internal.R 43.1 KB
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## Function for the rdp format
getval <- function(annotation_rdp, interest, threshold_annot){
  annotation_rdp = unlist(strsplit(annotation_rdp,"\t"))
  annotation = c(annotation_rdp[1])
  for(level in interest){
    idlevel=which(annotation_rdp == level)
    if(length(idlevel)>0){
      if(as.numeric(annotation_rdp[idlevel+1]) >= threshold_annot){
        annotation = c(annotation, gsub("\"", "", annotation_rdp[idlevel-1]))
      }
      else annotation = c(annotation, "NA")
    }
    else annotation = c(annotation, "NA")  
  }
  return(annotation)
}

## Read rdp file
read_rdp <- function(filename, threshold_annot)
{
  interest=c("phylum", "class", "order", "family", "genus")
  conn <- file(filename,open="r")
  linn <-readLines(conn)
  tab=t(sapply(1:length(linn), function(i) getval(linn[i], interest, threshold_annot)))
  close(conn)
  
  if(!TRUE%in%duplicated(tab[,1])) rownames(tab)=tab[,1];tab=tab[,-1]
  colnames(tab) = c("Phylum","Class","Order","Family","Genus")
  
  return(tab)
}









CheckCountsTable <- function(counts)
  {
    Error = NULL
    Warning = NULL
    numTest = FALSE%in%sapply(counts,is.numeric)
    if(ncol(counts)<=1){Error = "The number of columns of the counts table must be at least 2" }
    if(nrow(counts)<=1){Error = "The number of rows of the counts table must be at least 2" }
    if(numTest){Error = "The counts table must contain only numeric values" }
    if(!numTest)
    {
      if(0%in%colSums(counts)){Error = "At least one of the column of the counts table is 0" }
      if(min(counts)<0){Error = "The counts table must contain only positive values" }
    }
    if(TRUE%in%sapply(counts,is.na)){Warning = "NA values are considered as 0 is the counts table"; counts[sapply(counts,is.na)]=0}
    
  
    return(list(Error=Error,Warning=Warning,counts=counts))
  }
  
  CheckTaxoTable <- function(taxo)
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  {
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    Error = NULL
    Warning = NULL
    if(ncol(taxo)<=1){Error = "The number of columns of the taxonomy table must be at least 2" }
    if(nrow(taxo)<=1){Error = "The number of rows if the taxonomy table must be at least 2" }
    print(is.numeric(taxo[2,4]))
    if(TRUE%in%is.numeric(taxo)){Error = "The taxonomy table must contain only character" }

    for(i in 1:ncol(taxo))
    {
      level = levels(taxo[,i])
      nb = length(level)
      if(nb==1 && level=="NA"){ Error = "At least one column contains only NA"}
    }
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    return(list(Error=Error,Warning=Warning))
  }
  
  PercentAnnot <- function(counts,taxo)
  {
    Error = NULL  
    tmp = table(rownames(counts)%in%rownames(taxo))
    print(tmp)
    Percent = tmp["TRUE"]/sum(tmp)
    if(is.na(Percent)) Percent = 0
    print(Percent)
    if(Percent==0){Error = "Counts table and annotation do not matched" }
       
    return(list(Error=Error,Percent=Percent))
  }
  
  
  GetDataFromBIOM <-function(dataBIOM)
  {
    ## Counts table
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    counts = biom_data(dataBIOM)
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    counts = as.matrix(counts)
    counts = as.data.frame(counts)
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    CheckCounts = CheckCountsTable(counts)
    counts = CheckCounts$counts
    
    ## Taxonomy table
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    taxo = as.data.frame(observation_metadata(dataBIOM))
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    CheckTaxo = CheckTaxoTable(taxo)
    
    ## Pourcentage of annotation
    tmp = PercentAnnot(counts,taxo)
    
    return(list(counts=counts,taxo=taxo,CheckCounts=CheckCounts,CheckTaxo=CheckTaxo,Percent=tmp$Percent,CheckPercent=tmp$Error))
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  }
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  GetDataFromCT <-function(dataC,dataT)
  {
    
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    ## Counts table
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    counts = dataC
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    CheckCounts = CheckCountsTable(counts)
    counts = CheckCounts$counts
    
    ## Taxonomy table
    taxo = as.data.frame(dataT)
    CheckTaxo = CheckTaxoTable(taxo)
    
    ## Pourcentage of annotation
    tmp = PercentAnnot(counts,taxo)
    
    return(list(counts=counts,taxo=taxo,CheckCounts=CheckCounts,CheckTaxo=CheckTaxo,Percent=tmp$Percent,CheckPercent=tmp$Error))
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  }
  
  GetInteraction2 <- function(target)
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  { 
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    res=c()
    namesTarget = colnames(target)[2:ncol(target)]
    k=1
    for(i in 1:(length(namesTarget)-1))
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    { 
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      for(j in (i+1):length(namesTarget))
      { 
        res[k] = paste(namesTarget[i],":",namesTarget[j],sep="")
        k = k+1
      }
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    }
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    return(res)
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  }
  


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  ## Print the contrasts
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  PrintContrasts <- function (coefs, contrasts,contnames) 
  {
    contrasts = as.matrix(contrasts)
    out <-""
    
    for (i in 1:ncol(contrasts)) 
    {
      contrast <- contrasts[,i]
      contrast <- paste(ifelse(contrast > 0, "+ ", ""), contrast, sep = "")
      contrast <- gsub("( 1)|(1)", "", contrast)
      out = paste(out,paste("<b>",contnames[i], ":</b> <br/>", paste(contrast[contrast != 0], coefs[contrast != 0], collapse = " ", sep = " ")),"<br/>")
    }
    return(out)
    
  }
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  ## Get the counts for the selected taxonomy
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  GetCountsMerge <- function(input,dataInput,taxoSelect,target,design)
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  {
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    counts= NULL
    CheckTarget = FALSE
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    ## Counts and taxo tables
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    CT = dataInput$counts
    taxo = dataInput$taxo
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    ## Select cols in the target
    labels = target[,1]
    ind = which(colnames(CT)%in%labels)
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    if(length(ind)==length(labels))
    { 
      CT = CT[,ind]
      
      ## Order CT according to the target
      CT = OrderCounts(CT,labels)
#       ind0 = which(rowSums(CT)==0)
#       if(length(ind0)>0) CT = CT[-ind0,]
      
      ## Counts normalisation
      dds <- DESeqDataSetFromMatrix(countData=CT, colData=target, design=design)
      dds <- estimateSizeFactors(dds,locfunc=eval(as.name(input$locfunc)))

      CT = as.data.frame(round(counts(dds, normalized = TRUE)))
      ordOTU = order(rownames(taxo))
      indOTU_annot = which(rownames(CT)%in%rownames(taxo))
      counts_annot = CT[indOTU_annot[ordOTU],]
      
      if(taxoSelect=="OTU") counts = counts_annot
      else{
      taxoS = taxo[ordOTU,taxoSelect]
      counts = aggregate(counts_annot,by=list(Taxonomy = taxoS),sum)
      rownames(counts)=counts[,1];counts=counts[,-1]
      }
      
      ## Ordering the counts table according to the target labels 
      counts = OrderCounts(counts,labels)
      CheckTarget = TRUE
    }
    return(list(counts=counts,CheckTarget=CheckTarget))
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  }

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  ## Order the counts 
  OrderCounts <- function(counts,labels)
  {
    n = length(labels)
    CountsOrder = counts
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    for(i in 1:n)
    {
      
      ind = which(labels[i]==colnames(counts))
      CountsOrder[,i] = counts[,ind]
    }
    colnames(CountsOrder) = labels
    return(CountsOrder)
  }
  
  
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  ## Get the dds object of DESeq2
  Get_dds_object <- function(input,counts,target,design)
  {
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    dds <- DESeqDataSetFromMatrix(countData=counts, colData=target, design=design)
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    normFactors = rep(1,nrow(target))
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    ## Size factor estimation
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    #dds <- estimateSizeFactors(dds,locfunc=eval(as.name(input$locfunc)))
    #normalizationFactors(dds) <- normFactors
    sizeFactors(dds)<- normFactors
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    dds <- estimateDispersions(dds, fitType=input$fitType)
    dds <- nbinomWaldTest(dds)
    return(list(dds = dds,counts=counts,target=target,design=design))
  }

  ## Get the design according to the input
  GetDesign <- function(input)
  {
    InterVar = input$InterestVar
    Interaction = input$Interaction2
    alltmp = c(InterVar,Interaction)
    design = as.formula(paste("~", paste0(alltmp, collapse= "+")))
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    return(design)
  }
  


  ## Diagnostic Plots
  Plot_diag <- function(input,resDiff)
  {
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    #colors = c("dodgerblue","firebrick1","MediumVioletRed","SpringGreen")
    colors = c("SpringGreen","dodgerblue","black","firebrick1")
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    VarInt = input$VarInt
    dds = resDiff$dds
    counts = resDiff$counts
    target = resDiff$target
    group = as.data.frame(target[,VarInt])
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    rownames(group) = rownames(target)
    
    ## If more than 4 levels for one factor
    if(length(VarInt)>1)  maxFact =max(sapply(group,FUN=function(x) length(levels(x))))
    else maxFact = length(levels(group))
    if(maxFact>=4) colors = rainbow(maxFact) 
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    if(input$DiagPlot=="barplotTot") barplotTot(input,counts,group = group, col=colors)
    if(input$DiagPlot=="barplotNul") barPlotNul(input,counts, group = group, col=colors)
    if(input$DiagPlot=="densityPlot") densityPlotTot(input,counts, group = group, col=colors)
    if(input$DiagPlot=="MajTax") majTaxPlot(input,counts, group = group, col=colors)
    if(input$DiagPlot=="SERE") SEREplot(input,counts)
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    if(input$DiagPlot=="Sfactors") diagSFactors(input,dds,frame=1) 
    if(input$DiagPlot=="SfactorsVStot") diagSFactors(input,dds,frame=2) 
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    if(input$DiagPlot=="pcaPlot") PCAPlot_meta(input,dds, group,  type.trans = input$TransType, col = colors)
    if(input$DiagPlot=="pcoaPlot") PCoAPlot_meta(input,dds, group) 
    if(input$DiagPlot=="clustPlot") HCPlot(input,dds,group,type.trans=input$TransType)
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  }

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#   HCPlot <- function (input,dds,group,type.trans,col = c("lightblue", "orange", "MediumVioletRed", "SpringGreen")) 
#   {
#     counts = as.data.frame(round(counts(dds, normalized = TRUE)))
#     if (type.trans == "VST") counts.trans <- assay(varianceStabilizingTransformation(dds))
#     if (type.trans == "rlog") counts.trans <- assay(rlogTransformation(dds))
#     
#     hc <- hclust(dist(t(counts.trans)), method = "ward.D")
#     
#     type <- switch(input$typeHculst,
#                   "radial"="radial",
#                   "fan"="fan",
#                   "triangle"="cladogram",,
#                   "hori"= "hori",
#                   "verti"=NULL)
#     
#     par(cex=input$cexLabelDiag,mar=c(12,5,8,5))
#     if(input$colorHC && type=="hori") 
#     {
#       hc = dendrapply(as.dendrogram(hc),colLabdendo,group) 
#       plot(hc, xlab = "Euclidean distance, Ward criterion", main = "Cluster dendrogram")
#     }
#     
#     if(!input$colorHC && type=="hori") 
#     {
#       plot(hc, xlab = "Euclidean distance, Ward criterion", main = "Cluster dendrogram",hang=-1)
#     }
#     
#     if(type!="hori") 
#     { 
#       group = apply(group,1,paste, collapse = "-")
#       nb = length(unique(group))
#       plot(as.phylo(hc), type= type,label.offset = 1, tip.color = ifelse(input$colorHC, rainbow(nb)[as.integer(as.factor(group))], rep(1,nb)))
#     }
#     dev.off() 
#   }
  
  HCPlot <- function (input,dds,group,type.trans,col = c("lightblue", "orange", "MediumVioletRed", "SpringGreen")) 
  {
    
    ## Get the counts
    counts = as.data.frame(round(counts(dds, normalized = TRUE)))
    if (type.trans == "VST") counts.trans <- assay(varianceStabilizingTransformation(dds))
    if (type.trans == "rlog") counts.trans <- assay(rlogTransformation(dds))
    
    ## Get the group of leaf
    group = apply(group,1,paste, collapse = "-")    
    nb = length(unique((group)))
    
    ## Get the dendrogram
    hc <- hclust(dist(t(counts.trans)), method = "ward.D")
    dend = as.dendrogram(hc)
    
    ## Get the type of dendrogram
    type <- switch(input$typeHculst,
                   "fan"="fan",
                   "hori"= "hori")
    
    dend <- set(dend, "labels_cex", input$cexLabelDiag)
    if(input$colorHC) labels_colors(dend)<-rainbow(nb)[as.integer(as.factor(group))][order.dendrogram(dend)]
    
    if(type=="hori") 
    { 
      par(mar = c(8,4,4,2))
      plot(dend, main = "Cluster dendrogram")
    }  
    if(type!="hori")
    {
      par(mar = c(0.3,2,0.3,2))
      circlize_dendrogram(dend, labels_track_height = 0.2, dend_track_height = .3, main = "Cluster dendrogram")
    }
  }
  
  
  ## Color for the horizontal dendro
  colLabdendo <- function(n,group) {
    
    group = apply(group,1,paste, collapse = "-")
    
    nb = length(unique((group)))
    namesGrp = names(group)
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    if (is.leaf(n)) {
      a <- attributes(n)
      labCol <- rainbow(nb)[as.integer(as.factor(group))[which(namesGrp == a$label)]]
      attr(n, "nodePar") <- c(a$nodePar, lab.col = labCol)
    }
    return(n)
  }
  
  ## Diagnostic Plots Eigen value
  Plot_diag_Eigen <- function(input,resDiff)
  {
    colors = c("dodgerblue","firebrick1","MediumVioletRed","SpringGreen")
    VarInt = input$VarInt
    dds = resDiff$dds
    counts = resDiff$counts
    target = resDiff$target
    group = as.data.frame(target[,VarInt])
    
    ## If more than 4 levels for one factor
    maxFact =max(sapply(group,FUN=function(x) length(levels(x))))
    if(maxFact>=4) colors = rainbow(maxFact) 
    
    PCAPlot_meta(input,dds, group,  type.trans = input$TransType, col = colors, plot = "eigen") 
  }
  
  Plot_diag_pcoaEigen = function(input,resDiff)
  {
    colors = c("SpringGreen","dodgerblue","black","firebrick1")
    VarInt = input$VarInt
    dds = resDiff$dds
    target = resDiff$target
    group = as.data.frame(target[,VarInt])
    rownames(group) = rownames(target)
    PCoAPlot_meta(input,dds, group, col = colors, plot = "eigen") 
  }
  
  
  
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  ## barplot total
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  barplotTot <- function(input,counts, group, cex.names = 1, col = c("lightblue","orange", "MediumVioletRed", "SpringGreen")) 
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  {
    ncol1 <- ncol(group) == 1
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    par(cex=input$cexLabelDiag,mar=c(12,5,4,5))
    barplot(colSums(counts), cex.names = cex.names, main = "Total mapped read count per sample", ylab = "Total mapped read count", 
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            ylim = c(0, max(colSums(counts)) * 1.2), density = if (ncol1) {NULL}
            else {15}, 
            angle = if (ncol1) {NULL}
            else {c(-45, 0, 45, 90)[as.integer(group[, 2])]}, col = col[as.integer(group[, 1])], las = 2)
    legend("topright", levels(group[, 1]), fill = col[1:nlevels(group[,1])], bty = "n")
    if (!ncol1)  legend("topleft", levels(group[, 2]), density = 15,col = 1, angle = c(-45, 0, 45, 90)[1:nlevels(group[, 2])], bty = "n")
  
  }


  ## barplot Nul 
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  barPlotNul <-function (input,counts, group, cex.names = 1, col = c("lightblue","orange", "MediumVioletRed", "SpringGreen")) 
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  {
    
    percentage <- apply(counts, 2, function(x) {sum(x == 0)}) * 100/nrow(counts)
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    percentage.allNull <- (nrow(counts) - nrow(removeNulCounts(counts))) * 100/nrow(counts)
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    ncol1 <- ncol(group) == 1
    
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    par(cex=input$cexLabelDiag,mar=c(12,5,4,5))

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    barplot(percentage, las = 2, col = col[as.integer(group[,1])], 
            density = if (ncol1) {NULL}
            else {15}, 
            angle = if (ncol1) {NULL}
            else {c(-45, 0, 45, 90)[as.integer(group[, 2])]},
            cex.names = cex.names, ylab = "Proportion of null counts", 
            main = "Proportion of null counts per sample", 
            ylim = c(0, 1.2 * ifelse(max(percentage) == 0, 1, max(percentage))))
    
    abline(h = percentage.allNull, lty = 2, lwd = 2)
    legend("topright", levels(group[, 1]), fill = col[1:nlevels(group[,1])], bty = "n")
    if (!ncol1) legend("topleft", levels(group[, 2]), density = 15, col = 1, angle = c(-45, 0, 45, 90)[1:nlevels(group[, 2])], bty = "n")
  }


  ## Plot density
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  densityPlotTot <-function (input,counts, group, col = c("lightblue", "orange", "MediumVioletRed", "SpringGreen")) 
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  {
    
    counts <- removeNulCounts(counts)
    ncol1 <- ncol(group) == 1
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    par(cex=input$cexLabelDiag,mar=c(8,5,4,5))
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    plot(density(log2(counts[, 1] + 1)), las = 1, lwd = 2, main = "Density of counts distribution", 
         xlab = expression(log[2] ~ (raw ~ count + 1)), 
         ylim = c(0, max(apply(counts, 2, function(x) {max(density(log2(x + 1))$y)})) * 1.05), 
         lty = if (ncol1) {1}
         else{c(1, 2, 3, 4)[as.integer(group[, 2])[1]]}, 
         col = col[as.integer(group[, 1])[1]])
    
    for (i in 2:ncol(counts)) 
    {
      lines(density(log2(counts[, i] + 1)), col = col[as.integer(group[,1])[i]], lwd = 2, 
            lty = if (ncol1) {1}
            else {c(1, 2, 3, 4)[as.integer(group[, 2])[i]]})
    }
    legend("topright", levels(group[, 1]), lty = 1, col = col[1:nlevels(group[,1])], lwd = 2, bty = "n")
    if (!ncol1) legend("topleft", levels(group[, 2]), lty = c(1, 2, 3, 4)[1:nlevels(group[, 2])], col = 1, lwd = 2, bty = "n")
    
  }


  ## Table of maj. taxo
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  majTab <- function(input,counts,n)
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  {
    seqnames <- apply(counts, 2, function(x) {
      x <- sort(x, decreasing = TRUE)
      names(x)[1:n]
    })
    seqnames <- unique(unlist(as.character(seqnames)))
    sum <- apply(counts, 2, sum)
    counts <- counts[seqnames, ]
    sum <- matrix(sum, nrow(counts), ncol(counts), byrow = TRUE)
    p <- round(100 * counts/sum, digits = 3)
    return(p)
  }


  ## Plot maj. taxo
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  majTaxPlot <-function (input,counts, n = 3, group, cex.names = 1, col = c("lightblue",  "orange", "MediumVioletRed", "SpringGreen")) 
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  {
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    p = majTab(input,counts,n)
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    maj <- apply(p, 2, max)
    seqname <- rownames(p)[apply(p, 2, which.max)]
    ncol1 <- ncol(group) == 1
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    x <- barplot(maj, col = col[as.integer(group[, 1])], main = "Proportion of mapped reads from\nmost expressed sequence",
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                 ylim = c(0, max(maj) * 1.2), cex.main = 1, 
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                 cex.names = cex.names, las = 2, ylab = "Proportion of mapped reads", 
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                 density = if (ncol1) {NULL}
                 else {15}, 
                 angle = if (ncol1) {NULL}
                 else {c(-45, 0, 45, 90)[as.integer(group[, 2])]})
    
    legend("topright", levels(group[, 1]), fill = col[1:nlevels(group[,1])], bty = "n")
    if (!ncol1) legend("topleft", levels(group[, 2]), density = 15, col = 1, 
                       angle = c(-45, 0, 45, 90)[1:nlevels(group[, 2])], bty = "n")
    
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    for (i in 1:length(seqname)) text(x[i], maj[i]/2, seqname[i], cex=input$cexLabelDiag, srt = 90, adj = 0)
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  }
  

  ## plot SERE Coefs
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  SEREplot<-function(input,counts) 
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  {
    sere = SEREcoef(counts)
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    print(sere)
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    hc <- hclust(as.dist(sere), method = "ward.D")
    plot(hc, las = 2, hang = -1, xlab = "SERE distance, Ward criterion",main = "Cluster dendrogram\non SERE values")
    
  }
  
  
  ## Get the SERE COEF
  SEREcoef<-function(counts)
  {
    sere <- matrix(NA, ncol = ncol(counts), nrow = ncol(counts))
    for (i in 1:ncol(counts)) {
      for (j in 1:ncol(counts)) {
        sere[i, j] <- sigfun_Pearson_meta(counts[, c(i, j)])
      }
    }
    colnames(sere) <- rownames(sere) <- colnames(counts)
    sere <- round(sere, digits = 3)
    
    return(sere) 
  }
  

  ## function for the SERE coef
  sigfun_Pearson_meta <- function(observed) {
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    print("OK1")
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    laneTotals <- colSums(observed)
    total <- sum(laneTotals)
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    print("OK2")
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    fullObserved <- observed[rowSums(observed) > 0, ]
    fullLambda <- rowSums(fullObserved)/total
    fullLhat <- fullLambda > 0
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    print("OK3")
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    fullExpected <- outer(fullLambda, laneTotals)
    fullKeep <- which(fullExpected > 0)
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    print(fullKeep)
    print(fullExpected)
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    oeFull <- (fullObserved[fullKeep] - fullExpected[fullKeep])^2/fullExpected[fullKeep]
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    print(oeFull)
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    dfFull <- length(fullKeep) - sum(fullLhat != 0)
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    print(dfFull)
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    sqrt(sum(oeFull)/dfFull)
  }
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  ## Plots of size factors
  diagSFactors<-function (input,dds,frame=1) 
  {
    geomeans <- exp(rowMeans(log(counts(dds))))
    samples <- colnames(counts(dds))
    counts.trans <- log2(counts(dds)/geomeans)
    xmin <- min(counts.trans[is.finite(counts.trans)], na.rm = TRUE)
    xmax <- max(counts.trans[is.finite(counts.trans)], na.rm = TRUE)
    
    if(!is.na(input$NbcolSfactors)) parCols = as.numeric(input$NbcolSfactors)
    else parCols = ceiling(ncol(counts.trans)/3)
    
    parRows = ceiling(ncol(counts.trans)/parCols)
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    if(frame==1)
    {
      par(mfrow=c(parRows,parCols))
      for (j in 1:ncol(dds)) {
        hist(log2(counts(dds)[, j]/geomeans), nclass = 100, 
             xlab = expression(log[2] ~ (counts/geometric ~ mean)), las = 1, xlim = c(xmin, xmax), 
             main = paste("Size factors diagnostic - Sample ",samples[j], sep = ""), col = "skyblue")
        
        abline(v = log2(sizeFactors(dds)[j]), col = "red", lwd = 1.5)
      }
    }
    
    if(frame==2)
    {
      plot(sizeFactors(dds), colSums(counts(dds)), pch = 19, las = 1, 
           ylab = "Total number of reads", xlab = "Size factors", 
           main = "Diagnostic: size factors vs total number of reads")
      abline(lm(colSums(counts(dds)) ~ sizeFactors(dds) + 0), lty = 2, col = "grey")
    }
  }
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  ### PCoA
  PCoAPlot_meta <-function (input,dds, group_init,col = c("SpringGreen","dodgerblue","black","firebrick1"), plot = "pcoa") 
  {
    cval=c()
    # Set of shape
    shape=c(19,17,15,18)
    ## Var of interest
    VarInt  = input$VarInt
    ## Group
    group = as.character(apply(group_init,1,paste, collapse = "-"))
    
    ## Keep only some sample 
    val = c()
    for(i in 1:length(VarInt))
    { 
      Tinput = paste("input$","Mod",VarInt[i],sep="")
      expr=parse(text=Tinput)
      ## All the modalities for all the var of interest
      val = c(val,eval(expr))
    }
    if(length(VarInt)>1) Kval = apply(expand.grid(val,val),1,paste, collapse = "-")
    else Kval = val
    ind_kept = which(as.character(group)%in%Kval)
    ## Get the group corresponding to the modalities
    group = group[ind_kept]
    nb = length(unique((group)))
    group = as.factor(group)
    
    ## Get the norm data
    counts.norm = as.data.frame(round(counts(dds, normalized = TRUE)))
    
    # was removed
    counts.norm = counts.norm[,ind_kept]
    
    ## Get the distance
    dist.counts.norm = vegdist(t(counts.norm), method = input$DistPCOA)
    
    ## Do PCoA
    pco.counts.norm = dudi.pco(d = dist.counts.norm, scannf = FALSE,nf=3)
    
    ## Get eigen values
    eigen=(pco.counts.norm$eig/sum(pco.counts.norm$eig))*100
    
    
    ## xlim and ylim of the plot
    min = min(pco.counts.norm$li)
    max = max(pco.counts.norm$li)
    
    ## get condition set
    condition_set=val[which(val %in% unique(group_init$condition))]
    time_set=val[which(val %in% unique(group_init$time))]
    
    ## Colors
    if(length(col)<length(condition_set) * length(time_set))# && !input$colorgroup)
    {
      col = rainbow(length(condition_set) * length(time_set))
    }
    #else if(length(col)<length(condition_set) * length(time_set) && input$colorgroup){
    #  col = rep(col[1:length(condition_set)], length(time_set))
    #}
    print(condition_set)
    print(time_set)
    if (length(time_set) == 1 && length(condition_set) <= 4){
      cval = apply(expand.grid(condition_set,time_set),1,paste, collapse = "-")
      cval = sort(cval)
    }
    print(col)
    # to reactivate
    #pco.counts.norm$li = pco.counts.norm$li[ind_kept,]
    if (plot == "pcoa"){
      ## Plot axis, label and circles
      plot(pco.counts.norm$li[1:2], xlab=paste("PC1 : ",round(eigen[1],1),"%") , ylab=paste("PC2 : ",round(eigen[2],1),"%"),
           xlim=c(min+0.25*min,max+0.25*max), ylim=c(min-0.1,max+0.1), cex.axis=1, cex.lab=1,lwd=2, type="n")
      # Set different shapes
      if(input$labelPCOA == "Group"){
        print(cval)
        print(length(cval))
        if(!is.null(cval)){
          for (i in 1:length(cval)){
            points(pco.counts.norm$li[which(group==cval[i]),1:2],pch=shape[i],col=col[i], cex=input$cexpoint)
          }
          s.class(dfxy = pco.counts.norm$li, fac = group, col = col, label = levels(group),
                  add.plot = TRUE, cpoint = 0, cell=input$cexcircle, clabel=input$cexLabelDiag,  cstar = input$cexstar)
        }else s.class(dfxy = pco.counts.norm$li, fac = group, col = col, label = levels(group),
                      add.plot = TRUE, cpoint = input$cexpoint, cell=input$cexcircle, clabel=input$cexLabelDiag,  cstar = input$cexstar)
      }  
      else{
        s.label(pco.counts.norm$li, clabel = input$cexLabelDiag,boxes=FALSE, add.plot = TRUE)
        s.class(dfxy = pco.counts.norm$li, fac = group, col = col, label = levels(group), add.plot = TRUE, cpoint = 0, clabel = 0, cstar = input$cexstar, cell=input$cexcircle)
      }
    }else{
      barplot(eigen[1:7], xlab="Dimensions", ylab="Eigenvalues (%)", names.arg = 1:7, col = c(rep("black", 2), rep("grey", 5)), ylim=c(0,max(eigen)+5), cex.axis=1.2, cex.lab=1.4,cex.names=1.2)
    }
   
  }
  
  ### PCA
  PCAPlot_meta <-function (input,dds, group, n = min(500, nrow(counts(dds))), type.trans = c("VST", "rlog"), 
                           col = c("lightblue", "orange", "MediumVioletRed", "SpringGreen"),plot="pca") 
  {
    type.trans <- type.trans[1]
    
    if (type.trans == "VST") counts.trans <- assay(varianceStabilizingTransformation(dds))
    else counts.trans <- assay(rlogTransformation(dds))
    
    rv = apply(counts.trans, 1, var, na.rm = TRUE)
    pca = prcomp(t(counts.trans[order(rv, decreasing = TRUE),][1:n, ]))
    
   
    
    
    
    if(plot=="pca")
    { 
      prp <- pca$sdev^2 * 100/sum(pca$sdev^2)
      prp <- round(prp, 2)
      ncol1 <- ncol(group) == 1
      
      par(mfrow = c(1, 2))
      
      abs = range(pca$x[, 1])
      abs = abs(abs[2] - abs[1])/25
      ord = range(pca$x[, 2])
      ord = abs(ord[2] - ord[1])/25
      
      par(mar=c(8,5,4,5))
      plot(pca$x[, 1], pca$x[, 2], las = 1, cex = 2, col = col[as.integer(group[,1])], 
           pch = if (ncol1) {16}
           else {c(16:18, 25)[as.integer(group[, 2])]},
           xlab = paste0("PC1 (", prp[1], "%)"),
           ylab = paste0("PC2 (", prp[2], "%)"), 
           main = "Principal Component Analysis",
            )
      abline(h = 0, v = 0, lty = 2, col = "lightgray")
      text(pca$x[, 1] - ifelse(pca$x[, 1] > 0, abs, -abs), pca$x[,2] - ifelse(pca$x[, 2] > 0, ord, -ord), colnames(counts.trans), col = col[as.integer(group[, 1])])
      abs = range(pca$x[, 1])
      abs = abs(abs[2] - abs[1])/25
      ord = range(pca$x[, 3])
      ord = abs(ord[2] - ord[1])/25
      plot(pca$x[, 1], pca$x[, 3], las = 1, cex = 2, col = col[as.integer(group[, 1])], 
           pch = if (ncol1) {16}
           else {c(16:18, 25)[as.integer(group[, 2])]}, 
           xlab = paste0("PC1 (", prp[1], "%)"), 
           ylab = paste0("PC3 (", prp[3], "%)"), 
           main = "Principal Component Analysis")
      abline(h = 0, v = 0, lty = 2, col = "lightgray")
      text(pca$x[, 1] - ifelse(pca$x[, 1] > 0, abs, -abs), pca$x[,3] - ifelse(pca$x[, 3] > 0, ord, -ord), colnames(counts.trans), col = col[as.integer(group[, 1])],cex=input$cexLabelDiag)
    }
    
    if(plot=="eigen") barplot(pca$sdev^2, main = "Eigen values of the PCA", names.arg = 1:length(pca$sdev), xlab = "Axes")
  }
  
  
  
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  ############################################################
  ##
  ##              CREATE THE CONTRAST DATABASE
  ##
  ############################################################
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  BaseContrast <- function(input,names,namesfile)
  {  

    v_tmp = c()
    filesize = file.info(namesfile)[,"size"]
    
    for(i in 1:length(names))
    {  
      Tinput = paste("input$",names[i],sep="")
      expr=parse(text=Tinput)
      val = eval(expr) 
      v_tmp[i] = as.numeric(val)
    }
    
    if(filesize!=0)
    { 
      oldContrast = read.table(namesfile,header=TRUE)
      colnamesTmp = c(colnames(oldContrast),input$ContrastName)
      mat = cbind(oldContrast,v_tmp)
    }
    else{ colnamesTmp = input$ContrastName; mat = v_tmp}
    
    write.table(mat,namesfile,row.names=FALSE,col.names = colnamesTmp)
  }
  
  
  ## Remove nul counts
  removeNulCounts <-function (counts) 
  {
    return(counts[rowSums(counts) > 0, ])
  }
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  ############################################################
  ##
  ##              VISUALISATION PLOTS
  ##
  ############################################################
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  GetDataToPlot <- function(resDiff,VarInt,ind_taxo,aggregate=TRUE)
  {
    dds = resDiff$dds
    counts = as.data.frame(round(counts(dds, normalized = TRUE)))
    target = resDiff$target
    counts_tmp_combined = NULL
    prop_tmp_combined = NULL
    targetInt = NULL
    ## Select a subset within the taxonomy level (default is the 12 most abundant)
    nbKept = length(ind_taxo)
    Taxonomy = rownames(counts)
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    if (length(VarInt)>0 && nbKept>0)
    { 
      ## Create the variable to plot
      targetInt = as.data.frame(target[,VarInt])
      rownames(targetInt)=target[,1]  
      if(length(VarInt)>1) targetInt$AllVar = apply(targetInt,1,paste, collapse = "-")
      if(length(VarInt)<=1)  targetInt$AllVar = target[,VarInt]
      colnames(targetInt) = c(VarInt,"AllVar")
      ## Create the counts matrix only for the selected subset
      counts_tmp = counts[Taxonomy%in%ind_taxo,]
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      ## Be careful transposition !
      if(aggregate)
      { 
        counts_tmp_combined = aggregate(t(counts_tmp),by=list(targetInt$AllVar),sum)
        rownames(counts_tmp_combined) = counts_tmp_combined$Group.1
        counts_tmp_combined = as.matrix(counts_tmp_combined[,-1])
      }
      if(!aggregate)
      {  
        counts_tmp_combined = t(counts_tmp)
        prop_tmp_combined = counts_tmp_combined/colSums(counts)
        rownames(counts_tmp_combined) = targetInt$AllVar
        rownames(prop_tmp_combined) = targetInt$AllVar
      }
      ## Ordering the counts
      MeanCounts = apply(counts_tmp_combined,2,mean)
      ord = order(MeanCounts,decreasing=TRUE)
      counts_tmp_combined = as.matrix(counts_tmp_combined[,ord])
      if(!aggregate) prop_tmp_combined = as.matrix(prop_tmp_combined[,ord])
    }
    
      return(list(counts = counts_tmp_combined,targetInt=targetInt,prop=prop_tmp_combined))
    
    
  }
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  ###########################
  ## Plots for visualisation
  ###########################
  
  Plot_Visu_Barplot <- function(input,resDiff)
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  {
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    ## Get Input for BarPlot
    VarInt = input$VisuVarIntBP
    ind_taxo = input$selectTaxoPlotBP
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    counts_tmp_combined = GetDataToPlot(resDiff,VarInt,ind_taxo)$counts
    nbKept = length(ind_taxo)
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    if(!is.null(counts_tmp_combined) && nrow(counts_tmp_combined)>0)
    { 
      counts_tmp_combined = GetDataToPlot(resDiff,VarInt,ind_taxo)$counts
      Taxonomy = rownames(counts_tmp_combined)
      ## Create the data frame for the plot function
      dataBarPlot_mat = c()
      tmp_mat = matrix(0,ncol=3,nrow=nbKept)
      tmp_counts = c()
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        for(i in 1:(nrow(counts_tmp_combined)))
        {
          ## Taxo
          tmp_mat[1:nbKept,1] = colnames(counts_tmp_combined)
          
          ## Counts
  
          tmpProp = counts_tmp_combined[i,]
          if(input$CountsOrProp=="prop")
          { 
            tmpProp = round(tmpProp/sum(tmpProp),3)
            tmpProp = as.numeric(tmpProp/sum(tmpProp) * 100)
          }
          tmp_counts = c(tmp_counts,tmpProp)      
          
          ## Meta data
          tmp_mat[1:nbKept,3] = as.character(rep(rownames(counts_tmp_combined)[i],nbKept))
          
          ## Conbined the sample
          dataBarPlot_mat = rbind(dataBarPlot_mat,tmp_mat)
        }
        
        
        ## Add numeric vector to the dataframe
        dataBarPlot_mat = as.data.frame(dataBarPlot_mat)
        
        colnames(dataBarPlot_mat) = c("Taxonomy","Proportions","AllVar")
        dataBarPlot_mat[,2] = tmp_counts
  
        plotd3 <- nvd3Plot(Proportions ~ AllVar | Taxonomy, data = dataBarPlot_mat, type = input$SensPlotVisuBP, id = 'barplotTaxo',height = input$heightVisu,width=input$widthVisu)
        plotd3$chart(stacked = TRUE)
    } 
    else{ 
      ## Pb affichage quand data NULL
      dataNull = data.frame(x=c(1,2),y=c(1,2))
      plotd3 = nvd3Plot(x ~ y , data = dataNull, type = "multiBarChart", id = 'barplotTaxoNyll',height = input$heightVisu,width=input$widthVisu)
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    }
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    return(plotd3)
  }
  
  
  
######################################################
##
##            HEATMAP
##
######################################################
  
  
  Plot_Visu_Heatmap <- function(input,resDiff){
  
  VarInt = input$VisuVarIntHM
  ind_taxo = input$selectTaxoPlotHM
  
  counts_tmp_combined = GetDataToPlot(resDiff,VarInt,ind_taxo)$counts
  
  if(!is.null(counts_tmp_combined) && nrow(counts_tmp_combined)>0)
  { 
    ## Transform to log2
    counts_tmp_combined = log2(GetDataToPlot(resDiff,VarInt,ind_taxo)$counts+1)
   
    col <- switch(input$colors,
                  "green-blue"=colorRampPalette(brewer.pal(9,"GnBu"))(200),
                  "blue-white-red"=colorRampPalette(rev(brewer.pal(9, "RdBu")))(200),
                  "purple-white-orange"=colorRampPalette(rev(brewer.pal(9, "PuOr")))(200),
                  "red-yellow-green"= colorRampPalette(rev(brewer.pal(9,"RdYlGn")))(200))
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    ## Transpose matrix if Horizontal
    if(input$SensPlotVisuHM=="Horizontal") counts_tmp_combined = t(as.matrix(counts_tmp_combined))
         #print(counts_tmp_combined)
    return(heatmap.2(counts_tmp_combined, dendrogram = "none", Rowv = NA, Colv = NA, na.rm = TRUE, density.info="none", margins=c(12,8),trace="none",srtCol=45,
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                    col = col, scale = input$scaleHeatmap,cexRow = 0.6))
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#     return(d3heatmap(counts_tmp_combined, dendrogram = "none", Rowv = NA, Colv = NA, na.rm = TRUE, 
#                      width = 1500, height = 1000, show_grid = FALSE, colors = col, scale = input$scaleHeatmap,
#                      cexRow = 0.6))
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  }
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  }

  ######################################################
  ##
  ##            BOXPLOT
  ##
  ######################################################
  
  
  Plot_Visu_Boxplot <- function(input,resDiff){
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    gg = NULL
    ## Get Input for BoxPlot
    VarInt = input$VisuVarIntBoxP
    ind_taxo = input$selectTaxoPlotBoxP
    
    tmp_merge = GetDataToPlot(resDiff,VarInt,ind_taxo,aggregate=FALSE)
    counts_tmp_combined = tmp_merge$counts

    nbKept = length(ind_taxo)
    
    if(!is.null(counts_tmp_combined) && nrow(counts_tmp_combined)>0)
    { 
      Taxonomy = rownames(counts_tmp_combined)
    
      if(input$typeDataBox == "Relative") 
      { 
        counts_tmp_combined = tmp_merge$prop
      }
      if(input$typeDataBox == "Log2") counts_tmp_combined = log2(counts_tmp_combined+1)
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      if(nbKept==1) colnames(counts_tmp_combined)=ind_taxo
    

      ## Create the data frame for the plot function
      dataBarPlot_mat = c()
      tmp_mat = matrix(0,ncol=3,nrow=nbKept)
      tmp_counts = c()
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      for(i in 1:(nrow(counts_tmp_combined)))
      {
        ## Taxo
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