## ----include = FALSE----------------------------------------------------------
knitr::opts_chunk[["set"]](
  collapse = TRUE,
  comment = "#>"
#  fig.width = 5L,
#  fig.height = 5L
)


## ----message=FALSE, warning=FALSE---------------------------------------------
library(COTAN)
library(zeallot)
library(rlang)
library(data.table)
library(Rtsne)
library(qpdf)
library(GEOquery)

options(parallelly.fork.enable = TRUE)

## ----eval=TRUE, include=TRUE--------------------------------------------------
dataDir <- tempdir()

GEO <- "GSM2861514"
fName <- "GSM2861514_E175_Only_Cortical_Cells_DGE.txt.gz"

dataSetFile <- file.path(dataDir, GEO, fName)

dir.create(file.path(dataDir, GEO), showWarnings = FALSE)

if (!file.exists(dataSetFile)) {
  getGEOSuppFiles(GEO, makeDirectory = TRUE,
                  baseDir = dataDir, fetch_files = TRUE,
                  filter_regex = fName)
}

sample.dataset <- read.csv(dataSetFile, sep = "\t", row.names = 1L)

## -----------------------------------------------------------------------------
outDir <- dataDir

# Log-level 2 was chosen to showcase better how the package works
# In normal usage a level of 0 or 1 is more appropriate
setLoggingLevel(2L)

# This file will contain all the logs produced by the package
# as if at the highest logging level
setLoggingFile(file.path(outDir, "vignette_v2.log"))

message("COTAN uses the `torch` library when asked to `optimizeForSpeed`")
message("Run the command 'options(COTAN.UseTorch = FALSE)'",
        " in your session to disable `torch` completely!")

# this command does check whether the torch library is properly installed
c(useTorch, deviceStr) %<-% COTAN:::canUseTorch(TRUE, "cuda")
if (useTorch) {
  message("The `torch` library is available and ready to use")
  if (deviceStr == "cuda") {
    message("The `torch` library can use the `CUDA` GPU")
  } else {
    message("The `torch` library can only use the CPU")
    message("Please ensure you have the `OpenBLAS` libraries",
            " installed on the system")
  }
}

rm(useTorch, deviceStr)

## -----------------------------------------------------------------------------
cond <- "mouse_cortex_E17.5"

obj <- COTAN(raw = sample.dataset)
obj <- initializeMetaDataset(obj,
                             GEO = GEO,
                             sequencingMethod = "Drop_seq",
                             sampleCondition = cond)

logThis(paste0("Condition ", getMetadataElement(obj, datasetTags()[["cond"]])),
        logLevel = 1L)

## -----------------------------------------------------------------------------
plot(ECDPlot(obj))

## -----------------------------------------------------------------------------
plot(cellSizePlot(obj))

## -----------------------------------------------------------------------------
plot(genesSizePlot(obj))

## -----------------------------------------------------------------------------
plot(scatterPlot(obj))

## -----------------------------------------------------------------------------
cellsSizeThr <- 6000L
obj <- addElementToMetaDataset(obj, "Cells size threshold", cellsSizeThr)

cells_to_rem <- getCells(obj)[getCellsSize(obj) > cellsSizeThr]
obj <- dropGenesCells(obj, cells = cells_to_rem)

plot(cellSizePlot(obj))

## -----------------------------------------------------------------------------
genesSizeHighThr <- 3000L
obj <- addElementToMetaDataset(obj, "Num genes high threshold", genesSizeHighThr)

cells_to_rem <- getCells(obj)[getNumExpressedGenes(obj) > genesSizeHighThr]
obj <- dropGenesCells(obj, cells = cells_to_rem)

plot(genesSizePlot(obj))

## -----------------------------------------------------------------------------
genesSizeLowThr <- 500L
obj <- addElementToMetaDataset(obj, "Num genes low threshold", genesSizeLowThr)

cells_to_rem <- getCells(obj)[getNumExpressedGenes(obj) < genesSizeLowThr]
obj <- dropGenesCells(obj, cells = cells_to_rem)

plot(genesSizePlot(obj))

## -----------------------------------------------------------------------------
c(mitPlot, mitSizes) %<-% mitochondrialPercentagePlot(obj, genePrefix = "^Mt")

plot(mitPlot)

## -----------------------------------------------------------------------------
mitPercThr <- 1.0
obj <- addElementToMetaDataset(obj, "Mitoc. perc. threshold", mitPercThr)

cells_to_rem <- rownames(mitSizes)[mitSizes[["mit.percentage"]] > mitPercThr]
obj <- dropGenesCells(obj, cells = cells_to_rem)

c(mitPlot, mitSizes) %<-% mitochondrialPercentagePlot(obj, genePrefix = "^Mt")

plot(mitPlot)

## -----------------------------------------------------------------------------
genes_to_rem <- getGenes(obj)[grep("^Mt", getGenes(obj))]
cells_to_rem <- getCells(obj)[which(getCellsSize(obj) == 0L)]

obj <- dropGenesCells(obj, genes_to_rem, cells_to_rem)

## -----------------------------------------------------------------------------
logThis(paste("n cells", getNumCells(obj)), logLevel = 1L)

## -----------------------------------------------------------------------------
obj <- addElementToMetaDataset(obj, "Num drop B group", 0)

obj <- clean(obj)

c(pcaCellsPlot, pcaCellsData, genesPlot,
  UDEPlot, nuPlot, zoomedNuPlot) %<-% cleanPlots(obj)

plot(pcaCellsPlot)

## -----------------------------------------------------------------------------
plot(genesPlot)

## ----eval=TRUE, include=TRUE--------------------------------------------------
cells_to_rem <- rownames(pcaCellsData)[pcaCellsData[["groups"]] == "B"]
obj <- dropGenesCells(obj, cells = cells_to_rem)

obj <- addElementToMetaDataset(obj, "Num drop B group", 1)

obj <- clean(obj)

c(pcaCellsPlot, pcaCellsData, genesPlot,
  UDEPlot, nuPlot, zoomedNuPlot) %<-% cleanPlots(obj)

plot(pcaCellsPlot)

## -----------------------------------------------------------------------------
plot(UDEPlot)

## -----------------------------------------------------------------------------
plot(nuPlot)

## -----------------------------------------------------------------------------
plot(zoomedNuPlot)

## -----------------------------------------------------------------------------
UDELowThr <- 0.30
obj <- addElementToMetaDataset(obj, "Low UDE cells' threshold", UDELowThr)

obj <- addElementToMetaDataset(obj, "Num drop B group", 2)

cells_to_rem <- getCells(obj)[getNu(obj) < UDELowThr]
obj <- dropGenesCells(obj, cells = cells_to_rem)

## -----------------------------------------------------------------------------
obj <- clean(obj)

c(pcaCellsPlot, pcaCellsData, genesPlot,
  UDEPlot, nuPlot, zoomedNuPlot) %<-% cleanPlots(obj)

plot(pcaCellsPlot)

## -----------------------------------------------------------------------------
plot(scatterPlot(obj))

## -----------------------------------------------------------------------------
logThis(paste("n cells", getNumCells(obj)), logLevel = 1L)

## -----------------------------------------------------------------------------
obj <- proceedToCoex(obj, calcCoex = TRUE,
                     optimizeForSpeed = TRUE, cores = 6L, deviceStr = "cuda",
                     saveObj = FALSE, outDir = outDir)

## ----eval=FALSE, include=TRUE-------------------------------------------------
#  # saving the structure
#  saveRDS(obj, file = file.path(outDir, paste0(cond, ".cotan.RDS")))

## ----eval=FALSE, include=TRUE-------------------------------------------------
#  obj2 <- automaticCOTANObjectCreation(
#    raw = sample.dataset,
#    GEO = GEO,
#    sequencingMethod = "Drop_seq",
#    sampleCondition = cond,
#    calcCoex = TRUE, cores = 6L, optimizeForSpeed = TRUE,
#    saveObj = TRUE, outDir = outDir)

## -----------------------------------------------------------------------------
gdiDF <- calculateGDI(obj)

head(gdiDF)

# This will store only the $GDI column
obj <- storeGDI(obj, genesGDI = gdiDF)

## -----------------------------------------------------------------------------
genesList <- list(
  "NPGs" = c("Nes", "Vim", "Sox2", "Sox1", "Notch1", "Hes1", "Hes5", "Pax6"),
  "PNGs" = c("Map2", "Tubb3", "Neurod1", "Nefm", "Nefl", "Dcx", "Tbr1"),
  "hk"   = c("Calm1", "Cox6b1", "Ppia", "Rpl18", "Cox7c", "Erh", "H3f3a",
             "Taf1", "Taf2", "Gapdh", "Actb", "Golph3", "Zfr", "Sub1",
             "Tars", "Amacr")
)

GDIPlot(obj, cond = cond, genes = genesList, GDIThreshold = 1.40)

## -----------------------------------------------------------------------------
plot(heatmapPlot(obj, genesLists = genesList))

## -----------------------------------------------------------------------------
plot(genesHeatmapPlot(obj, primaryMarkers = c("Satb2", "Bcl11b", "Vim", "Hes1"),
                      pValueThreshold = 0.001, symmetric = TRUE))

## ----eval=FALSE, include=TRUE-------------------------------------------------
#  plot(genesHeatmapPlot(obj, primaryMarkers = c("Satb2", "Bcl11b", "Fezf2"),
#                        secondaryMarkers = c("Gabra3", "Meg3", "Cux1", "Neurod6"),
#                        pValueThreshold = 0.001, symmetric = FALSE))

## -----------------------------------------------------------------------------
print("Contingency Tables:")
contingencyTables(obj, g1 = "Satb2", g2 = "Bcl11b")

print("Corresponding Coex")
getGenesCoex(obj)["Satb2", "Bcl11b"]

## -----------------------------------------------------------------------------
# For the whole matrix
coex <- getGenesCoex(obj, zeroDiagonal = FALSE)
coex[1000L:1005L, 1000L:1005L]

## -----------------------------------------------------------------------------
# For a partial matrix
coex <- getGenesCoex(obj, genes = c("Satb2", "Bcl11b", "Fezf2"))
coex[1000L:1005L, ]

## ----eval=TRUE, include=TRUE--------------------------------------------------
layersGenes <- list(
  "L1"   = c("Reln",   "Lhx5"),
  "L2/3" = c("Satb2",  "Cux1"),
  "L4"   = c("Rorb",   "Sox5"),
  "L5/6" = c("Bcl11b", "Fezf2"),
  "Prog" = c("Vim",    "Hes1")
)
c(gSpace, eigPlot, pcaGenesClDF, treePlot) %<-%
  establishGenesClusters(obj, groupMarkers = layersGenes,
                         numGenesPerMarker = 25L, kCuts = 5L)

plot(eigPlot)

## ----eval=TRUE, include=TRUE--------------------------------------------------
plot(treePlot)

## ----eval=TRUE, include=TRUE--------------------------------------------------
colSelection <- vapply(pcaGenesClDF, is.numeric, logical(1L))
genesUmapPl <- UMAPPlot(pcaGenesClDF[, colSelection, drop = FALSE],
                        clusters = getColumnFromDF(pcaGenesClDF, "hclust"),
                        elements = layersGenes,
                        title = "Genes' clusters UMAP Plot",
                        numNeighbors = 32L, minPointsDist = 0.25)

plot(genesUmapPl)

## ----eval=FALSE, include=TRUE-------------------------------------------------
#  # This code is a little too computationally heavy to be used in an example
#  # So we stored the result and we can load it in the next section
#  
#  # default constructed checker is OK
#  advChecker <- new("AdvancedGDIUniformityCheck")
#  
#  c(splitClusters, splitCoexDF) %<-%
#    cellsUniformClustering(obj, initialResolution = 0.8, checker = advChecker,
#                           optimizeForSpeed = TRUE, deviceStr = "cuda",
#                           cores = 6L, saveObj = TRUE, outDir = outDir)
#  
#  obj <- addClusterization(obj, clName = "split",
#                           clusters = splitClusters, coexDF = splitCoexDF)
#  
#  table(splitClusters)

## ----eval=TRUE, include=TRUE--------------------------------------------------
data("vignette.split.clusters", package = "COTAN")
splitClusters <- vignette.split.clusters[getCells(obj)]

splitCoexDF <- DEAOnClusters(obj, clusters = splitClusters)

obj <- addClusterization(obj, clName = "split", clusters = splitClusters,
                         coexDF = splitCoexDF, override = FALSE)

## ----eval=TRUE, include=TRUE--------------------------------------------------
c(summaryData, summaryPlot) %<-%
  clustersSummaryPlot(obj, clName = "split", plotTitle = "split summary")

summaryData

## ----eval=TRUE, include=TRUE--------------------------------------------------
plot(summaryPlot)

## ----eval=TRUE, include=TRUE--------------------------------------------------
c(., scoreDF) %<-% clustersMarkersHeatmapPlot(obj, groupMarkers = layersGenes,
                                              clName = "split", kCuts = 5L)

## ----eval=FALSE, include=TRUE-------------------------------------------------
#  c(mergedClusters, mergedCoexDF) %<-%
#    mergeUniformCellsClusters(obj, clusters = splitClusters,
#                              checkers = advChecker,
#                              optimizeForSpeed = TRUE, deviceStr = "cuda",
#                              cores = 6L, saveObj = TRUE, outDir = outDir)
#  
#  # merges are:
#  #  1 <- 06 + 07
#  #  2 <- '-1' + 08 + 11
#  #  3 <- 09 + 10
#  #  4 <- 01
#  #  5 <- 02
#  #  6 <- 12 + 13
#  #  7 <- 03 + 05
#  #  8 <- 04
#  
#  obj <- addClusterization(obj, clName = "merge", override = TRUE,
#                           clusters = mergedClusters, coexDF = mergedCoexDF)
#  
#  table(mergedClusters)

## ----eval=FALSE, include=TRUE-------------------------------------------------
#  
#  checkersList <- list(advChecker,
#                       shiftCheckerThresholds(advChecker, 0.01),
#                       shiftCheckerThresholds(advChecker, 0.03))
#  
#  prevCheckRes <- data.frame()
#  
#  # In this case we want to re-use the already calculated merge checks
#  # Se we reload them from the output files. This, along a similar facility for
#  # the split method, is also useful in the cases the execution is interrupted
#  # prematurely...
#  #
#  if (TRUE) {
#    # read from the last file among those named all_check_results_XX.csv
#    mergeDir <- file.path(outDir, cond, "leafs_merge")
#    resFiles <- list.files(path = mergeDir, pattern = "all_check_results_.*csv",
#                           full.names = TRUE)
#  
#    prevCheckRes <- read.csv(resFiles[length(resFiles)],
#                             header = TRUE, row.names = 1L)
#  }
#  
#  c(mergedClusters2, mergedCoexDF2) %<-%
#    mergeUniformCellsClusters(obj, clusters = splitClusters,
#                              checkers = checkersList,
#                              allCheckResults = prevCheckRes,
#                              optimizeForSpeed = TRUE, deviceStr = "cuda",
#                              cores = 6L, saveObj = TRUE, outDir = outDir)
#  
#  # merges are:
#  #  1 <- '-1' + 06 + 09
#  #  2 <- 07
#  #  3 <- 03  + 04 + 05 + 10 + 13
#  #  4 <- 12
#  #  5 <- 01 + 08 + 11
#  #  6 <- 02
#  
#  obj <- addClusterization(obj, clName = "merge2", override = TRUE,
#                           clusters = mergedClusters2, coexDF = mergedCoexDF2)
#  
#  table(mergedClusters2)

## ----eval=TRUE, include=TRUE--------------------------------------------------
data("vignette.merge.clusters", package = "COTAN")
mergedClusters <- vignette.merge.clusters[getCells(obj)]

mergedCoexDF <- DEAOnClusters(obj, clusters = mergedClusters)

obj <- addClusterization(obj, clName = "merge", clusters = mergedClusters,
                         coexDF = mergedCoexDF, override = FALSE)

data("vignette.merge2.clusters", package = "COTAN")
mergedClusters2 <- vignette.merge2.clusters[getCells(obj)]

mergedCoexDF2 <- DEAOnClusters(obj, clusters = mergedClusters2)

obj <- addClusterization(obj, clName = "merge2", clusters = mergedClusters2,
                         coexDF = mergedCoexDF2, override = FALSE)

## ----eval=TRUE, include=TRUE--------------------------------------------------
invisible(
  clustersMarkersHeatmapPlot(obj, clName = "merge", condNameList = "split",
                             conditionsList = list(splitClusters)))

invisible(
  clustersMarkersHeatmapPlot(obj, clName = "merge2", condNameList = "split",
                             conditionsList = list(splitClusters)))

## -----------------------------------------------------------------------------
c(umapPlot, cellsPCA) %<-% cellsUMAPPlot(obj, clName = "merge2",
                                         dataMethod = "AdjBinarized",
                                         genesSel = "HGDI",
                                         colors = NULL, numNeighbors = 15L,
                                         minPointsDist = 0.2)

plot(umapPlot)

## -----------------------------------------------------------------------------
if (file.exists(file.path(outDir, paste0(cond, ".cotan.RDS")))) {
  #Delete file if it exists
  file.remove(file.path(outDir, paste0(cond, ".cotan.RDS")))
}
if (file.exists(file.path(outDir, paste0(cond, "_times.csv")))) {
  #Delete file if it exists
  file.remove(file.path(outDir, paste0(cond, "_times.csv")))
}
if (dir.exists(file.path(outDir, cond))) {
  unlink(file.path(outDir, cond), recursive = TRUE)
}
if (dir.exists(file.path(outDir, GEO))) {
  unlink(file.path(outDir, GEO), recursive = TRUE)
}

# stop logging to file
setLoggingFile("")
file.remove(file.path(outDir, "vignette_v2.log"))

options(parallelly.fork.enable = FALSE)

## -----------------------------------------------------------------------------
Sys.time()

## -----------------------------------------------------------------------------
sessionInfo()

