# `ideal`-report

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

```{r setup, include=FALSE, eval = TRUE, echo = FALSE}
opts_chunk$set(
  echo=input$report_echo,
  error = TRUE # to continue generating the report
  
)
```

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This can be done by using the `r BiocStyle::CRANpkg("webshot")` package and calling `webshot::install_phantomjs()`.
Alternatively, the more recent `r BiocStyle::CRANpkg("webshot2")` package uses the headless Chrome browser (via the `r BiocStyle::CRANpkg("chromote")` package, requiring Google Chrome or other Chromium-based browser).

# Data Setup

Following objects were provided to the initial call of the function:

```{r}
dds_obj
res_obj
head(annotation_obj)
head(countmatrix)
expdesign
```

The data provided was used to construct the following objects

```{r}
head(values$countmatrix)
values$expdesign
values$dds_obj
values$res_obj
head(values$annotation_obj)
```

```{r}
design(values$dds_obj)

values$cur_species

input$idtype

# load according annotation package? use annospecies_df

if(!is.null(mcols(values$dds_obj)$dispGeneEst))
  plotDispEsts(values$dds_obj)

if(!is.null(values$dds_obj))
  paste0(nrow(values$dds_obj), " genes - ",ncol(values$dds_obj)," samples")


if(!is.null(values$annotation_obj))
  paste0(nrow(values$annotation_obj), " genes - ",ncol(values$annotation_obj)," ID types")

DEregu <- sum(values$res_obj$padj < input$FDR & values$res_obj$log2FoldChange != 0, na.rm = TRUE)
if(!is.null(values$res_obj))
  paste0(DEregu, " DE genes - out of ",nrow(values$res_obj),"")
```

# Counts Overview

```{r}
if(input$countstable_unit=="raw_counts")
  cur_mat <- counts(values$dds_obj,normalized=FALSE)
if(input$countstable_unit=="normalized_counts")
  cur_mat <- counts(values$dds_obj,normalized=TRUE)
if(input$countstable_unit=="vst_counts")
  cur_mat <- vst(values$dds_obj)
if(input$countstable_unit=="log10_counts")
  cur_mat <- log10(1 + counts(values$dds_obj,normalized=TRUE))

input$countstable_unit

head(cur_mat)
```

```{r}
t1 <- rowSums(counts(values$dds_obj))
t2 <- rowMeans(counts(values$dds_obj,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")
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$dds_obj),
    "genes have at least a sample with more than",thresh_rowsums,"counts\n")
cat(abs_t2,"genes have more than",thresh_rowmeans,"counts (normalized) on average\n")
cat(paste0(round(rel_t2,3),"%"), "of the",nrow(values$dds_obj),
    "genes have more than",thresh_rowsums,"counts (normalized) on average\n")
cat("Counts are ranging from", min(counts(values$dds_obj)),
    "to",max(counts(values$dds_obj)))
```

# Extract results

```{r}
if(!is.null(values$res_obj)) {
  print(design_factors())
  
  print(input$choose_expfac)

  fac1 <- input$choose_expfac
  nrl <- length(levels(colData(values$dds_obj)[,fac1]))

  print(input$fac1_c1)
  print(input$fac1_c2)
  print(values$res_obj)

  print(sub(".*p-value: (.*)","\\1",mcols(values$res_obj, use.names=TRUE)["pvalue","description"]))
  print(summary(values$res_obj,alpha = input$FDR))

  cur_res <- values$res_obj
  
  if(!is.null(values$annotation_obj)) {
    cur_res$symbol <- values$annotation_obj$gene_name[match(rownames(cur_res),
                                                          rownames(values$annotation_obj))]
  }
}
```

```{r}
if(!is.null(values$res_obj)) {
  res_df <- as.data.frame(values$res_obj)
  res_df <- dplyr::filter(res_df, !is.na(pvalue))

  p1 <- ggplot(res_df, aes_string("pvalue")) +
    geom_histogram(binwidth = 0.01, boundary = 0) + theme_bw()
  # for visual estimation of the false discovery proportion in the first bin
  alpha <- binw <- input$FDR
  pi0 <- 2*mean(res_df$pvalue > 0.5)
  p1 <- p1 + geom_hline(yintercept = pi0 * binw * nrow(res_df), col = "steelblue") + 
    geom_vline(xintercept = alpha, col = "red")
  
  p1 <- p1 + ggtitle(
    label = "p-value histogram",
    subtitle = paste0(
      "Expected nulls = ", pi0 * binw * nrow(res_df), 
      " - #elements in the selected bins = ", sum(res_df$pvalue < alpha)
    ))

  print(p1)
  
  res_df <- mutate(
    res_df, 
    stratum = cut(baseMean, include.lowest = TRUE, 
                  breaks = signif(quantile(baseMean, probs = seq(0,1, length.out = 10)),2)))
  
  p2 <- ggplot(res_df, aes_string("pvalue")) +
    geom_histogram(binwidth = 0.01, boundary = 0) + 
    facet_wrap(~stratum) + 
    theme_bw()
      
  p2 <- p2 + ggtitle(
    label = "p-value histogram",
    subtitle = "stratified on the different value classes of mean expression values")
  
  phi <- input$FDR
  res_df <- mutate(res_df, rank = rank(pvalue))
  m <- nrow(res_df)
  
  p3 <- ggplot(filter(res_df, rank <= 6000), 
              aes(x = rank, y = pvalue)) + 
    geom_line() + 
    geom_abline(slope = phi/m, col = "red") + 
    theme_bw()
  
  p3 <- p3 + ggtitle(
    label = "Schweder-Spjotvoll plot",
    subtitle = paste0(
      "Intersection point at rank ", with(arrange(res_df,rank), last(which(pvalue <= phi * rank / m))))
    )
  
  p4 <- ggplot(res_df, aes_string("log2FoldChange")) +
    geom_histogram(binwidth = 0.1) + theme_bw()
  p4 <- p4 + ggtitle("Histogram of the log2 fold changes")
  print(p4)

  mydf <- as.data.frame(values$res_obj[order(values$res_obj$padj),])#[1:500,]
  rownames(mydf) <- createLinkENS(rownames(mydf),species = annoSpecies_df$ensembl_db[match(input$speciesSelect,annoSpecies_df$species)]) ## TODO: check what are the species from ensembl and
      ## TODO: add a check to see if wanted?
  mydf$symbol <- createLinkGeneSymbol(mydf$symbol)
  datatable(mydf, escape = FALSE)                       
}
```

# Summary Plots

```{r}
if(!is.null(values$res_obj)) {
  print(plot_ma(values$res_obj,annotation_obj = values$annotation_obj))
  
  if(!is.null(input$ma_brush)){
    if(!is.null(values$annotation_obj))
        print(plot_ma(values$res_obj,annotation_obj = values$annotation_obj,FDR = input$FDR) +
        coord_cartesian(xlim = c(input$ma_brush$xmin,input$ma_brush$xmax),
                        ylim = c(input$ma_brush$ymin,input$ma_brush$ymax)) +
        geom_text(aes_string(label="genename"),size=3,hjust=0.25, vjust=-0.75))
      else
        print(plot_ma(values$res_obj,annotation_obj = values$annotation_obj,FDR = input$FDR) +
        coord_cartesian(xlim = c(input$ma_brush$xmin,input$ma_brush$xmax),
                        ylim = c(input$ma_brush$ymin,input$ma_brush$ymax)))
  }
  
  print(plot_volcano(values$res_obj, FDR = input$FDR))
  
  print(head(curData()))
  
  if(!(is.null(input$ma_brush)) & !is.null(values$dds_obj)) { 

      brushedObject <- curData()

      selectedGenes <- as.character(brushedObject$ID)
      toplot <- assay(values$dds_obj)[selectedGenes,]
      rownames(toplot) <- values$annotation_obj$gene_name[match(rownames(toplot),rownames(values$annotation_obj))]

      if(input$pseudocounts) toplot <- log2(1+toplot)

      mat_rowscale <- function(x)
      {
        m <- apply(x, 1, mean, na.rm = TRUE)
        s <- apply(x, 1, sd, na.rm = TRUE)
        return((x - m)/s)
      }

      if(input$rowscale) toplot <- mat_rowscale(toplot)
      
      mycolss <- c("#313695","#4575b4","#74add1","#abd9e9","#e0f3f8","#fee090","#fdae61","#f46d43","#d73027","#a50026") # to be consistent with red/blue usual coding
      

      pheatmap(toplot,cluster_cols = as.logical(input$heatmap_colv))
      
      heatmaply(toplot,Colv = as.logical(input$heatmap_colv),colors = mycolss)
  }
}
```

# Gene Finder

```{r}
if(length(input$color_by) > 0 &
   (length(input$avail_symbols)>0 | length(input$avail_ids)>0)
  ){
  
  if(length(input$avail_symbols)>0) {
        # got the symbol, look for the id
        mysyms <- input$avail_symbols
        myids <- values$annotation_obj$gene_id[match(mysyms, values$annotation_obj$gene_name)]
      } else {
        myids <- input$avail_ids
        # make it optional if annot is available
        if(!is.null(values$annotation_obj)) {
          mysims <- values$annotation_obj$gene_name[match(myids, values$annotation_obj$gene_id)]
        } else {
          mysims <- ""
        }
      }
  
  print(length(myids))
  
  for(myid in myids) {
      p <- ggplotCounts(values$dds_obj, myid, intgroup = input$color_by,annotation_obj=values$annotation_obj)
      if(input$ylimZero_genefinder)
        p <- p + ylim(0.1, NA)
      print(p)
  }
  
  if("symbol" %in% names(values$res_obj)) {
    print(plot_ma(values$res_obj,
                intgenes = input$avail_symbols,annotation_obj = values$annotation_obj,FDR = input$FDR))
      } else {
        print(plot_ma(values$res_obj,
                intgenes = input$avail_ids,annotation_obj = values$annotation_obj,FDR = input$FDR))
      }

  if(!is.null(values$genelist_ma)) {
    print(values$genelist_ma)
    if("symbol" %in% names(values$res_obj)) {
        print(plot_ma(values$res_obj,
                intgenes = values$genelist_ma$`Gene Symbol`,annotation_obj = values$annotation_obj,FDR = input$FDR))
      }
  }
  datatable(cur_combires())
}
# if selected, maplot with annot plus table
```

# Functional Analysis

```{r}
if(!is.null(values$res_obj)) {
  values$genelistUP()
  values$genelistDOWN()
  values$genelistUPDOWN()
  as.character(values$genelist1$`Gene Symbol`)
  as.character(values$genelist2$`Gene Symbol`)

  gplots::venn(gll())

  UpSetR::upset(fromList(gll()))
}

# 3x5 tables if available

# here only for goana
if(!is.null(values$gse_up)) {
  mytbl <- values$gse_up
  rownames(mytbl) <- createLinkGO(rownames(mytbl))
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$gse_down)) {
  mytbl <- values$gse_down
  rownames(mytbl) <- createLinkGO(rownames(mytbl))
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$gse_updown)) {
  mytbl <- values$gse_updown
  rownames(mytbl) <- createLinkGO(rownames(mytbl))
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$gse_list1)) {
  mytbl <- values$gse_list1
  rownames(mytbl) <- createLinkGO(rownames(mytbl))
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$gse_list2)) {
  mytbl <- values$gse_list2
  rownames(mytbl) <- createLinkGO(rownames(mytbl))
  datatable(mytbl,escape=FALSE)
}

# here only for topgo
if(!is.null(values$topgo_up)) {
  mytbl <- values$topgo_up
  mytbl$GO.ID <- createLinkGO(mytbl$GO.ID)
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$topgo_down)) {
  mytbl <- values$topgo_down
  mytbl$GO.ID <- createLinkGO(mytbl$GO.ID)
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$topgo_updown)) {
  mytbl <- values$topgo_updown
  mytbl$GO.ID <- createLinkGO(mytbl$GO.ID)
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$topgo_list1)) {
  mytbl <- values$topgo_list1
  mytbl$GO.ID <- createLinkGO(mytbl$GO.ID)
  datatable(mytbl,escape=FALSE)
}
if(!is.null(values$topgo_list2)) {
  mytbl <- values$topgo_list2
  mytbl$GO.ID <- createLinkGO(mytbl$GO.ID)
  datatable(mytbl,escape=FALSE)
}

# similarly for goseq...
if(!is.null(values$gse_updown_goseq)) {
  mytbl <- values$gse_updown_goseq
  mytbl$category <- createLinkGO(mytbl$category)
  datatable(mytbl,escape=FALSE)
}
 
# for the selected lines, also do heatmaps?
```


# Signatures explorer

```{r}
if(!is.null(values$gene_signatures) & 
   !(is.null(values$vst_obj)) & 
   !(is.null(values$anno_vec)) &
   !is.null(input$sig_selectsig)) {
  
  print(input$sig_selectsig)
  
  sig_sigmembers <- values$gene_signatures[[input$sig_selectsig]]
  
  id_type_data <- input$sig_id_data
  id_type_sigs <- input$sig_id_sigs
  
  head(values$anno_vec)
  
  sig_members <- values$gene_signatures[[input$sig_selectsig]]
  print(sig_members)
  
  sig_heatmap(
          values$vst_obj,
          my_signature = values$gene_signatures[[input$sig_selectsig]],
          res_data = values$res_obj,
          FDR = input$FDR,
          de_only = input$sig_useDEonly,
          annovec = values$anno_vec,
          # anno_colData = colData(values$vst_obj)[,input$sig_annocoldata, drop = FALSE],
          title = names(values$gene_signatures)[match(input$sig_selectsig,names(values$gene_signatures))],
          cluster_rows = input$sig_clusterrows,
          cluster_cols = input$sig_clustercols,
          center_mean = input$sig_centermean,
          scale_row = input$sig_scalerow
        )
}
```

# About `ideal`

`ideal` is a Bioconductor package containing a Shiny application for
 interactive and reproducible Differential Expression analysis.

`ideal` is developed in the Bioinformatics Division (led by Harald Binder) 
of the Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI)
at the University Medical Center of the Johannes Gutenberg University Mainz.

## Developers

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

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

## Code availability

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

## Citation info

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

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

# Session Info

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

```{r, echo = FALSE}
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$a(href="https://github.com/federicomarini/ideal", "ideal"), br(),
        "ideal is a project developed by Federico Marini in the Bioinformatics division of the ",
        tags$a(href="http://www.unimedizin-mainz.de/imbei","IMBEI"),br(),
        "Development of the pcaExplorer package is on ",
        tags$a(href="https://github.com/federicomarini/ideal", "GitHub")
      )
    )
  )
}
```

```{r, echo = FALSE}
footertemplate()
```
