# Section title

This content has been loaded from the template report `.Rmd` file. Please edit it at your best convenience!

```{r setup, include=FALSE, eval = TRUE, echo = FALSE}
opts_chunk$set(
  echo=input$report_echo
)
```


# Overview on the data

The data provided was used to construct the following objects

```{r}
values$mydds

values$myrlt

head(values$myannotation)
```

The following design were used:

```{r}
DT::datatable(as.data.frame(colData(values$mydds)))
```

An overview of the table for the features is shown here, by displaying the `r input$countstable_unit`

```{r}
if(input$countstable_unit=="raw_counts")
  currentMat <- counts(values$mydds,normalized=FALSE)
if(input$countstable_unit=="normalized_counts")
  currentMat <- counts(values$mydds,normalized=TRUE)
if(input$countstable_unit=="rlog_counts")
  currentMat <- assay(values$myrlt)
if(input$countstable_unit=="log10_counts")
  currentMat <- log10(1 + counts(values$mydds,normalized=TRUE))
```


```{r}
DT::datatable(currentMat)
```

This is how the samples cluster if we use euclidean distance on the rlog transformed values

```{r}
if (!is.null(input$color_by)){
  expgroups <- as.data.frame(colData(values$myrlt)[,input$color_by])
  # expgroups <- interaction(expgroups)
  rownames(expgroups) <- colnames(values$myrlt)
  colnames(expgroups) <- input$color_by

  pheatmap(as.matrix(dist(t(assay(values$myrlt)))),annotation_col = expgroups)
} else {
  pheatmap(as.matrix(dist(t(assay(values$myrlt)))))
}

```


This is an overview of the number of available reads in each sample (normally these are only uniquely aligned reads)


```{r}
rr <- colSums(counts(values$mydds))/1e6
if(is.null(names(rr)))
  names(rr) <- paste0("sample_",1:length(rr))
rrdf <- data.frame(Reads=rr,Sample=names(rr),stringsAsFactors = FALSE)
if (!is.null(input$color_by)) {
  selGroups <- as.data.frame(colData(values$mydds)[input$color_by])
  rrdf$Group <- interaction(selGroups)
  p <- ggplot(rrdf,aes_string("Sample",weight="Reads")) + geom_bar(aes_string(fill="Group")) + theme_bw()
  p
} else {
  p <- ggplot(rrdf,aes_string("Sample",weight="Reads")) + geom_bar() + theme_bw()
  p
}

print(colSums(counts(values$mydds)))
summary(colSums(counts(values$mydds))/1e6)      
```


This is a quick info on the number of detected genes

```{r}
t1 <- rowSums(counts(values$mydds))
t2 <- rowMeans(counts(values$mydds,normalized=TRUE))

thresh_rowsums <- input$threshold_rowsums
thresh_rowmeans <- input$threshold_rowmeans
abs_t1 <- sum(t1 > thresh_rowsums)
rel_t1 <- 100 * mean(t1 > thresh_rowsums)
abs_t2 <- sum(t2 > thresh_rowmeans)
rel_t2 <- 100 * mean(t2 > thresh_rowmeans)

cat("Number of detected genes:\n")
# TODO: parametrize the thresholds
cat(abs_t1,"genes have at least a sample with more than",thresh_rowsums,"counts\n")
cat(paste0(round(rel_t1,3),"%"), "of the",nrow(values$mydds),"genes have at least a sample with more than",thresh_rowsums,"counts\n")
cat(abs_t2,"genes have more than",thresh_rowmeans,"counts (normalized) on average\n")
cat(paste0(round(rel_t2,3),"%"), "of the",nrow(values$mydds),"genes have more than",thresh_rowsums,"counts (normalized) on average\n")
cat("Counts are ranging from", min(counts(values$mydds)),"to",max(counts(values$mydds)))
```


# PCA on the samples

This plot shows how the samples are related to each other by plotting PC `r input$pc_x` vs PC `r input$pc_y`, using the top `r input$pca_nrgenes` most variant genes

```{r}
res <- pcaplot(values$myrlt,intgroup = input$color_by,ntop = input$pca_nrgenes,
                     pcX = as.integer(input$pc_x),pcY = as.integer(input$pc_y),
                     text_labels = input$sample_labels,
                     point_size = input$pca_point_size, title="Samples PCA - zoom in",
                     ellipse = input$pca_ellipse, ellipse.prob = input$pca_cislider
      )
res <- res + theme_bw()
res
```


The scree plot helps determining the number of underlying principal components

```{r}
rv <- rowVars(assay(values$myrlt))
select <- order(rv, decreasing = TRUE)[seq_len(min(input$pca_nrgenes,length(rv)))]
pca <- prcomp(t(assay(values$myrlt)[select, ]))

res <- pcascree(pca,type = input$scree_type, pc_nr = input$scree_pcnr, title="Scree plot for the samples PCA")
res <- res + theme_bw()
res
```



The genes with the highest loadings in the selected principal components are the following

```{r}
rv <- rowVars(assay(values$myrlt))
select <- order(rv, decreasing = TRUE)[seq_len(min(input$pca_nrgenes,length(rv)))]
pca <- prcomp(t(assay(values$myrlt)[select, ]))

par(mfrow=c(2,1))
hi_loadings(pca,whichpc = as.integer(input$pc_x),topN = input$ntophiload,annotation = values$myannotation)
hi_loadings(pca,whichpc = as.integer(input$pc_y),topN = input$ntophiload,annotation = values$myannotation)
```



# PCA on the genes

This plot illustrates how the top `r input$pca_nrgenes` variant genes are distributed in PC `r input$pc_x` vs PC `r input$pc_y`

```{r}
if(!is.null(input$color_by)) {
        expgroups <- as.data.frame(colData(values$myrlt)[,input$color_by])
        expgroups <- interaction(expgroups)
        expgroups <- factor(expgroups,levels=unique(expgroups))

      } else {
        expgroups <- colnames(values$myrlt)
      }
      colGroups <- colSel()[factor(expgroups)]

res <- genespca(values$myrlt,
                ntop = input$pca_nrgenes,
                choices = c(as.integer(input$pc_x),as.integer(input$pc_y)),
                biplot = TRUE,
                arrowColors = factor(colGroups,levels=unique(colGroups)),
                groupNames = expgroups,
                alpha=input$pca_point_alpha,coordEqual=FALSE,useRownamesAsLabels=FALSE,labels.size=input$pca_label_size,
                point_size=input$pca_point_size,varname.size=input$pca_varname_size, scaleArrow = input$pca_scale_arrow,
                annotation=values$myannotation)
res
```


For the selected genes, this is the overall profile across all samples

```{r}
if(!is.null(input$pcagenes_brush) & length(input$color_by)>0)
      geneprofiler(values$myrlt,
                   genelist = curData_brush()$ids,
                   intgroup = input$color_by,
                   plotZ = input$zprofile)
```


And here is an interactive heatmap for that subset

```{r}

if(!is.null(input$pcagenes_brush))
{
  brushedObject <- curData_brush()
  if(nrow(brushedObject) > 1){
    selectedGenes <- brushedObject$ids
    toplot <- assay(values$myrlt)[selectedGenes,]
    rownames(toplot) <- values$myannotation$gene_name[match(rownames(toplot),rownames(values$myannotation))]
  
    mycolss <- c("#313695","#4575b4","#74add1","#abd9e9","#e0f3f8","#fee090","#fdae61","#f46d43","#d73027","#a50026") # to be consistent with red/blue usual coding
  
    d3heatmap(toplot,Colv = as.logical(input$heatmap_colv),colors = mycolss)
  }
}

```


# Shortlisted genes


This gene was selected in the interactive session.

```{r}
anno_id <- rownames(values$myrlt)
      anno_gene <- values$myannotation$gene_name

      # if(is.null(input$color_by) & input$genefinder!="")
      #   return(ggplot() + annotate("text",label="Select a factor to plot your gene",0,0) + theme_bw())
      # if(is.null(input$color_by) & input$genefinder=="")
      #   return(ggplot() + annotate("text",label="Select a gene and a factor to plot gene",0,0) + theme_bw())
      # if(input$genefinder=="")
      #   return(ggplot() + annotate("text",label="Type in a gene name/id",0,0) + theme_bw())
      # if(!input$genefinder %in% anno_gene & !input$genefinder %in% anno_id)
      #   return(ggplot() + annotate("text",label="Gene not found...",0,0) + theme_bw())
if(input$genefinder!="") {

  if (input$genefinder %in% anno_id) {
    selectedGene <- rownames(values$myrlt)[match(input$genefinder,rownames(values$myrlt))]
    selectedGeneSymbol <- values$myannotation$gene_name[match(selectedGene,rownames(values$myannotation))]
    }
    if (input$genefinder %in% anno_gene) {
    selectedGeneSymbol <- values$myannotation$gene_name[which(values$myannotation$gene_name==input$genefinder)]
    if (length(selectedGeneSymbol) > 1) return(ggplot() + annotate("text",label=paste0("Type in a gene name/id of the following:\n",paste(selectedGene,collapse=", ")),0,0) + theme_bw())
    selectedGene <- rownames(values$myannotation)[which(values$myannotation$gene_name==input$genefinder)]
    }
    genedata <- plotCounts(values$mydds,gene=selectedGene,intgroup = input$color_by,returnData = TRUE)
    onlyfactors <- genedata[,match(input$color_by,colnames(genedata))]
    genedata$plotby <- interaction(onlyfactors)
    
    if(input$plot_style=="boxplot"){
    res <- ggplot(genedata,aes_string(x="plotby",y="count",fill="plotby")) +
      geom_boxplot(outlier.shape = NA,alpha=0.7) + theme_bw()
    if(input$ylimZero){
      res <- res + scale_y_log10(name="Normalized counts - log10 scale",limits=c(0.4,NA))
    } else {
      res <- res + scale_y_log10(name="Normalized counts - log10 scale")
    }
    
    res <- res +
      labs(title=paste0("Normalized counts for ",selectedGeneSymbol," - ",selectedGene)) +
      scale_x_discrete(name="") +
      geom_jitter(aes_string(x="plotby",y="count"),position = position_jitter(width = 0.1)) +
      scale_fill_discrete(name="Experimental\nconditions")
    # exportPlots$genesBoxplot <- res
    res
    
    } else if(input$plot_style=="violin plot"){
    res <- ggplot(genedata,aes_string(x="plotby",y="count",fill="plotby")) +
      geom_violin(aes_string(col="plotby"),alpha = 0.6) + theme_bw()
    if(input$ylimZero){
      res <- res + scale_y_log10(name="Normalized counts - log10 scale",limits=c(0.4,NA))
    } else {
      res <- res + scale_y_log10(name="Normalized counts - log10 scale")
    }
    
    res <- res +
      labs(title=paste0("Normalized counts for ",selectedGeneSymbol," - ",selectedGene)) +
      scale_x_discrete(name="") +
      geom_jitter(aes_string(x="plotby",y="count"),alpha = 0.8,position = position_jitter(width = 0.1)) +
      scale_fill_discrete(name="Experimental\nconditions") + scale_color_discrete(guide="none")
    # exportPlots$genefinder <- res
    res
  }
}
```

Repeat the same chunk of code and change the identifier of the gene to obtain the similar plot for the other candidates.



# Functional interpretation of the principal components

These tables report the functional categories enriched in the genes with the top and bottom loadings in the selected principal components.

```{r}
if(!is.null(values$mypca2go))
{
  goe <- values$mypca2go[[paste0("PC",input$pc_x)]][["posLoad"]]
  kable(goe, caption=paste0("Functional categories enriched in ","PC",input$pc_x, "- positive loadings"))
}

if(!is.null(values$mypca2go))
{
  goe <- values$mypca2go[[paste0("PC",input$pc_x)]][["negLoad"]]
  kable(goe, caption=paste0("Functional categories enriched in ","PC",input$pc_x, "- negative loadings"))
}

if(!is.null(values$mypca2go))
{
  goe <- values$mypca2go[[paste0("PC",input$pc_y)]][["posLoad"]]
  kable(goe, caption=paste0("Functional categories enriched in ","PC",input$pc_y, "- positive loadings"))
}

if(!is.null(values$mypca2go))
{
  goe <- values$mypca2go[[paste0("PC",input$pc_y)]][["negLoad"]]
  kable(goe, caption=paste0("Functional categories enriched in ","PC",input$pc_y, "- negative loadings"))
}
```




# Multifactor exploration of the dataset

```{r}

if(input$composemat > 0){
  pcmat <- obj3()[[1]]
  tcol <- obj3()[[2]]
  tcol2 <- obj3()[[3]]
  pres <- prcomp(t(pcmat),scale=FALSE)

  plot.index <- c(as.integer(input$pc_x_multifac),as.integer(input$pc_y_multifac))
  offset <- ncol(pcmat)/2
  gene.no <- offset
  pcx <- pres$x
  # set.seed(11)
    # for (i in 1:ncol(pcx)) {
    #   pcx[,i] <- pcx[,i] + rnorm(nrow(pcx),sd=diff(range(pcx[,i]))/100)
    # }
    plot(pcx[(offset+1):ncol(pcmat),plot.index[1]][1:gene.no],pcx[(offset+1):ncol(pcmat),plot.index[2]][1:gene.no],xlim=range(pcx[,plot.index[1]]),ylim=range(pcx[,plot.index[2]]),pch=20,col=tcol,cex=0.3)#,type="n")
    #plot(0,type="n",xlim=range(pres$x[,plot.index]),ylim=range(pres$x[,plot.index]))
    lcol <- ifelse(tcol != tcol2,"black","grey")
    for (i in 1:gene.no) {
      lines(pcx[c(i,offset+i),plot.index[1]],pcx[c(i,offset+i),plot.index[2]],col=lcol[i])
    }
    points(pcx[1:offset,plot.index[1]][1:gene.no],pcx[1:offset,plot.index[2]][1:gene.no],pch=20,col=tcol,cex=0.3)
    points(pcx[(offset+1):ncol(pcmat),plot.index[1]][1:gene.no],pcx[(offset+1):ncol(pcmat),plot.index[2]][1:gene.no],pch=20,col=tcol2,cex=0.3)}
```



# About pcaExplorer


`pcaExplorer` is a Bioconductor package containing a Shiny application for
analyzing expression data in different conditions and experimental factors. 

`pcaExplorer` guides the user in exploring the Principal Components of the data, 
providing tools and functionality to detect outlier samples, genes that show 
particular patterns, and additionally provides a functional interpretation of 
the principal components for further quality assessment and hypothesis generation
on the input data. 

Thanks to its interactive/reactive design, it is designed to become a practical
companion to any RNA-seq dataset analysis, making exploratory data analysis 
accessible also to the bench biologist, while providing additional insight also
for the experienced data analyst.

`pcaExplorer` was developed in the Bioinformatics Division led by Harald Binder 
at the IMBEI (Institut für Medizinische Biometrie, Epidemiologie und Informatik) 
in the University Medical Center of the Johannes Gutenberg University Mainz.




## Developers

`pcaExplorer` is currently maintained by Federico Marini at the IMBEI (www.imbei.uni-mainz.de).
You can contact him by clicking on the button below.

<a href="mailto:mailto:marinif@uni-mainz.de" class="btn btn-primary">Federico Marini</a>

## Code

`pcaExplorer` is a part of the Bioconductor project (www.bioconductor.org).
All code for `pcaExplorer`, especially for the development version, is available
on <a href="https://github.com/federicomarini/pcaExplorer" target="_blank">GitHub</a>.



# Citation info

If you use `pcaExplorer` for your analysis, please cite it as here below:

```{r}
citation("pcaExplorer")
```





# Session Information

```{r}
sessionInfo()
```


```{r}
library(shiny)
footertemplate <- function(){
  tags$div(
    class = "footer",
    style = "text-align:center",
    tags$div(
      class = "foot-inner",
      list(
        hr(),
        "This report was generated with", tags$a(href="http://bioconductor.org/packages/pcaExplorer/", "pcaExplorer"), br(),
        "pcaExplorer is a project developed by Federico Marini in the Bioinformatics division of the ",
        tags$a(href="http://www.unimedizin-mainz.de/imbei","IMBEI"),br(),
        "Development of the pcaExplorer package is on ",
        tags$a(href="https://github.com/federicomarini/pcaExplorer", "GitHub")
      )
    )
  )
}
```

```{r}
footertemplate()
```

