %>% 
  mutate(mapped_reads=as.numeric(mapped_reads)) %>% 
  tidyr::unite("sample",indv:time,sep = "_",remove = FALSE) %>% 
  dplyr::select(sample, indv:time, read_type,reads, mapped_reads)

Ind3_summary  <- read.csv("data/Ind3_summary.txt", row.names = 1)%>% 
dplyr::rename("sample"=X1,"reads"=X2,"mapped"=X3)%>% 
  mutate(sample=gsub(pattern = "flagstat_first/trimmed_","total_",sample)) %>% 
  mutate(sample= gsub(pattern= "flagstat_noM/trimmed_","nuclear_",sample)) %>% 
  mutate(sample=gsub(pattern = "filt_files/trimmed_","other_",sample)) %>% 
  mutate(sample = gsub("24h","_24h",sample), 
       sample = gsub("3h","_3h",sample)) %>%
  separate(sample, into=c("read_type","indv","trt","time",NA,"neat")) %>% 
  mutate(read_type=if_else(read_type=="other",neat,read_type)) %>% 
  mutate(trt= gsub('77DX','DOX',trt),
         trt= gsub('77E','EPI', trt),
         trt=gsub('77DA','DNR',trt),
         trt=gsub('77M','MTX',trt),
         trt=gsub('77T','TRZ',trt),
         trt=gsub('77V','VEH',trt)) %>% 
  separate(reads,into=c("reads",NA),sep= " ") %>% 
   mutate(reads=as.numeric(reads)) %>% 
  separate(mapped, into= c("mapped_reads", NA,NA, "percent_mapped_reads",NA)) %>% 
  mutate(mapped_reads=as.numeric(mapped_reads)) %>% 
  tidyr::unite("sample",indv:time,sep = "_",remove = FALSE) %>% 
  dplyr::select(sample, indv:time, read_type,reads, mapped_reads)

Ind6_summary  <- read.csv("data/Ind6_summary.txt", row.names = 1)%>% 
  dplyr::rename("sample"=X1,"reads"=X2,"mapped"=X3)%>% 
  mutate(sample=gsub(pattern = "flagstat_first/trimmed_","total_",sample)) %>% 
  mutate(sample= gsub(pattern= "flagstat_noM/trimmed_","nuclear_",sample)) %>% 
  mutate(sample=gsub(pattern = "filt_files/trimmed_","other_",sample)) %>% 
  mutate(sample = gsub("24h","_24h",sample), 
       sample = gsub("3h","_3h",sample)) %>%
  separate(sample, into=c("read_type","indv","trt","time",NA,"neat")) %>% 
  mutate(read_type=if_else(read_type=="other",neat,read_type)) %>% 
  mutate(trt= gsub('71DX','DOX',trt),
         trt= gsub('71E','EPI', trt),
         trt=gsub('71DA','DNR',trt),
         trt=gsub('71M','MTX',trt),
         trt=gsub('71T','TRZ',trt),
         trt=gsub('71V','VEH',trt)) %>% 
  separate(reads,into=c("reads",NA),sep= " ") %>% 
   mutate(reads=as.numeric(reads)) %>% 
  separate(mapped, into= c("mapped_reads", NA,NA, "percent_mapped_reads",NA)) %>% 
  mutate(mapped_reads=as.numeric(mapped_reads)) %>% 
  tidyr::unite("sample",indv:time,sep = "_",remove = FALSE) %>% 
  dplyr::select(sample, indv:time, read_type,reads, mapped_reads)

Final_four_counts <- Ind1_summary %>% rbind(Ind2_summary) %>% rbind(Ind3_summary) %>% rbind(Ind6_summary)

FF_bound <- Final_four_counts %>% 
  mutate(time=factor(time, levels =c("3h","24h"))) %>% 
  pivot_longer(cols = reads:mapped_reads, names_to="count_type", values_to = "read_num") %>% 
  mutate(read_type=case_when(read_type=="fin"~"unique",.default = read_type)) %>% 
  tidyr::unite("Total_reads",read_type:count_type,sep = "_", remove = TRUE) %>% 
  tidyr::unite("Total_reads_time",Total_reads:time,sep = "_", remove = FALSE) %>% 
  mutate(Total_reads=factor(Total_reads, levels = c("total_reads", "total_mapped_reads","nuclear_reads","nuclear_mapped_reads","unique_reads", "unique_mapped_reads", "nodup_reads", "nodup_mapped_reads"))) %>% 
  dplyr::filter(Total_reads != "nuclear_reads") %>% 
dplyr::filter(Total_reads != "nodup_reads") %>% 
dplyr::filter(Total_reads != "unique_reads") %>% 
  dplyr::mutate(trt=factor(trt, levels = c("DOX", "EPI","DNR", "MTX","TRZ","VEH"))) #%>% 
FF_bound %>% 
  ggplot(., aes(x=Total_reads, y=read_num, group=interaction(indv,time)))+
  geom_col(aes(fill=trt), position="dodge")+
  theme_minimal()+
 scale_fill_manual(values=drug_pal)+
  facet_wrap(~time+trt+indv,nrow = 6, ncol = 12 )+
  theme(strip.text = element_text(face = "bold",  hjust = 0, size = 8),
        strip.background = element_rect(fill = "white", linetype = "solid",
                                        color = "black", linewidth = 1),
        panel.spacing = unit(1, 'points'),
        axis.text.x=element_text(angle = 90, vjust = 0.5, hjust=1))

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
FF_bound %>% 
 ggplot(., aes(x=Total_reads, y=read_num, col=indv, group=interaction(time,Total_reads_time)))+
  geom_boxplot(aes(fill=trt)) + 
  geom_point(aes(col=indv))+
   theme_bw()+
  facet_wrap(~trt+time,nrow = 3, ncol = 6 )+
 scale_fill_manual(values=drug_pal)+
  theme(strip.text = element_text(face = "bold",  hjust = 0, size = 8),
        strip.background = element_rect(fill = "white", linetype = "solid",
                                        color = "black", linewidth = 1),
        panel.spacing = unit(1, 'points'),
        axis.text.x=element_text(angle = 90, vjust = 0.5, hjust=1))

Final_four_counts %>% 
  mutate(time=factor(time, levels =c("3h","24h"))) %>% 
pivot_longer(cols = reads:mapped_reads, names_to="count_type", values_to = "read_num") %>% 
  dplyr::filter(read_type=="nodup"& count_type=="mapped_reads") %>% 
  mutate(trt= factor(trt, levels = c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes(x=trt,y=read_num,group=(interaction(time,trt))))+
  geom_boxplot(aes(fill=trt))+
   geom_point(aes(col=indv, size =3))+
  # geom_hline(yintercept = 20)+
  facet_wrap(time~.)+
   scale_fill_manual(values=drug_pal)+
  scale_color_brewer(palette = "Dark2")+
  ggtitle("Reads across treatment and time")+
  theme_bw()+
  theme(strip.text = element_text(face = "bold",  hjust = .5, size = 8),
        strip.background = element_rect(fill = "white", linetype = "solid",
                                        color = "black", linewidth = 1),
        panel.spacing = unit(1, 'points'))

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
allTSSE <- readRDS( "data/all_TSSE_scores.RDS")
allTSSE %>% as.data.frame() %>% 
  rownames_to_column("sample") %>% 
  separate(sample, into = c("indv","trt","time"), sep= "_") %>%
  mutate(trt= factor(trt, levels = c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  mutate(time = factor(time, levels = c("3","24"),labels = c("3 hours","24 hours"))) %>% 
  dplyr::filter(indv !=4 &indv !=5) %>% 
  ggplot(., aes(x= time, y= V1, group = indv))+

   # geom_col(position= "dodge",aes(fill=trt)) %>%
  geom_jitter(aes(col = trt, size = 1.5, alpha = 0.5) ,  position=position_jitter(0.25))+
  geom_hline(yintercept=5, linetype = 3)+
    geom_hline(yintercept=7, col = "blue")+
  facet_wrap(~indv)+
   theme_bw()+
  ggtitle("TSS enrichment scores")+
   scale_color_manual(values=drug_pal)+
   theme(strip.text = element_text(face = "bold",  hjust = .5, size = 8),
        strip.background = element_rect(fill = "white", linetype = "solid",
                                        color = "black", linewidth = 1))

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
Ind1_TSS_peaks_plot <- readRDS("data/Ind1_TSS_peaks.RDS")
Ind2_TSS_peaks_plot <- readRDS("data/Ind2_TSS_peaks.RDS")
Ind3_TSS_peaks_plot <- readRDS("data/Ind3_TSS_peaks.RDS")
Ind6_TSS_peaks_plot <- readRDS("data/Ind6_TSS_peaks.RDS")

a1<- plotAvgProf(Ind1_TSS_peaks_plot[c(1,3,5,7,9,11)], xlim=c(-3000, 3000), ylab = "Count Frequency")+ ggtitle("3 hour Individual 1" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:08:47 AM 
b1 <- plotAvgProf(Ind2_TSS_peaks_plot[c(1,3,5,7,9,11)], xlim=c(-3000, 3000), ylab = "Count Frequency")+ ggtitle("3 hour Individual 2" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:09:01 AM 
c1 <- plotAvgProf(Ind3_TSS_peaks_plot[c(1,3,5,7,9,11)], xlim=c(-3000, 3000), ylab = "Count Frequency")+ ggtitle("3 hour Individual 3" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:09:11 AM 
d1 <- plotAvgProf(Ind6_TSS_peaks_plot[c(1,3,5,7,9,11)], xlim=c(-3000, 3000), ylab = "Count Frequency")+ ggtitle("3 hour Individual 6" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:09:16 AM 
a2 <- plotAvgProf(Ind1_TSS_peaks_plot[c(2,4,6,8,10,12)], xlim=c(-3000, 3000),ylab = "Count Frequency")+ ggtitle("24 hour Individual 1" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:09:16 AM 
b2 <- plotAvgProf(Ind2_TSS_peaks_plot[c(2,4,6,8,10,12)], xlim=c(-3000, 3000),ylab = "Count Frequency")+ ggtitle("24 hour Individual 2" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:09:34 AM 
c2 <- plotAvgProf(Ind3_TSS_peaks_plot[c(2,4,6,8,10,12)], xlim=c(-3000, 3000),ylab = "Count Frequency")+ ggtitle("24 hour Individual 3" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:10:05 AM 
d2 <- plotAvgProf(Ind6_TSS_peaks_plot[c(2,4,6,8,10,12)], xlim=c(-3000, 3000),ylab = "Count Frequency")+ ggtitle("24 hour Individual 6" )+coord_cartesian(xlim=c(-2000,2000))
>> plotting figure...            2024-09-27 11:10:20 AM 
plot_grid(a1,a2, b1,b2,c1,c2,d1,d2, axis="l",align = "hv",nrow=4, ncol=2)

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
# a2+coord_cartesian(xlim=c(-2000,2000))

Analysis using the four selected individuals and the peaks generated from just that set:

# Peaks_bed_file <- rtracklayer::BEDFile("~/ATAC_downloads/finalfour_bl_filt_peaks.bed")
# import(Peaks_bed_file) %>% as.data.frame()
# write_delim(all_four_filt_counts, "data/all_four_filtered_counts.txt",delim = "\t")
  all_four_filt_counts <- read.delim("data/all_four_filtered_counts.txt",header=TRUE) %>% 
  GRanges() %>% 
  keepStandardChromosomes(pruning.mode = "coarse") %>% 
  as.data.frame()
# all_four_filt_counts %>% 
names(all_four_filt_counts) = gsub(pattern = "_S.*", replacement = "", x = names(all_four_filt_counts))

names(all_four_filt_counts) = gsub(pattern = "ind1.trimmed.filt_files.trimmed_|ind2.trimmed.filt_files.trimmed_|ind3.trimmed.filt_files.trimmed_|ind6.trimmed.filt_files.trimmed_", replacement = "", x = names(all_four_filt_counts))


names(all_four_filt_counts) = gsub(pattern = "ind1.trimmed.filt_files.trimmed_|ind2.trimmed.filt_files.trimmed_|ind3.trimmed.filt_files.trimmed_|ind6.trimmed.filt_files.trimmed_", replacement = "", x = names(all_four_filt_counts))

summary of peaks by sample

##check commandline file for how I pulled the wc -l on all files.
# peakcount_new4 <- read_delim("~/ATAC_downloads/master_peaks/4_indonly/peakcount_new4.txt", 
# +     delim = "\t", escape_double = FALSE, 
# # +     col_names = FALSE, trim_ws = TRUE)
# peakcount_new4 %>% 
#   mutate(X1=gsub("75","1_", X1), 
#          X1=gsub("87","2_",X1),
#          X1=gsub("77","3_",X1),
#          X1=gsub("71","6_",X1),
#          X1=gsub("DA","DNR_",X1),
#          X1=gsub("DX","DOX_",X1),
#          X1=gsub("E","EPI_",X1),
#          X1=gsub("T","TRZ_",X1),
#          X1=gsub("M","MTX_",X1),
#          X1=gsub("V","VEH_",X1)) %>% 
#   separate(X1, into=c("indv","trt","time"), sep= "_", remove = TRUE) %>% 
#   dplyr::rename(peak_number=X2) %>% write_delim(., file="data/Final_four_data/Peak_count_ff.txt", delim = "\t")

peakcount_ff <- read_delim("data/Final_four_data/Peak_count_ff.txt",delim= "\t")

peakcount_ff %>% 
  mutate(time = factor(time, levels = c("3h", "24h"), labels= c("3 hours","24 hours"))) %>% 
  mutate(trt = factor(trt, levels = c("DOX","EPI", "DNR", "MTX", "TRZ", "VEH"))) %>% 
  mutate(indv=factor(indv)) %>% 
   ggplot(., aes(x=trt,y=peak_number))+
  geom_boxplot(aes(fill=trt))+
   geom_point(aes(col=indv, size =3))+
  facet_wrap(time~.)+
     scale_fill_manual(values=drug_pal)+
  scale_color_brewer(palette = "Dark2")+
  ggtitle("Peak number across treatment and time")+
  theme_bw()+
  theme(strip.text = element_text(face = "bold",  hjust = .5, size = 8),
        strip.background = element_rect(fill = "white", linetype = "solid",
                                        color = "black", linewidth = 1),
        panel.spacing = unit(1, 'points'))

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
PCA4mat <- all_four_filt_counts%>% 
  dplyr::select(Geneid,Ind1_75DA24h:Ind6_71V3h) %>% 
  column_to_rownames("Geneid") %>% 
  as.matrix()

annotation_mat <- data.frame(timeset=colnames(PCA4mat)) %>% 
  mutate(sample = timeset) %>% 
  mutate(timeset=gsub("Ind1_75","1_",timeset)) %>% 
  mutate(timeset=gsub("Ind2_87","2_",timeset)) %>% 
  mutate(timeset=gsub("Ind3_77","3_",timeset)) %>% 
  mutate(timeset=gsub("Ind6_71","4_",timeset)) %>% 
  mutate(timeset = gsub("24h","_24h",timeset), 
       timeset = gsub("3h","_3h",timeset)) %>%
  separate(timeset, into = c("indv","trt","time"), sep= "_") %>% 
  mutate(trt= case_match(trt, 'DX' ~'DOX', 'E'~'EPI', 'DA'~'DNR', 'M'~'MTX', 'T'~'TRZ', 'V'~'VEH',.default = trt)) %>% 
  # mutate(indv = factor(indv, levels = c("1", "2", "3", "4", "5", "6"))) %>%
  mutate(time = factor(time, levels = c("3h", "24h"), labels= c("3 hours","24 hours"))) %>% 
  mutate(trt = factor(trt, levels = c("DOX","EPI", "DNR", "MTX", "TRZ", "VEH"))) 

PCA4_info <- (prcomp(t(PCA4mat), scale. = TRUE)) 
PCA4_info_anno <- PCA4_info$x %>% cbind(.,annotation_mat)
# autoplot(PCA_info)
summary(PCA4_info)
Importance of components:
                            PC1      PC2       PC3      PC4      PC5      PC6
Standard deviation     271.5384 161.7523 112.03795 84.95590 77.83204 62.46278
Proportion of Variance   0.4275   0.1517   0.07278  0.04185  0.03512  0.02262
Cumulative Proportion    0.4275   0.5792   0.65195  0.69380  0.72892  0.75154
                            PC7      PC8      PC9     PC10    PC11     PC12
Standard deviation     55.50414 54.53991 50.63479 45.33378 42.9600 39.54174
Proportion of Variance  0.01786  0.01725  0.01486  0.01192  0.0107  0.00907
Cumulative Proportion   0.76940  0.78665  0.80151  0.81343  0.8241  0.83319
                           PC13     PC14     PC15     PC16     PC17     PC18
Standard deviation     38.68789 36.80855 36.40024 34.44726 33.89483 33.36466
Proportion of Variance  0.00868  0.00786  0.00768  0.00688  0.00666  0.00645
Cumulative Proportion   0.84187  0.84973  0.85741  0.86429  0.87095  0.87740
                           PC19     PC20     PC21     PC22     PC23     PC24
Standard deviation     33.17758 32.55288 31.31915 30.91018 30.44559 30.19378
Proportion of Variance  0.00638  0.00614  0.00569  0.00554  0.00537  0.00529
Cumulative Proportion   0.88378  0.88993  0.89561  0.90115  0.90653  0.91181
                           PC25    PC26     PC27     PC28     PC29     PC30
Standard deviation     29.89402 29.6621 29.11618 28.74140 28.45611 28.05219
Proportion of Variance  0.00518  0.0051  0.00492  0.00479  0.00469  0.00456
Cumulative Proportion   0.91699  0.9221  0.92701  0.93180  0.93649  0.94106
                           PC31     PC32     PC33     PC34    PC35     PC36
Standard deviation     27.94847 27.87613 27.64626 27.04068 25.9458 25.73918
Proportion of Variance  0.00453  0.00451  0.00443  0.00424  0.0039  0.00384
Cumulative Proportion   0.94559  0.95009  0.95452  0.95876  0.9627  0.96651
                           PC37    PC38     PC39    PC40     PC41     PC42
Standard deviation     25.52298 25.2772 24.66403 24.2316 23.83300 23.52718
Proportion of Variance  0.00378  0.0037  0.00353  0.0034  0.00329  0.00321
Cumulative Proportion   0.97028  0.9740  0.97751  0.9809  0.98421  0.98742
                           PC43     PC44     PC45    PC46     PC47      PC48
Standard deviation     22.30846 21.65858 21.00631 20.3327 18.66373 5.022e-13
Proportion of Variance  0.00289  0.00272  0.00256  0.0024  0.00202 0.000e+00
Cumulative Proportion   0.99031  0.99303  0.99558  0.9980  1.00000 1.000e+00
# %>% as.tibble() %>% 
#   kable(., caption = "Summary of PCA variables") %>% 
#   kable_paper("striped", full_width = TRUE) %>%
#   kable_styling(full_width = FALSE, font_size = 16) %>%
#   scroll_box(height = "500px")
# cpm(PCAmat, log=TRUE)
pca_plot(PCA4_info_anno, col_var='trt', shape_var = 'time')+ggtitle("PCA of raw counts by time and treatment")

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
pca_plot(PCA4_info_anno, col_var='trt', shape_var = 'indv')+ggtitle("PCA of raw counts by treatment and individual")

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
FCmatrix_full <-   PCA4mat %>%
  as.data.frame() %>% 
  rename_with(.,~gsub(pattern = "Ind1_75", replacement = "1_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind2_87", replacement = "2_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind3_77", replacement = "3_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind6_71", replacement = "6_",.)) %>% 
  rename_with(.,~gsub( "DX" ,'DOX',.)) %>% 
  rename_with(.,~gsub( "DA" ,'DNR',.)) %>% 
  rename_with(.,~gsub( "E" ,'EPI',.)) %>% 
  rename_with(.,~gsub( "T" ,'TRZ',.)) %>%
  rename_with(.,~gsub( "M" ,'MTX',.)) %>% 
  rename_with(.,~gsub( "V" ,'VEH',.)) %>% 
  rename_with(.,~gsub("24h","_24h",.)) %>% 
  rename_with(.,~gsub("3h","_3h",.)) %>% 
  as.matrix() %>% 
  cpm(., log = TRUE) %>% 
  cor()

filmat_groupmat_col <- data.frame(timeset = colnames(FCmatrix_full))

counts_corr_mat <-filmat_groupmat_col %>%
  # mutate(sample = timeset) %>% 
  separate(timeset, into = c("indv","trt","time"), sep= "_") %>% 
  mutate(class = if_else(trt == "DNR", "AC", 
                         if_else(trt == "DOX", "AC", 
                                 if_else(trt == "EPI", "AC", "nAC")))) %>%
  mutate(TOP2i = if_else(trt == "DNR", "yes", 
                         if_else(trt == "DOX", "yes", 
                                 if_else(trt == "EPI", "yes", 
                                         if_else(trt == "MTX", "yes", "no"))))) 

                         
 mat_colors <- list( 
   trt= c("#F1B72B","#8B006D","#DF707E","#3386DD","#707031","#41B333"),
   indv=c("#1B9E77", "#D95F02" ,"#7570B3", "#E6AB02"),
   time=c("pink", "chocolate4"),
   class=c("yellow1","darkorange1"), 
   TOP2i =c("darkgreen","lightgreen"))                        
                         
names(mat_colors$trt)   <- unique(counts_corr_mat$trt)                      
names(mat_colors$indv) <- unique(counts_corr_mat$indv)
names(mat_colors$time) <- unique(counts_corr_mat$time)
names(mat_colors$class) <- unique(counts_corr_mat$class)
names(mat_colors$TOP2i) <- unique(counts_corr_mat$TOP2i)

# mat_col_ff <-
#   data.frame(
#     time = c(rep("3 hours", 5), rep("24 hours", 5)),
#     class = (c(
#       "AC", "AC", "AC", "nAC","nAC",  "AC", "AC", "AC", "nAC","nAC" 
#     )))
# rownames(mat_col_ff) <- colnames(FCmatrix_ff)
# 
# mat_colors_ff <-
#   list(
#     time = c("pink", "chocolate4"),
    # class = c("yellow1", "lightgreen"))

# names(mat_colors_ff$time) <- unique(mat_col_ff$time)
# names(mat_colors_ff$class) <- unique(mat_col_ff$class)
# names(mat_colors_FC$TOP2i) <- unique(mat_col_FC$TOP2i)
# corrFC_ff <- cor(FCmatrix_ff)

htanno_full <-  HeatmapAnnotation(df = counts_corr_mat, col = mat_colors)
Heatmap(FCmatrix_full, top_annotation = htanno_full)

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
# saveRDS(x4_filtered,"data/Final_four_data/ATAC_filtered_raw_counts_allsamples.RDS")
l4cpm <- cpm(PCA4mat, log=TRUE)  ### for determining the basic cutoffs
dim(l4cpm)
[1] 172481     48
row_means <- rowMeans(l4cpm)
x4_filtered <- PCA4mat[row_means > 0,]
dim(x4_filtered)
[1] 155560     48
filt4_matrix_lcpm <- cpm(x4_filtered, log=TRUE)

PCA4_info_filter <- (prcomp(t(filt4_matrix_lcpm), scale. = TRUE))
summary(PCA4_info_filter)
Importance of components:
                            PC1      PC2       PC3      PC4      PC5      PC6
Standard deviation     186.3046 145.1035 112.75334 92.12518 74.32713 65.16030
Proportion of Variance   0.2231   0.1353   0.08173  0.05456  0.03551  0.02729
Cumulative Proportion    0.2231   0.3585   0.44020  0.49476  0.53027  0.55757
                            PC7      PC8      PC9     PC10     PC11     PC12
Standard deviation     62.14680 60.93584 55.20555 51.92794 50.29842 49.63894
Proportion of Variance  0.02483  0.02387  0.01959  0.01733  0.01626  0.01584
Cumulative Proportion   0.58240  0.60627  0.62586  0.64319  0.65945  0.67529
                           PC13     PC14     PC15     PC16     PC17     PC18
Standard deviation     48.89413 46.56074 46.04427 45.27015 44.82248 44.19051
Proportion of Variance  0.01537  0.01394  0.01363  0.01317  0.01291  0.01255
Cumulative Proportion   0.69066  0.70460  0.71823  0.73140  0.74432  0.75687
                           PC19     PC20     PC21     PC22    PC23     PC24
Standard deviation     43.76294 43.54048 42.37991 41.72939 41.5544 40.40543
Proportion of Variance  0.01231  0.01219  0.01155  0.01119  0.0111  0.01049
Cumulative Proportion   0.76918  0.78137  0.79291  0.80411  0.8152  0.82570
                           PC25    PC26     PC27     PC28     PC29     PC30
Standard deviation     40.23834 39.2449 38.71749 38.53516 37.86304 37.48148
Proportion of Variance  0.01041  0.0099  0.00964  0.00955  0.00922  0.00903
Cumulative Proportion   0.83611  0.8460  0.85565  0.86519  0.87441  0.88344
                           PC31     PC32     PC33     PC34     PC35    PC36
Standard deviation     36.76445 36.31594 36.08503 35.40489 35.25544 34.3866
Proportion of Variance  0.00869  0.00848  0.00837  0.00806  0.00799  0.0076
Cumulative Proportion   0.89213  0.90061  0.90898  0.91704  0.92503  0.9326
                          PC37     PC38     PC39     PC40     PC41     PC42
Standard deviation     33.9352 33.63268 33.55568 32.46461 31.17213 31.02734
Proportion of Variance  0.0074  0.00727  0.00724  0.00678  0.00625  0.00619
Cumulative Proportion   0.9400  0.94730  0.95454  0.96132  0.96756  0.97375
                           PC43     PC44     PC45     PC46     PC47      PC48
Standard deviation     30.58641 30.12543 28.78207 27.44490 25.66405 7.625e-13
Proportion of Variance  0.00601  0.00583  0.00533  0.00484  0.00423 0.000e+00
Cumulative Proportion   0.97976  0.98560  0.99092  0.99577  1.00000 1.000e+00
pca_var_plot(PCA4_info_filter)

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
pca_final_four <- calc_pca(t(filt4_matrix_lcpm))
pca_final_four_anno <- data.frame(annotation_mat, pca_final_four$x)


pca_final_four_anno %>%
  ggplot(.,aes(x = PC1, y = PC2, col=trt, shape=time, group=indv))+
  geom_point(size= 5)+
  scale_color_manual(values=drug_pal)+
   ggrepel::geom_text_repel(aes(label = indv))+
   ggtitle(expression("PCA of log"[2]*"(cpm) new filtered peak set"))+
  theme_bw()+
  guides(col="none", size =4)+
  labs(y = "PC 2 (13.5%)", x ="PC 1 (22.3%)")+
  theme(plot.title=element_text(size= 14,hjust = 0.5),
        axis.title = element_text(size = 12, color = "black"))

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23
pca_final_four_anno %>%
  ggplot(.,aes(x = PC3, y = PC4, col=trt, shape=time, group=indv))+
  geom_point(size= 5)+
  scale_color_manual(values=drug_pal)+
   ggrepel::geom_text_repel(aes(label = indv))+
   ggtitle(expression("PCA of log"[2]*"(cpm) new filtered peak set"))+
  theme_bw()+
  guides(col="none", size =4)+
  labs(y = "PC 4 (5.5%)", x ="PC 3 (8.2%)")+
  theme(plot.title=element_text(size= 14,hjust = 0.5),
        axis.title = element_text(size = 12, color = "black"))

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

We go from 172481, 48 peaks to155560, 48 peaks using rowMeans (log2cpm) > 0 across all samples. This is my new high confidence peak set.

Contribution of variance in PCA plot

pending code

facs <- c("indv", "trt", "time")
names(facs) <- c("Individual", "Treatment", "Time")
drug1 <- c("DOX","EPI", "DNR", "MTX", "TRZ", "VEH")##for changing shapes and colors
time <- rep(c("24h", "3h"),24) %>% factor(., levels = c("3h","24h"))
##gglistmaking
for (f in facs) {
  # PC1 v PC2
  pca_plot(pca_final_four_anno, col_var = f, shape_var = time,
           title = names(facs)[which(facs == f)])
  print(last_plot())
  
  # Plot f versus PC1 and PC2
  f_v_pc1 <- arrangeGrob(plot_versus_pc(pca_final_four_anno, 1, f))
  f_v_pc2 <- arrangeGrob(plot_versus_pc(pca_final_four_anno, 2, f))
  grid.arrange(f_v_pc1, f_v_pc2, ncol = 2, top = names(facs)[which(facs == f)])
  # summary(plot_versus_pc(PCA_info_anno_all, 1, f))
  # summary(plot_versus_pc(PCA_info_anno_all, 2, f))
 
}

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

Comparing the peak sets, old and new

# write_delim((filt4_matrix_lcpm %>% as.data.frame(.) %>% rownames_to_column("Peakid")), "data/Final_four_data/LCPM_matrix_ff.txt", delim = "/t")
# filt4_matrix_lcpm <- read_delim("data/Final_four_data/LCPM_matrix_ff.txt",delim = "/t")
new_peaks_list <- row.names(filt4_matrix_lcpm) %>% as.data.frame() %>% separate_wider_delim(., cols =.,names=c("chr","start","end"), delim = ".", cols_remove = FALSE) %>%  dplyr::rename("peakid"=4)
new_peaks_gr <- new_peaks_list %>%
  dplyr::filter(chr!="chrY") %>% 
  GRanges() %>% 
   keepStandardChromosomes(., pruning.mode = "coarse")
stdChr <- standardChromosomes(new_peaks_gr)
peakAnnoList_n45_motif <- readRDS("data/peakAnnoList_n45_motif.RDS")
old_peaks_gr <- peakAnnoList_n45_motif$background_gr %>% as.GRanges()
old_peaks_gr$old_width <- width(old_peaks_gr)
a <- join_overlap_intersect(old_peaks_gr,new_peaks_gr,suffix = c("old", "new")) %>% 
  as.data.frame()

a %>% 
  dplyr::select(id,peakid,width,old_width) %>% 
  mutate(check =if_else(id ==peakid, "TRUE","FALSE")) %>% 
  dplyr::filter(check=="FALSE") %>% 
  mutate(width_ol=if_else(width<old_width,"smaller","larger")) %>%
  dplyr::rename("old_peak_id"="id","new_peak_id"="peakid") %>% 
  mutate(diff= old_width - width) %>% 
  kable(., caption = "summary of newpeaks that are smaller than old peaks, but are basically the same") %>% 
  kable_paper("striped", full_width = FALSE) %>%
  kable_styling(
    full_width = FALSE,
    position = "left",
    bootstrap_options = c("striped", "hover")
  ) %>%
  scroll_box(width = "100%", height = "400px")
summary of newpeaks that are smaller than old peaks, but are basically the same
old_peak_id new_peak_id width old_width check width_ol diff
chr1.821114.821933 chr1.821114.821757 644 821 FALSE smaller 177
chr1.920872.921848 chr1.920872.921733 862 978 FALSE smaller 116
chr1.960487.960762 chr1.960492.960762 271 277 FALSE smaller 6
chr1.961274.961788 chr1.961274.961703 430 516 FALSE smaller 86
chr1.979643.980042 chr1.979686.980042 357 401 FALSE smaller 44
chr1.1024609.1025381 chr1.1024609.1025344 736 774 FALSE smaller 38
chr1.1041893.1042721 chr1.1042221.1042721 501 830 FALSE smaller 329
chr1.1043580.1044925 chr1.1044510.1044915 406 1347 FALSE smaller 941
chr1.1050736.1051096 chr1.1050794.1051096 303 362 FALSE smaller 59
chr1.1069144.1069685 chr1.1069144.1069625 482 543 FALSE smaller 61
chr1.1122855.1123919 chr1.1122855.1123828 974 1066 FALSE smaller 92
chr1.1217896.1218634 chr1.1217914.1218541 628 740 FALSE smaller 112
chr1.1231222.1233459 chr1.1231222.1232501 1280 2239 FALSE smaller 959
chr1.1307178.1309120 chr1.1307243.1309120 1878 1944 FALSE smaller 66
chr1.1340102.1340799 chr1.1340206.1340601 396 699 FALSE smaller 303
chr1.1371553.1372351 chr1.1371659.1372320 662 800 FALSE smaller 138
chr1.1381391.1382163 chr1.1381510.1382163 654 774 FALSE smaller 120
chr1.1406544.1408158 chr1.1406544.1408077 1534 1616 FALSE smaller 82
chr1.1419289.1421201 chr1.1419325.1419926 602 1914 FALSE smaller 1312
chr1.1435372.1435871 chr1.1435372.1435855 484 501 FALSE smaller 17
chr1.1479180.1480142 chr1.1479180.1480138 959 964 FALSE smaller 5
chr1.1535413.1536277 chr1.1535548.1536277 730 866 FALSE smaller 136
chr1.1537637.1538609 chr1.1537912.1538606 695 974 FALSE smaller 279
chr1.1550814.1551382 chr1.1550842.1551382 541 570 FALSE smaller 29
chr1.1554541.1555602 chr1.1554541.1555504 964 1063 FALSE smaller 99
chr1.1628373.1629316 chr1.1628477.1629294 818 945 FALSE smaller 127
chr1.1678292.1679123 chr1.1678465.1679123 659 833 FALSE smaller 174
chr1.1760355.1760840 chr1.1760576.1760840 265 487 FALSE smaller 222
chr1.1796812.1797274 chr1.1796843.1797274 432 464 FALSE smaller 32
chr1.1826457.1827708 chr1.1827153.1827708 556 1253 FALSE smaller 697
chr1.1873128.1873487 chr1.1873139.1873487 349 361 FALSE smaller 12
chr1.1890818.1892543 chr1.1890818.1892336 1519 1727 FALSE smaller 208
chr1.1980529.1981576 chr1.1980529.1981342 814 1049 FALSE smaller 235
chr1.2046528.2046745 chr1.2046542.2046745 204 219 FALSE smaller 15
chr1.2099928.2100159 chr1.2099966.2100135 170 233 FALSE smaller 63
chr1.2108390.2108851 chr1.2108429.2108851 423 463 FALSE smaller 40
chr1.2121076.2121580 chr1.2121078.2121580 503 506 FALSE smaller 3
chr1.2179778.2180788 chr1.2179979.2180774 796 1012 FALSE smaller 216
chr1.2190449.2190881 chr1.2190487.2190881 395 434 FALSE smaller 39
chr1.2204323.2204898 chr1.2204373.2204898 526 577 FALSE smaller 51
chr1.2214027.2214452 chr1.2214131.2214452 322 427 FALSE smaller 105
chr1.2228943.2229752 chr1.2228949.2229752 804 811 FALSE smaller 7
chr1.2240101.2240543 chr1.2240101.2240542 442 444 FALSE smaller 2
chr1.2279018.2279871 chr1.2279093.2279871 779 855 FALSE smaller 76
chr1.2446428.2446977 chr1.2446428.2446954 527 551 FALSE smaller 24
chr1.2925986.2926908 chr1.2925986.2926863 878 924 FALSE smaller 46
chr1.3066258.3066748 chr1.3066258.3066708 451 492 FALSE smaller 41
chr1.3114717.3115158 chr1.3114790.3115158 369 443 FALSE smaller 74
chr1.3146492.3147615 chr1.3146983.3147615 633 1125 FALSE smaller 492
chr1.3164665.3165177 chr1.3164665.3165159 495 514 FALSE smaller 19
chr1.3378423.3378961 chr1.3378423.3378869 447 540 FALSE smaller 93
chr1.3410241.3411141 chr1.3410241.3410926 686 902 FALSE smaller 216
chr1.3424432.3425134 chr1.3424432.3425112 681 704 FALSE smaller 23
chr1.3471185.3472241 chr1.3471265.3472241 977 1058 FALSE smaller 81
chr1.3491182.3492583 chr1.3491182.3492272 1091 1403 FALSE smaller 312
chr1.3491182.3492583 chr1.3492412.3492583 172 1403 FALSE smaller 1231
chr1.3501475.3502639 chr1.3501476.3501969 494 1166 FALSE smaller 672
chr1.3501475.3502639 chr1.3502068.3502639 572 1166 FALSE smaller 594
chr1.3529400.3529760 chr1.3529405.3529760 356 362 FALSE smaller 6
chr1.3565863.3566425 chr1.3565983.3566425 443 564 FALSE smaller 121
chr1.3567806.3568284 chr1.3567877.3568284 408 480 FALSE smaller 72
chr1.3585881.3586600 chr1.3586032.3586600 569 721 FALSE smaller 152
chr1.3611029.3612045 chr1.3611051.3612045 995 1018 FALSE smaller 23
chr1.3613317.3614136 chr1.3613317.3614126 810 821 FALSE smaller 11
chr1.3625399.3625896 chr1.3625399.3625845 447 499 FALSE smaller 52
chr1.3643071.3643436 chr1.3643071.3643418 348 367 FALSE smaller 19
chr1.3649839.3650242 chr1.3649859.3650242 384 405 FALSE smaller 21
chr1.3713083.3713447 chr1.3713084.3713447 364 366 FALSE smaller 2
chr1.4650520.4650770 chr1.4650520.4650755 236 252 FALSE smaller 16
chr1.4654752.4656772 chr1.4655205.4656772 1568 2022 FALSE smaller 454
chr1.4710778.4711657 chr1.4710778.4711524 747 881 FALSE smaller 134
chr1.4749935.4750589 chr1.4750133.4750589 457 656 FALSE smaller 199
chr1.5423150.5423597 chr1.5423163.5423597 435 449 FALSE smaller 14
chr1.5551482.5552010 chr1.5551482.5551970 489 530 FALSE smaller 41
chr1.5652075.5652782 chr1.5652105.5652782 678 709 FALSE smaller 31
chr1.5757923.5758663 chr1.5757962.5758663 702 742 FALSE smaller 40
chr1.6016163.6016620 chr1.6016177.6016620 444 459 FALSE smaller 15
chr1.6206990.6208123 chr1.6206990.6208117 1128 1135 FALSE smaller 7
chr1.6235407.6236232 chr1.6235707.6236232 526 827 FALSE smaller 301
chr1.6238710.6239018 chr1.6238721.6239018 298 310 FALSE smaller 12
chr1.6290089.6290453 chr1.6290089.6290438 350 366 FALSE smaller 16
chr1.6453884.6455160 chr1.6453904.6454980 1077 1278 FALSE smaller 201
chr1.6455309.6455871 chr1.6455500.6455871 372 564 FALSE smaller 192
chr1.6489915.6491040 chr1.6489915.6491017 1103 1127 FALSE smaller 24
chr1.6519972.6520623 chr1.6520072.6520623 552 653 FALSE smaller 101
chr1.6612747.6614536 chr1.6612747.6614525 1779 1791 FALSE smaller 12
chr1.6666804.6667424 chr1.6666804.6667023 220 622 FALSE smaller 402
chr1.6770647.6771281 chr1.6770655.6771281 627 636 FALSE smaller 9
chr1.6904594.6905061 chr1.6904594.6905029 436 469 FALSE smaller 33
chr1.6925805.6926224 chr1.6925805.6926158 354 421 FALSE smaller 67
chr1.6962028.6963541 chr1.6962149.6963541 1393 1515 FALSE smaller 122
chr1.7075459.7075727 chr1.7075463.7075727 265 270 FALSE smaller 5
chr1.7207782.7208486 chr1.7207782.7208352 571 706 FALSE smaller 135
chr1.7438711.7439094 chr1.7438777.7439094 318 385 FALSE smaller 67
chr1.7500578.7501069 chr1.7500713.7501069 357 493 FALSE smaller 136
chr1.7526392.7526950 chr1.7526394.7526950 557 560 FALSE smaller 3
chr1.7539588.7540154 chr1.7539588.7540093 506 568 FALSE smaller 62
chr1.7614248.7615016 chr1.7614248.7614910 663 770 FALSE smaller 107
chr1.7704462.7704924 chr1.7704512.7704924 413 464 FALSE smaller 51
chr1.7719629.7719973 chr1.7719660.7719967 308 346 FALSE smaller 38
chr1.7990049.7990678 chr1.7990106.7990678 573 631 FALSE smaller 58
chr1.8025629.8027358 chr1.8025629.8027230 1602 1731 FALSE smaller 129
chr1.8062015.8062412 chr1.8062015.8062372 358 399 FALSE smaller 41
chr1.8202264.8202849 chr1.8202264.8202839 576 587 FALSE smaller 11
chr1.8276278.8276855 chr1.8276278.8276811 534 579 FALSE smaller 45
chr1.8317889.8318454 chr1.8317983.8318454 472 567 FALSE smaller 95
chr1.8357465.8358203 chr1.8357465.8358089 625 740 FALSE smaller 115
chr1.8367445.8368077 chr1.8367456.8368077 622 634 FALSE smaller 12
chr1.8371025.8371725 chr1.8371027.8371725 699 702 FALSE smaller 3
chr1.8373527.8374304 chr1.8373542.8374304 763 779 FALSE smaller 16
chr1.8402136.8402645 chr1.8402144.8402645 502 511 FALSE smaller 9
chr1.8409093.8409572 chr1.8409093.8409465 373 481 FALSE smaller 108
chr1.8447733.8448153 chr1.8447781.8448153 373 422 FALSE smaller 49
chr1.8464406.8464711 chr1.8464406.8464704 299 307 FALSE smaller 8
chr1.8552777.8553506 chr1.8552816.8553506 691 731 FALSE smaller 40
chr1.8816891.8818206 chr1.8816900.8818206 1307 1317 FALSE smaller 10
chr1.8862587.8862832 chr1.8862612.8862832 221 247 FALSE smaller 26
chr1.8943305.8943820 chr1.8943491.8943820 330 517 FALSE smaller 187
chr1.9112795.9113342 chr1.9112795.9113308 514 549 FALSE smaller 35
chr1.9121887.9122488 chr1.9122044.9122488 445 603 FALSE smaller 158
chr1.9192634.9193134 chr1.9192638.9193134 497 502 FALSE smaller 5
chr1.9249509.9250292 chr1.9249509.9250291 783 785 FALSE smaller 2
chr1.9270444.9270753 chr1.9270585.9270753 169 311 FALSE smaller 142
chr1.9280437.9280883 chr1.9280439.9280883 445 448 FALSE smaller 3
chr1.9294939.9295417 chr1.9294939.9295230 292 480 FALSE smaller 188
chr1.9349886.9350815 chr1.9349901.9350815 915 931 FALSE smaller 16
chr1.9384465.9385244 chr1.9384465.9385222 758 781 FALSE smaller 23
chr1.9412968.9414140 chr1.9412968.9414073 1106 1174 FALSE smaller 68
chr1.9477638.9478334 chr1.9477638.9478064 427 698 FALSE smaller 271
chr1.9588320.9589673 chr1.9588430.9589673 1244 1355 FALSE smaller 111
chr1.9687212.9688612 chr1.9687212.9688546 1335 1402 FALSE smaller 67
chr1.9690004.9690404 chr1.9690010.9690404 395 402 FALSE smaller 7
chr1.9861272.9861633 chr1.9861272.9861615 344 363 FALSE smaller 19
chr1.9867260.9867962 chr1.9867260.9867936 677 704 FALSE smaller 27
chr1.9874716.9875727 chr1.9874716.9875655 940 1013 FALSE smaller 73
chr1.10046037.10046857 chr1.10046067.10046857 791 822 FALSE smaller 31
chr1.10057444.10058186 chr1.10057444.10057924 481 744 FALSE smaller 263
chr1.10108148.10109234 chr1.10108148.10108997 850 1088 FALSE smaller 238
chr1.10225669.10225944 chr1.10225669.10225934 266 277 FALSE smaller 11
chr1.10337444.10337792 chr1.10337444.10337778 335 350 FALSE smaller 15
chr1.10415310.10415689 chr1.10415310.10415675 366 381 FALSE smaller 15
chr1.10430094.10431279 chr1.10430118.10431279 1162 1187 FALSE smaller 25
chr1.10450221.10450657 chr1.10450223.10450657 435 438 FALSE smaller 3
chr1.10530388.10531196 chr1.10530388.10531178 791 810 FALSE smaller 19
chr1.10639387.10641914 chr1.10639524.10641887 2364 2529 FALSE smaller 165
chr1.10676833.10677740 chr1.10676833.10677719 887 909 FALSE smaller 22
chr1.10705625.10706383 chr1.10705793.10706383 591 760 FALSE smaller 169
chr1.10788609.10789054 chr1.10788609.10789048 440 447 FALSE smaller 7
chr1.10835880.10836301 chr1.10835885.10836301 417 423 FALSE smaller 6
chr1.11187424.11188227 chr1.11187464.11188227 764 805 FALSE smaller 41
chr1.11262074.11262946 chr1.11262074.11262931 858 874 FALSE smaller 16
chr1.11287024.11287231 chr1.11287053.11287231 179 209 FALSE smaller 30
chr1.11331503.11332031 chr1.11331503.11332009 507 530 FALSE smaller 23
chr1.11374481.11375152 chr1.11374579.11375152 574 673 FALSE smaller 99
chr1.11653121.11654958 chr1.11653182.11654958 1777 1839 FALSE smaller 62
chr1.11702025.11702641 chr1.11702043.11702625 583 618 FALSE smaller 35
chr1.11957125.11957472 chr1.11957147.11957472 326 349 FALSE smaller 23
chr1.11990456.11991565 chr1.11990456.11991507 1052 1111 FALSE smaller 59
chr1.12038619.12039412 chr1.12038619.12039354 736 795 FALSE smaller 59
chr1.12076503.12076909 chr1.12076523.12076909 387 408 FALSE smaller 21
chr1.12079442.12079874 chr1.12079452.12079874 423 434 FALSE smaller 11
chr1.12124730.12125435 chr1.12124730.12125430 701 707 FALSE smaller 6
chr1.12276366.12276746 chr1.12276366.12276663 298 382 FALSE smaller 84
chr1.12397501.12398046 chr1.12397501.12398002 502 547 FALSE smaller 45
chr1.12511561.12512466 chr1.12511561.12512409 849 907 FALSE smaller 58
chr1.12584779.12586273 chr1.12584820.12586190 1371 1496 FALSE smaller 125
chr1.12636532.12636959 chr1.12636532.12636952 421 429 FALSE smaller 8
chr1.13543536.13544390 chr1.13543536.13544366 831 856 FALSE smaller 25
chr1.13725712.13726316 chr1.13725712.13726267 556 606 FALSE smaller 50
chr1.13828628.13829298 chr1.13828628.13829247 620 672 FALSE smaller 52
chr1.14098494.14098908 chr1.14098494.14098892 399 416 FALSE smaller 17
chr1.14346092.14346513 chr1.14346104.14346490 387 423 FALSE smaller 36
chr1.14479781.14480379 chr1.14479837.14480379 543 600 FALSE smaller 57
chr1.14515230.14515633 chr1.14515230.14515572 343 405 FALSE smaller 62
chr1.14910102.14910514 chr1.14910112.14910514 403 414 FALSE smaller 11
chr1.14942784.14943479 chr1.14942784.14943454 671 697 FALSE smaller 26
chr1.15046373.15047153 chr1.15046373.15047093 721 782 FALSE smaller 61
chr1.15106354.15107288 chr1.15106354.15107281 928 936 FALSE smaller 8
chr1.15139584.15140520 chr1.15139584.15140472 889 938 FALSE smaller 49
chr1.15168566.15169038 chr1.15168566.15168945 380 474 FALSE smaller 94
chr1.15243310.15243831 chr1.15243332.15243831 500 523 FALSE smaller 23
chr1.15272341.15272967 chr1.15272355.15272967 613 628 FALSE smaller 15
chr1.15337579.15338469 chr1.15337579.15338214 636 892 FALSE smaller 256
chr1.15337579.15338469 chr1.15338279.15338469 191 892 FALSE smaller 701
chr1.15377324.15377665 chr1.15377324.15377592 269 343 FALSE smaller 74
chr1.15444247.15444866 chr1.15444247.15444825 579 621 FALSE smaller 42
chr1.15492433.15493101 chr1.15492486.15493101 616 670 FALSE smaller 54
chr1.15584537.15585349 chr1.15584583.15585349 767 814 FALSE smaller 47
chr1.15681096.15681510 chr1.15681114.15681505 392 416 FALSE smaller 24
chr1.15684056.15684415 chr1.15684056.15684404 349 361 FALSE smaller 12
chr1.15700408.15700790 chr1.15700415.15700790 376 384 FALSE smaller 8
chr1.15771851.15772471 chr1.15772211.15772471 261 622 FALSE smaller 361
chr1.15792047.15792404 chr1.15792061.15792404 344 359 FALSE smaller 15
chr1.15800148.15800973 chr1.15800171.15800973 803 827 FALSE smaller 24
chr1.15860112.15860659 chr1.15860112.15860587 476 549 FALSE smaller 73
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chrX.154600370.154600755 chrX.154600370.154600680 311 387 FALSE smaller 76
chrX.154729514.154730201 chrX.154729514.154730150 637 689 FALSE smaller 52
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chrX.154774967.154775961 chrX.154775513.154775961 449 996 FALSE smaller 547
b <- filter_by_overlaps(old_peaks_gr,new_peaks_gr)
b.a <- filter_by_overlaps(new_peaks_gr, old_peaks_gr) 
b.b <- filter_by_non_overlaps(new_peaks_gr, old_peaks_gr)
b.c <- filter_by_non_overlaps(old_peaks_gr, new_peaks_gr)

new_peaks_gr %>% as.data.frame %>% summarize(mean =mean(width))
      mean
1 606.7886

number of peaks in old data set: 151666
number of peaks in new data set: 155557
number of overlaps in between both sets: 149895
number of peaks in new set that do not overlap old set: 5678
number of peaks in old set that do not overlap new set: 1985

Differential analysis

group <- c( rep(c(1,2,3,4,5,6,7,8,9,10,11,12),4))
group <- factor(group, levels =c("1","2","3","4","5","6","7","8","9","10","11","12"))
short_names <- paste0(pca_final_four_anno$indv,"_",pca_final_four_anno$trt,"_",pca_final_four_anno$time)

dge <- DGEList.data.frame(counts = x4_filtered, group = group, genes = row.names(x4_filtered))

group_1 <- c(rep(c("DNR_24","DNR_3","DOX_24","DOX_3","EPI_24","EPI_3","MTX_24","MTX_3","TRZ_24","TRZ_3","VEH_24", "VEH_3"),4))

dge$group$indv <- pca_final_four_anno$indv
dge$group$time <- pca_final_four_anno$time
dge$group$trt <- pca_final_four_anno$trt

indv <- pca_final_four_anno$indv
efit4 <- readRDS("data/Final_four_data/efit4_filt_bl.RDS")
# mm <- model.matrix(~0 +group_1)  
# colnames(mm) <-  c("DNR_24", "DNR_3", "DOX_24","DOX_3","EPI_24", "EPI_3","MTX_24", "MTX_3", "TRZ_24","TRZ_3","VEH_24", "VEH_3")
# 
# y <- voom(dge$counts, mm,plot =TRUE)
# # 
# corfit <- duplicateCorrelation(y, mm, block = indv)
# # 
# v <- voom(dge$counts, mm, block = indv, correlation = corfit$consensus)
# # 
# fit <- lmFit(v, mm, block = indv, correlation = corfit$consensus)
# colnames(mm) <- c("DNR_24","DNR_3","DOX_24","DOX_3","EPI_24","EPI_3","MTX_24","MTX_3","TRZ_24","TRZ_3","VEH_24", "VEH_3")
#
#
# cm <- makeContrasts(
#   DNR_3.VEH_3 = DNR_3-VEH_3,
#   DOX_3.VEH_3 = DOX_3-VEH_3,
#   EPI_3.VEH_3 = EPI_3-VEH_3,
#   MTX_3.VEH_3 = MTX_3-VEH_3,
#   TRZ_3.VEH_3 = TRZ_3-VEH_3,
#   DNR_24.VEH_24 =DNR_24-VEH_24,
#   DOX_24.VEH_24= DOX_24-VEH_24,
#   EPI_24.VEH_24= EPI_24-VEH_24,
#   MTX_24.VEH_24= MTX_24-VEH_24,
#   TRZ_24.VEH_24= TRZ_24-VEH_24,
#   levels = mm)

# vfit <- lmFit(y, mm)

# vfit<- contrasts.fit(vfit, contrasts=cm)
# 
# efit4 <- eBayes(vfit)
# saveRDS(efit4,"data/Final_four_data/efit4_filt_bl.RDS")
results = decideTests(efit4)
summary(results)
       DNR_3.VEH_3 DOX_3.VEH_3 EPI_3.VEH_3 MTX_3.VEH_3 TRZ_3.VEH_3
Down          5925         448        2959          22           0
NotSig      146648      155046      151480      155536      155560
Up            2987          66        1121           2           0
       DNR_24.VEH_24 DOX_24.VEH_24 EPI_24.VEH_24 MTX_24.VEH_24 TRZ_24.VEH_24
Down           27539         21704         23244          1585             0
NotSig         92353        107936        107084        149730        155560
Up             35668         25920         25232          4245             0
# V.DNR_3.top_ff= topTable(efit4, coef=1, adjust.method="BH", number=Inf, sort.by="p") %>% rownames_to_column("peak")
# V.DOX_3.top_ff= topTable(efit4, coef=2, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.EPI_3.top_ff= topTable(efit4, coef=3, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.MTX_3.top_ff= topTable(efit4, coef=4, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.TRZ_3.top_ff= topTable(efit4, coef=5, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.DNR_24.top_ff= topTable(efit4, coef=6, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.DOX_24.top_ff= topTable(efit4, coef=7, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.EPI_24.top_ff= topTable(efit4, coef=8, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.MTX_24.top_ff= topTable(efit4, coef=9, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# V.TRZ_24.top_ff= topTable(efit4, coef=10, adjust.method="BH", number=Inf, sort.by="p")%>% rownames_to_column("peak")
# 
# toplist_full_ff  <- list(V.DNR_3.top_ff, V.DOX_3.top_ff,V.EPI_3.top_ff,V.MTX_3.top_ff,V.TRZ_3.top_ff,V.DNR_24.top_ff, V.DOX_24.top_ff,V.EPI_24.top_ff,V.MTX_24.top_ff,V.TRZ_24.top_ff)
# names(toplist_full_ff) <- c("DNR_3", "DOX_3","EPI_3","MTX_3","TRZ_3","DNR_24", "DOX_24","EPI_24","MTX_24","TRZ_24")
# toplist_ff <-map_df(toplist_full_ff, ~as.data.frame(.x), .id="trt_time")
# #
# toplist_ff <- toplist_ff %>%
#   separate(trt_time, into= c("trt","time"), sep = "_") %>%
#   mutate(trt=factor(trt, levels = c("DOX","EPI","DNR","MTX","TRZ"))) %>%
#   mutate(time = factor(time, levels = c("3", "24"), labels = c("3 hours", "24 hours")))
# saveRDS(toplist_ff,"data/Final_four_data/toplist_ff.RDS")
toplist_ff <- readRDS("data/Final_four_data/toplist_ff.RDS")
median_3_lfc <- read_csv("data/Final_four_data/median_3_lfc.csv")
median_24_lfc <- read_csv("data/Final_four_data/median_24_lfc.csv")
### making median lfc

median_df <- toplist_ff %>% 
  pivot_wider(., id_cols=c(time,peak), names_from = trt, values_from = logFC) %>% 
  rowwise() %>% 
  mutate(median_lfc= median(c_across(DNR:TRZ)))

median_3_lfc <-   median_df %>%
    dplyr::filter(time == "3 hours") %>% 
  ungroup() %>% 
  dplyr::select(time, peak,median_lfc) %>% 
  dplyr::rename("med_3h_lfc"=median_lfc)
  

median_24_lfc <- median_df %>%
    dplyr::filter(time == "24 hours") %>% 
  ungroup() %>% 
  dplyr::select(time, peak,median_lfc) %>% 
  dplyr::rename("med_24h_lfc"=median_lfc)
  
write_csv(median_3_lfc, "data/Final_four_data/median_3_lfc.csv")
write_csv(median_24_lfc, "data/Final_four_data/median_24_lfc.csv")

Correlation of LFC

FCmatrix_ff <- subset(efit4$coefficients)

colnames(FCmatrix_ff) <-
  c("DNR\n3h",
    "DOX\n3h",
    "EPI\n3h",
    "MTX\n3h",
    "TRZ\n3h",
    "DNR\n24h",
    "DOX\n24h",
    "EPI\n24h",
    "MTX\n24h",
    "TRZ\n24h"
     )


mat_col_ff <-
  data.frame(
    time = c(rep("3 hours", 5), rep("24 hours", 5)),
    class = (c(
      "AC", "AC", "AC", "nAC","nAC",  "AC", "AC", "AC", "nAC","nAC" 
    )))
rownames(mat_col_ff) <- colnames(FCmatrix_ff)

mat_colors_ff <-
  list(
    time = c("pink", "chocolate4"),
    class = c("yellow1", "lightgreen"))

names(mat_colors_ff$time) <- unique(mat_col_ff$time)
names(mat_colors_ff$class) <- unique(mat_col_ff$class)
# names(mat_colors_FC$TOP2i) <- unique(mat_col_FC$TOP2i)
corrFC_ff <- cor(FCmatrix_ff)

htanno_ff <-  HeatmapAnnotation(df = mat_col_ff, col = mat_colors_ff)
Heatmap(corrFC_ff, top_annotation = htanno_ff)

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V.DNR_3.top= topTable(efit4, coef=1, adjust.method="BH", number=Inf, sort.by="p")
V.DOX_3.top= topTable(efit4, coef=2, adjust.method="BH", number=Inf, sort.by="p")
V.EPI_3.top= topTable(efit4, coef=3, adjust.method="BH", number=Inf, sort.by="p")
V.MTX_3.top= topTable(efit4, coef=4, adjust.method="BH", number=Inf, sort.by="p")
V.TRZ_3.top= topTable(efit4, coef=5, adjust.method="BH", number=Inf, sort.by="p")
V.DNR_24.top= topTable(efit4, coef=6, adjust.method="BH", number=Inf, sort.by="p")
V.DOX_24.top= topTable(efit4, coef=7, adjust.method="BH", number=Inf, sort.by="p")
V.EPI_24.top= topTable(efit4, coef=8, adjust.method="BH", number=Inf, sort.by="p")
V.MTX_24.top= topTable(efit4, coef=9, adjust.method="BH", number=Inf, sort.by="p")
V.TRZ_24.top= topTable(efit4, coef=10, adjust.method="BH", number=Inf, sort.by="p")

# volcanoplot(efit2,coef = c(1,2,3,4,5,6),style = "p-value",
#                           # highlight = 8,
#                           # names = efit2$genes$SYMBOL,
#                           hl.col = "red",xlab = "Log2 Fold Change",
#                           ylab = NULL,pch = 16,cex = 0.35,
#                           main = "test")
# 

plot_filenames <- c("V.DNR_3.top","V.DOX_3.top","V.EPI_3.top","V.MTX_3.top",
                    "V.TRZ_.top","V.DNR_24.top","V.DOX_24.top","V.EPI_24.top",
                    "V.MTX_24.top","V.TRZ_24.top")
plot_files <- c( V.DNR_3.top,V.DOX_3.top,V.EPI_3.top,V.MTX_3.top,
                    V.TRZ_3.top,V.DNR_24.top,V.DOX_24.top,V.EPI_24.top,
                    V.MTX_24.top,V.TRZ_24.top)

volcanosig <- function(df, psig.lvl) {
    df <- df %>% 
    mutate(threshold = ifelse(adj.P.Val > psig.lvl, "A", ifelse(adj.P.Val <= psig.lvl & logFC<=0,"B","C")))
      # ifelse(adj.P.Val <= psig.lvl & logFC >= 0,"B", "C")))
    ##This is where I could add labels, but I have taken out
    # df <- df %>% mutate(genelabels = "")
    # df$genelabels[1:topg] <- df$rownames[1:topg]
    
  ggplot(df, aes(x=logFC, y=-log10(P.Value))) + 
    geom_point(aes(color=threshold))+
    # geom_text_repel(aes(label = genelabels), segment.curvature = -1e-20,force = 1,size=2.5,
    # arrow = arrow(length = unit(0.015, "npc")), max.overlaps = Inf) +
    #geom_hline(yintercept = -log10(psig.lvl))+
    xlab(expression("Log"[2]*" FC"))+
    ylab(expression("-log"[10]*"P Value"))+
    scale_color_manual(values = c("black", "red","blue"))+
    theme_cowplot()+
    ylim(0,20)+
    xlim(-6,6)+
    theme(legend.position = "none",
              plot.title = element_text(size = rel(1.5), hjust = 0.5),
              axis.title = element_text(size = rel(0.8))) 
}
#v1<- volcanosig(V.DA24.top, 0.01,0)
v1 <- volcanosig(V.DNR_3.top, 0.01)+ ggtitle("DNR 3 hour")
v2 <- volcanosig(V.DNR_24.top, 0.01)+ ggtitle("DNR 24 hour")+ylab("")
v3 <- volcanosig(V.DOX_3.top, 0.01)+ ggtitle("DOX 3 hour")
v4 <- volcanosig(V.DOX_24.top, 0.01)+ ggtitle("DOX 24 hour")+ylab("")
v5 <- volcanosig(V.EPI_3.top, 0.01)+ ggtitle("EPI 3 hour")
v6 <- volcanosig(V.EPI_24.top, 0.01)+ ggtitle("EPI 24 hour")+ylab("")
v7 <- volcanosig(V.MTX_3.top, 0.01)+ ggtitle("MTX 3 hour")
v8 <- volcanosig(V.MTX_24.top, 0.01)+ ggtitle("MTX 24 hour")+ylab("")
v9 <- volcanosig(V.TRZ_3.top, 0.01)+ ggtitle("TRZ 3 hour")
v10 <- volcanosig(V.TRZ_24.top, 0.01)+ ggtitle("TRZ 24 hour")+ylab("")
# volcanoplot(efit2,coef = 10,style = "p-value",
#                           highlight = 8,
#                           names = efit2$genes$SYMBOL,
#                           hl.col = "red",xlab = "Log2 Fold Change",
#                           ylab = NULL,pch = 16,cex = 0.35,
#                           main = "Using Trastuzumab 24 hour data and volcanoplot function")
plot_grid(v1,v2,  rel_widths =c(.8,1))

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plot_grid(v3,v4,  rel_widths =c(.8,1))

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plot_grid(v5,v6,  rel_widths =c(.8,1))

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plot_grid(v7,v8,  rel_widths =c(.8,1))

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plot_grid(v9,v10,  rel_widths =c(.8,1))

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DNR_3_top3_ff <- row.names(V.DNR_3.top[1:3,])

log_filt_ff <- filt4_matrix_lcpm %>% 
  as.data.frame() %>% 
  rename_with(.,~gsub(pattern = "Ind1_75", replacement = "1_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind2_87", replacement = "2_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind3_77", replacement = "3_",.)) %>% 
  rename_with(.,~gsub(pattern = "Ind6_71", replacement = "6_",.)) %>% 
  rename_with(.,~gsub( "DX" ,'DOX',.)) %>% 
  rename_with(.,~gsub( "DA" ,'DNR',.)) %>% 
  rename_with(.,~gsub( "E" ,'EPI',.)) %>% 
  rename_with(.,~gsub( "T" ,'TRZ',.)) %>%
  rename_with(.,~gsub( "M" ,'MTX',.)) %>% 
  rename_with(.,~gsub( "V" ,'VEH',.)) %>% 
  rename_with(.,~gsub("24h","_24h",.)) %>% 
  rename_with(.,~gsub("3h","_3h",.))
  



row.names(log_filt_ff) <- row.names(filt4_matrix_lcpm)

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% DNR_3_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 3 hour DNR")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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8acb1e8 reneeisnowhere 2024-09-23
DOX_3_top3_ff <- row.names(V.DOX_3.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% DOX_3_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 3 hour DOX")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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8acb1e8 reneeisnowhere 2024-09-23
EPI_3_top3_ff <- row.names(V.EPI_3.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% EPI_3_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 3 hour EPI")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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MTX_3_top3_ff <- row.names(V.MTX_3.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% MTX_3_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 3 hour MTX")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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TRZ_3_top3_ff <- row.names(V.TRZ_3.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% TRZ_3_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 3 hour TRZ")+
 scale_fill_manual(values = drug_pal)+
  theme_bw()

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DNR_24_top3_ff <- row.names(V.DNR_24.top[1:3,])


# row.names(log_filt_23s) <- row.names(lcpm_h3_23s_filtered)
log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% DNR_24_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 24 hour DNR")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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DOX_24_top3_ff <- row.names(V.DOX_24.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% DOX_24_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 24 hour DOX")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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EPI_24_top3_ff <- row.names(V.EPI_24.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% EPI_24_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 24 hour EPI")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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MTX_24_top3_ff <- row.names(V.MTX_24.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% MTX_24_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 24 hour MTX")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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8acb1e8 reneeisnowhere 2024-09-23
TRZ_24_top3_ff <- row.names(V.TRZ_24.top[1:3,])

log_filt_ff %>% 
  dplyr::filter(row.names(.) %in% TRZ_24_top3_ff) %>% 
  mutate(Peak = row.names(.)) %>% 
  pivot_longer(cols = !Peak, names_to = "sample", values_to = "counts") %>% 
  separate("sample", into = c("indv","trt","time")) %>% 
  mutate(time=factor(time, levels = c("3h","24h"))) %>% 
  mutate(trt=factor(trt, levels= c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>% 
  ggplot(., aes (x = time, y=counts))+
  geom_boxplot(aes(fill=trt))+
  facet_wrap(Peak~.)+
  ggtitle("top 3 DAR in 24 hour TRZ")+
  scale_fill_manual(values = drug_pal)+
  theme_bw()

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8acb1e8 reneeisnowhere 2024-09-23

Cormotif

# indv_n45 <- df_names_n45$indv
# indv <- factor(indv, levels = c(1,2,3,4,5,6))
# time_n45 <- df_names_n45$time
# time <- factor(time, levels =c("3h","24"))
# trt_n45 <- df_names_n45$trt
# label_n45 <- paste0(indv_n45,"_",trt_n45,"_",time_n45)
group_fac_ff <- group
groupid_ff <- as.numeric(group_fac_ff)

compid_ff <- data.frame(c1= c(2,4,6,8,10,1,3,5,7,9), c2 = c( 12,12,12,12,12,11,11,11,11,11))

y_TMM_cpm_ff <- cpm(x4_filtered, log = TRUE)

colnames(y_TMM_cpm_ff) <- short_names
# y_TMM_cpm
set.seed(31415)
# cormotif_initial_ff <- cormotiffit(exprs = y_TMM_cpm_ff, groupid = groupid_ff, compid = compid_ff, K=1:8, max.iter = 500, runtype = "logCPM")
# 
# saveRDS(cormotif_initial_ff,"data/Final_four_data/cormotif_ff_4_run.RDS")

# cormotif_four_ff <- cormotiffit(exprs = y_TMM_cpm_ff, groupid = groupid_ff, compid = compid_ff, K=4, max.iter = 500, runtype = "logCPM")

# saveRDS(cormotif_four_ff,"data/Final_four_data/cormotif_only4_run.RDS")

cormotif_initial_ff <-  readRDS("data/Final_four_data/cormotif_ff_4_run.RDS")
gene_prob_tran_ff <- cormotif_initial_ff$bestmotif$p.post
rownames(gene_prob_tran_ff) <- rownames(x4_filtered)
motif_prob_ff <- cormotif_initial_ff$bestmotif$clustlike
rownames(motif_prob_ff) <- rownames(gene_prob_tran_ff)
# write.csv(motif_prob_n45_he,"data/cormotif_probability_45_list_he.csv")
myColors <-  rev(c("#FFFFFF", "#E6E6E6" ,"#CCCCCC", "#B3B3B3", "#999999", "#808080", "#666666","#4C4C4C", "#333333", "#191919","#000000"))
Cormotif::plotIC(cormotif_initial_ff)

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plot.new()
legend('center',fill=myColors, legend =rev(c("0", "0.1", "0.2", "0.3", "0.4",  "0.5", "0.6", "0.7", "0.8","0.9", "1")), box.col="white",title = "Probability\nlegend", horiz=FALSE,title.cex=.8)

Cormotif::plotMotif(cormotif_initial_ff)

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Study breakdown: 1 = DNR_3, 2 = DOX_3, 3 = EPI_3, 4 = MTX_3, 5 = TRZ_3
6 = DNR_24, 7 = DOX_24, 8 = EPI_24, 9 = MTX_24, 10 = TRZ_24

# motif_prob_ff <- read.csv("data/cormotif_probability_45_list_he.csv")

# motif_prob_ff %>% 
#   as.data.frame() %>% 
#   dplyr::filter(V1<.5 & V2>.5 & V3 <.5& V4<0.5)

background_peaks <- motif_prob_ff %>% 
  as.data.frame() %>% 
  rownames_to_column("Peakid") %>% 
  dplyr::select(Peakid) %>% 
  separate(Peakid, into=c("chr","start","end"),remove = FALSE)
 
NR_ff <- motif_prob_ff %>%
  as.data.frame() %>% 
  dplyr::filter(V1>.5 & V2<.5 & V3 <.5& V4<0.5) %>% 
  rownames_to_column("Peakid") %>% 
  dplyr::select(Peakid) %>% 
  separate(Peakid, into=c("chr","start","end"),remove = FALSE)
 
LR_ff <- motif_prob_ff %>%
   as.data.frame() %>% 
  dplyr::filter(V1<.5 & V2>.5 & V3 <.5& V4<0.5) %>% 
  rownames_to_column("Peakid") %>% 
  dplyr::select(Peakid) %>% 
  separate(Peakid, into=c("chr","start","end"),remove = FALSE)
 
 
ESR_ff <- motif_prob_ff %>%
  as.data.frame() %>% 
  dplyr::filter(V1<.5 & V2<.5 & V3 >.5& V4<0.5) %>% 
  rownames_to_column("Peakid") %>% 
  dplyr::select(Peakid) %>% 
  separate(Peakid, into=c("chr","start","end"),remove = FALSE)
 

EAR_ff <- motif_prob_ff %>%
  as.data.frame() %>% 
  dplyr::filter(V1<.5 & V2<.5 & V3 <.5& V4>0.5) %>% 
  rownames_to_column("Peakid") %>% 
  dplyr::select(Peakid) %>% 
  separate(Peakid, into=c("chr","start","end"),remove = FALSE)

Number of Peaks by main grouping:
-EAR group has 7454 peaks.
-ESR group has 15781 peaks.
-LR group has 42609 peaks.
-NR group has 86331 peaks.
–Total number of peaks is 155560 –Total number of peaks in clusters is 152175
-percent of peaks classified into clusters: 97.8239907
-number of peaks not classified: 3385

Peak distribution characteristics

txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene
EAR_ff_gr <- EAR_ff %>% GRanges()
ESR_ff_gr <- ESR_ff %>% GRanges()
LR_ff_gr <- LR_ff %>% GRanges()
NR_ff_gr <- NR_ff %>% GRanges()
background_gr <- background_peaks %>% GRanges()
# mylist <- list(EAR_ff_gr,ESR_ff_gr,LR_ff_gr,NR_ff_gr,background_gr)
# peakAnnoList<- lapply(mylist, annotatePeak, tssRegion =c(-2000,2000), TxDb= txdb)
# names(peakAnnoList) <- c("EAR","ESR","LR","NR","background")
# saveRDS(peakAnnoList, "data/Final_four_data/peakAnnoList_ff_motif.RDS")

peakAnnoList_motif <- readRDS("data/Final_four_data/peakAnnoList_ff_motif.RDS")
plotAnnoBar(peakAnnoList_motif[c(4,1:3)])+
  ggtitle ("Genomic Feature Distribution, Four groups")

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#### HOw I assigned expressed NG TSS to new peak file
### export new peak file as .bed file
a4gr <- all_four_filt_counts %>% 
  dplyr::select(seqnames:Geneid) %>% 
  dplyr::rename("Peakid"=Geneid) %>% 
  GRanges()
 
# write_delim((as.data.frame(a4gr)),delim = "\t",file = "data/Final_four_data/all_four_filt.bed", col_name=FALSE)## write tab delim datafile without column headers

###Do the same with TSS file
#  ensembl <- useEnsembl(biomart = "genes", dataset = "hsapiens_gene_ensembl")
# my_attributes <- c('chromosome_name', 'start_position', 'end_position','entrezgene_id', 'ensembl_gene_id', 'hgnc_symbol')
# searchAttributes(mart = ensembl, pattern = "refseq")
# searchDatasets(mart=ensembl,pattern="NCBI")
# gene_list <- getBM(attributes=my_attributes,
#       filters = "hgnc_symbol",
#         values = S13Table$SYMBOL,
#       mart = ensembl)
# write.csv(gene_ref,"data/gene_ref.csv")
# listFilters(ensembl)
####
# gene_ref <- read.csv("data/gene_ref.csv")
# gene_full_list <- gene_list %>% 
#   mutate(chromosome_name=as.numeric(chromosome_name))%>% 
#   # filter(!is.na(chromosome_name)) #%>% 
#   na.omit(.)
#   dplyr::select(chromosome_name,start_position,end_position,entrezgene_id,ensembl_gene_id)
### I performed the bedtools using cli. I moved the files to the correct directory on my hardrive to do this.
# bedtools closest -a all_four_filt.bed -b All_TSS_NGs.bed -D b >assigned_newNGbyTSS.bed
#### This file only has the tss, I had to link the geneIDs and symbols and names using the lookup_table (contains TSS, Start and end, ENTREZID, ensembl_id,and SYBOL)
 lookup_table <- read.csv("data/Final_four_data/expressedgene_lookuptable.csv", row.names = 1)
NG_assigned_table <- read_delim("data/Final_four_data/assigned_newNGbyTSS.bed", col_names = FALSE)

TSS_NG_table <- NG_assigned_table %>% 
  dplyr::rename("chr"=X1,
         "start"=X2,
         "end"= X3,
         "Peakid"=X6,
         "NG_chr"=X7,
         "NG_start"=X8 ,
         "NG_end"=X9 ,
         "dist_to_NG"=X13) %>% 
  dplyr::select(chr:end,Peakid:NG_end,dist_to_NG) %>%
  left_join(., lookup_table, by= c("NG_chr"="NG_chr","NG_end"="NG_start")) %>% 
  dplyr::rename("NG_end"="NG_end.y", "NG_TSS"=NG_end) %>% 
  dplyr::select(chr:NG_start,NG_end,NG_TSS,ENTREZID:SYMBOL,dist_to_NG) #%>%
  # write_delim(.,"data/Final_four_data/TSS_assigned_NG.txt",delim = "\t",col_names = TRUE)

Collapsed_new_peaks <- TSS_NG_table %>% 
  group_by(chr, start, end, Peakid) %>%
    summarise(chr=paste(unique(chr)),
              start=paste(unique(start)),
              end=paste(unique(end)),
              NG_chr=paste(NG_chr,collapse=":"),
              NG_start=paste(NG_start,collapse=":"),
              NG_end=paste(NG_end,collapse=":"),
              NG_TSS=paste(NG_TSS,collapse=":"),
              NCBI_gene = paste(unique(ENTREZID),collapse=","),
              ensembl_ID= paste(unique(ensembl_id),collapse = ","),
              SYMBOL= paste(unique(SYMBOL),collapse = ",") , 
              dist_to_NG =min(dist_to_NG))
 # write_delim(Collapsed_new_peaks,"data/Final_four_data/collapsed_new_peaks.txt",delim = "\t",col_names = TRUE)

Eight group peak characteristics

TSS_assigned_NG <- read_delim("data/Final_four_data/TSS_assigned_NG.txt", delim = "\t", col_names = TRUE)
Collapsed_new_peaks <- read_delim("data/Final_four_data/collapsed_new_peaks.txt", delim = "\t", col_names = TRUE)

open_3med <- median_3_lfc %>% 
  dplyr::filter(med_3h_lfc > 0)

close_3med <- median_3_lfc %>% 
  dplyr::filter(med_3h_lfc < 0)

open_24med <- median_24_lfc %>% 
  dplyr::filter(med_24h_lfc > 0)

close_24med <- median_24_lfc %>% 
  dplyr::filter(med_24h_lfc < 0)

medA <- median_3_lfc %>% 
  left_join(median_24_lfc, by=c("peak"="peak")) %>% 
  dplyr::filter(med_3h_lfc > 0 & med_24h_lfc>0)

medB <- median_3_lfc %>% 
  left_join(median_24_lfc, by=c("peak"="peak")) %>% 
  dplyr::filter(med_3h_lfc < 0 & med_24h_lfc < 0)
 
medC <- median_3_lfc %>% 
  left_join(median_24_lfc, by=c("peak"="peak")) %>% 
  dplyr::filter(med_3h_lfc > 0& med_24h_lfc <0)
  

medD <- median_3_lfc %>% 
 left_join(median_24_lfc, by=c("peak"="peak"))%>% 
  dplyr::filter(med_3h_lfc < 0 & med_24h_lfc > 0)
 

EAR_open <- EAR_ff %>%
  dplyr::filter(Peakid %in% open_3med$peak)
  
EAR_open_gr <- EAR_open %>% GRanges()

EAR_close <- EAR_ff %>%
  dplyr::filter(Peakid %in% close_3med$peak) 

EAR_close_gr <- EAR_close %>% GRanges()

LR_open <- LR_ff %>%
  dplyr::filter(Peakid %in% open_24med$peak) 

LR_open_gr <- LR_open %>% GRanges()

LR_close <- LR_ff %>%
  dplyr::filter(Peakid %in% close_24med$peak) 

LR_close_gr <- LR_close %>% GRanges()

NR_gr <- NR_ff %>% 
   GRanges()

ESR_open <- ESR_ff %>% 
  dplyr::filter(Peakid %in% medA$peak)  
 
ESR_open_gr <- ESR_open %>% GRanges()

ESR_close <- ESR_ff %>% 
  dplyr::filter(Peakid %in% medB$peak)  

ESR_close_gr <- ESR_close %>% GRanges()

ESR_C <- ESR_ff %>% 
  dplyr::filter(Peakid %in% medC$peak) 

ESR_D <- ESR_ff %>% 
  dplyr::filter(Peakid %in% medD$peak) 


ESR_OC <- ESR_C %>% 
  rbind(ESR_D)
ESR_OC_gr <- ESR_OC %>% GRanges()

mylist_new <- list(EAR_open_gr, EAR_close_gr,ESR_open_gr, ESR_close_gr,ESR_OC_gr, LR_open_gr, LR_close_gr,NR_gr, background_gr)
names(mylist_new) <- c("EAR_open", "EAR_close","ESR_open", "ESR_close","ESR_OC", "LR_open", "LR_close","NR","background")

# peakAnnoList<- lapply(mylist_new, annotatePeak, tssRegion =c(-2000,2000), TxDb= txdb)
# names(peakAnnoList) <- c("EAR_open", "EAR_close","ESR_open", "ESR_close","ESR_OC", "LR_open", "LR_close","NR","background")
# saveRDS(peakAnnoList, "data/Final_four_data/peakAnnoList_ff_8motif.RDS")
peakAnnoList_8_motif <- readRDS("data/Final_four_data/peakAnnoList_ff_8motif.RDS")
plotAnnoBar(peakAnnoList_8_motif[c(8,1:7)])+
  ggtitle ("Genomic Feature Distribution, eight groups")

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8acb1e8 reneeisnowhere 2024-09-23

Overlapping peaks with H3K27ac data

raodah_fullpeaks <- readRDS( "data/Final_four_data/All_Raodahpeaks.RDS") %>%
  GRanges %>% 
  GenomeInfoDb::keepStandardChromosomes(., pruning.mode = "coarse") %>% 
  dplyr::filter(seqnames!="chrM") %>% 
  dplyr::filter(seqnames!="chrY")
  
  

overlap_ac <- join_overlap_intersect((new_peaks_gr %>%  dplyr::filter(seqnames !="chrY")),raodah_fullpeaks) %>% as.data.frame()
# overlap_ac %>% 
#   distinct(peakid)
# saveRDS(overlap_ac, "data/Final_four_data/overlapping_ac_atac_peaks.RDS")

abs LFC across clusters

toplist_ff <- readRDS("data/Final_four_data/toplist_ff.RDS")
mean_lfc <-   toplist_ff %>%
  mutate(EAR_open = if_else(peak %in% EAR_open$Peakid, "y", "no")) %>%
  mutate(EAR_close = if_else(peak %in% EAR_close$Peakid, "y", "no")) %>%
  mutate(ESR_open = if_else(peak %in% ESR_open$Peakid, "y", "no")) %>%
  mutate(ESR_close= if_else(peak %in% ESR_close$Peakid, "y", "no")) %>%
  # mutate(ESR_OC= if_else(peak %in% ESR_OC$Peakid, "y", "no")) %>%
  mutate(ESR_C= if_else(peak %in% ESR_C$Peakid, "y", "no")) %>%
  mutate(ESR_D= if_else(peak %in% ESR_D$Peakid, "y", "no")) %>%
    mutate(LR_open= if_else(peak %in% LR_open$Peakid, "y", "no")) %>%
    mutate(LR_close= if_else(peak %in% LR_close$Peakid, "y", "no")) %>%
    mutate(NR = if_else(peak %in% NR_ff$Peakid, "y", "no"))%>%
  mutate(trt = factor(trt,levels=c("DOX","EPI","DNR","MTX","TRZ","VEH"))) %>%
  group_by(trt, time) %>%
  mutate(absFC = (logFC)) %>%
  dplyr::select(trt, time, absFC, EAR_open:NR) %>%
  dplyr::summarize(
    EAR_op = mean(absFC[EAR_open == "y"]),
    EAR_cl = mean(absFC[EAR_close == "y"]),
    ESR_op = mean(absFC[ESR_open == "y"]),
    ESR_cl = mean(absFC[ESR_close == "y"]),
    # ESR_OC = mean(absFC[ESR_OC == "y"]),
    ESR_c = mean(absFC[ESR_C == "y"]),
    ESR_d = mean(absFC[ESR_D == "y"]),
    LR_op = mean(absFC[LR_open == "y"]),
    LR_cl = mean(absFC[LR_close == "y"]),
    NR = mean(absFC[NR == "y"])
  ) %>%
  as.data.frame()


mean_lfc %>%
  ungroup() %>%
  pivot_longer(!c(trt, time), names_to = "Motif",
               values_to = "meanLFC") %>%
  # mutate(Set = dplyr::case_match(Motif,
  #                        "EAR open"~  EAR_op ,
  #                         EAR_cm ~"EAR close" ,
  #                         ESR_om ~"ESR open",
  #                         ESR_cm ~ "ESR close",
  #                         ESR_ocm ~ "ESR open/close",
  #                         LR_om~ "LR open",
  #                         LR_cm~ "LR close",
  #                         NRm ~ "No Response",
  #                         .default = Motif)) %>%
  ggplot(., aes(x = time,y = meanLFC,col = trt,
    group = trt
  )) +
  geom_point(size = 2) +
  geom_line(linewidth = 2) +
  ggpubr::fill_palette(drug_pal) +
  # guides(fill=guide_legend(title = "Treatment"))+
  facet_wrap( ~ Motif, nrow = 2) +
  theme_bw() +
  xlab("Time (hours)") +
  scale_color_manual(values = drug_pal) +
  ylab(" Avg. Log Fold Change") +
  theme_bw() +
  # ggtitle(" average |Log Fold| for genes across treatment each motif")+
  theme(
    plot.title = element_text(size = rel(1.0), hjust = 0.5),
    axis.title = element_text(size = 15, color = "black"),
    axis.line = element_line(linewidth = 1.0),
    strip.background = element_rect(fill = "transparent"),
    axis.text = element_text(
      size = 10,
      color = "black",
      angle = 0
    ),
    strip.text.x = element_text(
      size = 11,
      color = "black",
      face = "bold"
    )
  )

Version Author Date
8acb1e8 reneeisnowhere 2024-09-23

sessionInfo()
R version 4.4.1 (2024-06-14 ucrt)
Platform: x86_64-w64-mingw32/x64
Running under: Windows 10 x64 (build 19045)

Matrix products: default


locale:
[1] LC_COLLATE=English_United States.utf8 
[2] LC_CTYPE=English_United States.utf8   
[3] LC_MONETARY=English_United States.utf8
[4] LC_NUMERIC=C                          
[5] LC_TIME=English_United States.utf8    

time zone: America/Chicago
tzcode source: internal

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

other attached packages:
 [1] cowplot_1.1.3                           
 [2] ComplexHeatmap_2.20.0                   
 [3] ggrepel_0.9.6                           
 [4] plyranges_1.24.0                        
 [5] ggsignif_0.6.4                          
 [6] genomation_1.36.0                       
 [7] eulerr_7.0.2                            
 [8] devtools_2.4.5                          
 [9] usethis_3.0.0                           
[10] ggpubr_0.6.0                            
[11] BiocParallel_1.38.0                     
[12] Cormotif_1.50.0                         
[13] affy_1.82.0                             
[14] scales_1.3.0                            
[15] VennDiagram_1.7.3                       
[16] futile.logger_1.4.3                     
[17] gridExtra_2.3                           
[18] ggfortify_0.4.17                        
[19] edgeR_4.2.1                             
[20] limma_3.60.4                            
[21] rtracklayer_1.64.0                      
[22] org.Hs.eg.db_3.19.1                     
[23] TxDb.Hsapiens.UCSC.hg38.knownGene_3.18.0
[24] GenomicFeatures_1.56.0                  
[25] AnnotationDbi_1.66.0                    
[26] Biobase_2.64.0                          
[27] GenomicRanges_1.56.1                    
[28] GenomeInfoDb_1.40.1                     
[29] IRanges_2.38.1                          
[30] S4Vectors_0.42.1                        
[31] BiocGenerics_0.50.0                     
[32] ChIPseeker_1.40.0                       
[33] RColorBrewer_1.1-3                      
[34] broom_1.0.6                             
[35] kableExtra_1.4.0                        
[36] lubridate_1.9.3                         
[37] forcats_1.0.0                           
[38] stringr_1.5.1                           
[39] dplyr_1.1.4                             
[40] purrr_1.0.2                             
[41] readr_2.1.5                             
[42] tidyr_1.3.1                             
[43] tibble_3.2.1                            
[44] ggplot2_3.5.1                           
[45] tidyverse_2.0.0                         
[46] workflowr_1.7.1                         

loaded via a namespace (and not attached):
  [1] fs_1.6.4                               
  [2] matrixStats_1.4.1                      
  [3] bitops_1.0-8                           
  [4] enrichplot_1.24.4                      
  [5] doParallel_1.0.17                      
  [6] httr_1.4.7                             
  [7] profvis_0.3.8                          
  [8] tools_4.4.1                            
  [9] backports_1.5.0                        
 [10] utf8_1.2.4                             
 [11] R6_2.5.1                               
 [12] lazyeval_0.2.2                         
 [13] GetoptLong_1.0.5                       
 [14] urlchecker_1.0.1                       
 [15] withr_3.0.1                            
 [16] preprocessCore_1.66.0                  
 [17] cli_3.6.3                              
 [18] formatR_1.14                           
 [19] scatterpie_0.2.4                       
 [20] labeling_0.4.3                         
 [21] sass_0.4.9                             
 [22] Rsamtools_2.20.0                       
 [23] systemfonts_1.1.0                      
 [24] yulab.utils_0.1.7                      
 [25] DOSE_3.30.5                            
 [26] svglite_2.1.3                          
 [27] R.utils_2.12.3                         
 [28] sessioninfo_1.2.2                      
 [29] plotrix_3.8-4                          
 [30] BSgenome_1.72.0                        
 [31] impute_1.78.0                          
 [32] rstudioapi_0.16.0                      
 [33] RSQLite_2.3.7                          
 [34] shape_1.4.6.1                          
 [35] generics_0.1.3                         
 [36] gridGraphics_0.5-1                     
 [37] TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2
 [38] BiocIO_1.14.0                          
 [39] vroom_1.6.5                            
 [40] gtools_3.9.5                           
 [41] car_3.1-2                              
 [42] GO.db_3.19.1                           
 [43] Matrix_1.7-0                           
 [44] fansi_1.0.6                            
 [45] abind_1.4-8                            
 [46] R.methodsS3_1.8.2                      
 [47] lifecycle_1.0.4                        
 [48] whisker_0.4.1                          
 [49] yaml_2.3.10                            
 [50] carData_3.0-5                          
 [51] SummarizedExperiment_1.34.0            
 [52] gplots_3.1.3.1                         
 [53] qvalue_2.36.0                          
 [54] SparseArray_1.4.8                      
 [55] blob_1.2.4                             
 [56] promises_1.3.0                         
 [57] crayon_1.5.3                           
 [58] miniUI_0.1.1.1                         
 [59] lattice_0.22-6                         
 [60] KEGGREST_1.44.1                        
 [61] magick_2.8.5                           
 [62] pillar_1.9.0                           
 [63] knitr_1.48                             
 [64] fgsea_1.30.0                           
 [65] rjson_0.2.23                           
 [66] boot_1.3-31                            
 [67] codetools_0.2-20                       
 [68] fastmatch_1.1-4                        
 [69] glue_1.7.0                             
 [70] getPass_0.2-4                          
 [71] ggfun_0.1.6                            
 [72] remotes_2.5.0                          
 [73] data.table_1.16.0                      
 [74] vctrs_0.6.5                            
 [75] png_0.1-8                              
 [76] treeio_1.28.0                          
 [77] gtable_0.3.5                           
 [78] cachem_1.1.0                           
 [79] xfun_0.47                              
 [80] mime_0.12                              
 [81] S4Arrays_1.4.1                         
 [82] tidygraph_1.3.1                        
 [83] iterators_1.0.14                       
 [84] statmod_1.5.0                          
 [85] ellipsis_0.3.2                         
 [86] nlme_3.1-166                           
 [87] ggtree_3.12.0                          
 [88] bit64_4.0.5                            
 [89] rprojroot_2.0.4                        
 [90] bslib_0.8.0                            
 [91] affyio_1.74.0                          
 [92] KernSmooth_2.23-24                     
 [93] colorspace_2.1-1                       
 [94] DBI_1.2.3                              
 [95] seqPattern_1.36.0                      
 [96] tidyselect_1.2.1                       
 [97] processx_3.8.4                         
 [98] bit_4.0.5                              
 [99] compiler_4.4.1                         
[100] curl_5.2.2                             
[101] git2r_0.33.0                           
[102] httr2_1.0.4                            
[103] xml2_1.3.6                             
[104] DelayedArray_0.30.1                    
[105] shadowtext_0.1.4                       
[106] caTools_1.18.3                         
[107] callr_3.7.6                            
[108] rappdirs_0.3.3                         
[109] digest_0.6.37                          
[110] rmarkdown_2.28                         
[111] XVector_0.44.0                         
[112] htmltools_0.5.8.1                      
[113] pkgconfig_2.0.3                        
[114] MatrixGenerics_1.16.0                  
[115] highr_0.11                             
[116] fastmap_1.2.0                          
[117] GlobalOptions_0.1.2                    
[118] htmlwidgets_1.6.4                      
[119] rlang_1.1.4                            
[120] UCSC.utils_1.0.0                       
[121] shiny_1.9.1                            
[122] farver_2.1.2                           
[123] jquerylib_0.1.4                        
[124] jsonlite_1.8.8                         
[125] GOSemSim_2.30.2                        
[126] R.oo_1.26.0                            
[127] RCurl_1.98-1.16                        
[128] magrittr_2.0.3                         
[129] GenomeInfoDbData_1.2.12                
[130] ggplotify_0.1.2                        
[131] patchwork_1.3.0                        
[132] munsell_0.5.1                          
[133] Rcpp_1.0.13                            
[134] ape_5.8                                
[135] viridis_0.6.5                          
[136] stringi_1.8.4                          
[137] ggraph_2.2.1                           
[138] zlibbioc_1.50.0                        
[139] MASS_7.3-61                            
[140] pkgbuild_1.4.4                         
[141] plyr_1.8.9                             
[142] parallel_4.4.1                         
[143] Biostrings_2.72.1                      
[144] graphlayouts_1.1.1                     
[145] splines_4.4.1                          
[146] circlize_0.4.16                        
[147] hms_1.1.3                              
[148] locfit_1.5-9.10                        
[149] ps_1.8.0                               
[150] igraph_2.0.3                           
[151] pkgload_1.4.0                          
[152] reshape2_1.4.4                         
[153] futile.options_1.0.1                   
[154] XML_3.99-0.17                          
[155] evaluate_1.0.0                         
[156] lambda.r_1.2.4                         
[157] BiocManager_1.30.25                    
[158] foreach_1.5.2                          
[159] tzdb_0.4.0                             
[160] tweenr_2.0.3                           
[161] httpuv_1.6.15                          
[162] polyclip_1.10-7                        
[163] clue_0.3-65                            
[164] gridBase_0.4-7                         
[165] ggforce_0.4.2                          
[166] xtable_1.8-4                           
[167] restfulr_0.0.15                        
[168] tidytree_0.4.6                         
[169] rstatix_0.7.2                          
[170] later_1.3.2                            
[171] viridisLite_0.4.2                      
[172] aplot_0.2.3                            
[173] memoise_2.0.1                          
[174] GenomicAlignments_1.40.0               
[175] cluster_2.1.6                          
[176] timechange_0.3.0