Defines functions flow_auto_qc

Documented in flow_auto_qc

#' Automatic quality control of flow cytometry data.
#' For a set of FCS files, \emph{flow_auto_qc} performs a complete and automatic
#' quality control. It consists in the detection and removal of anomalies by
#' checking three properties of flow cytometry: 1) flow rate, 2) signal
#' acquisition, 3) dynamic range.
#' @param fcsfiles It can be a character vector with the filenames of the FCS
#'   files, a flowSet or a flowFrame.
#' @param remove_from Select from which of the three steps the anomalies have to
#'   be excluded in the high quality FCS file. The default option \code{"all"}
#'   removes the anomalies from all the three steps. Alternatively, you can use:
#'   \code{"FR_FS", "FR_FM", "FS_FM", "FR", "FS", "FM"}, to remove the anomalies
#'   only on a subset of the steps where \emph{FR} stands for the flow rate,
#'   \emph{FS} stands for signal acquisition and \emph{FM} stands for dynamic
#'   range.
#' @param output Set it to 1 to return a flowFrame or a flowSet with high
#'   quality events only. Set it to 2 to return a flowFrame or a flowSet with an
#'   additional parameter where the low quality events have a value higher than
#'   10,000. Set it to 3 to return  a list with the IDs of low quality cells.
#'   Set it to any other value if no R object has to be returned. Default is
#'   \code{1}.
#' @param timeCh Character string corresponding to the name of the Time Channel
#'   in the set of FCS files. By default is \code{NULL} and the name is
#'   retrieved automatically.
#' @param second_fractionFR The fraction of a second that is used to split the
#'   time channel in order to recreate the flow rate. Set it to
#'   \code{"timestep"} if you wish to recreate the flow rate at the maximum
#'   resolution allowed by the flow cytometry instrument. Usually, for FCS files
#'   the timestep corresponds to 0.01, however, to shorten the running time of
#'   the analysis the fraction used by default is 0.1, corresponding to 1/10 of
#'   a second.
#' @param alphaFR The level of statistical significance used to accept anomalies
#'   detected by the ESD method. The default value is \code{0.01}.
#' @param decompFR Logical indicating whether the flow rate should be decomposed
#'   in the trend and cyclical components. Default is \code{TRUE} and the ESD
#'   outlier detection will be executed on the trend component penalized by the
#'   magnitude of the cyclical component. If it is \code{FALSE} the ESD outlier
#'   detection will be executed on the original flow rate.
#' @param ChExcludeFS Character vector with the names or name patterns of the
#'   channels that you want to exclude from the signal acquisition check. The
#'   default option, \code{c("FSC", "SSC")}, excludes the scatter parameters. If
#'   you want to include all the parameters in the analysis use \code{NULL}.
#' @param outlier_binsFS logical indicating whether outlier bins (not events)
#'   have to be removed before the changepoint detection of the signal
#'   acquisition check. The default is \code{FALSE}.
#' @param pen_valueFS The value of the penalty for the changepoint detection
#'   algorithm. This can be a numeric value or text giving the formula to use;
#'   for instance, you can use the character string \code{"1.5*log(n)"}, where n
#'   indicates the number of cells in the FCS file. The higher the penalty value
#'   the less strict is the detection of the anomalies. The default is
#'   \code{500}.
#' @param max_cptFS The maximum number of changepoints that can be detected for
#'   each channel. The default is \code{3}.
#' @param ChExcludeFM Character vector with the names or name patterns of the
#'   channels that you want to exclude from the signal acquisition check. The
#'   default option, \code{c("FSC", "SSC")}, excludes the scatter parameters. If
#'   you want to include all the parameters in the analysis use \code{NULL}.
#' @param sideFM Select whether the dynamic range check has to be executed on
#'   both limits, the upper limit or the lower limit. Use one of the options:
#'   \code{"both", "upper", "lower"}. The default is \code{"both"}.
#' @param neg_valuesFM Scalar indicating the method to use for the removal of
#'   the anomalies from the lower limit of the dynamic range. Use \code{1} to
#'   remove negative outliers or use \code{2} to truncate the negative values to
#'   the cut-off indicated in the FCS file.
#' @param html_report Suffix to be added to the FCS filename to name the HTML
#'   report of the quality control. The default is \code{"_QC"}. If you do not
#'   want to generate a report use \code{FALSE}.
#' @param mini_report Name for the TXT file containing the percentage of
#'   anomalies detected in the set of FCS files analyzed. The default is
#'   \code{"_QCmini"}. If you do not want to generate the mini report use
#'   \code{FALSE}.
#' @param fcs_QC Suffix to be added for the filename of the new FCS containing a
#'   new parameter where the low quality events only have a value higher than
#'   10,000. The default is \code{"_QC"}. If you do not want to generate the
#'   quality controlled FCS file use \code{FALSE}.
#' @param fcs_highQ Suffix to be added for the filename of the new FCS
#'   containing only the events that passed the quality control. The default is
#'   \code{FALSE} and hence the high quality FCS file is not generated.
#' @param fcs_lowQ Suffix to be added for the filename of the new FCS containing
#'   only the events that did not pass the quality control. The default is
#'   \code{FALSE} and hence the low quality FCS file is not generated.
#' @param folder_results Character string used to name the directory that
#'   contains the results. The default is \code{"resultsQC"}. If you intend to
#'   return the results in the working directory use \code{FALSE}.
#' @return A complete quality control is performed on flow cytometry data in FCS
#'   format. By default the analysis returns:
#'   1. a flowFrame or flowSet object containing new FCS files with only high
#'   quality events
#'   and a directory named \var{resultsQC} containing:
#'   1. a set of new FCS files with a new parameter to gate out the low quality
#'   events a value larger than 10,000 is assigned to them only,
#'   2. a set of HTML reports, one for each FCS file, that include graphs and
#'   table indicating where the anomalies were detected,
#'   3. a single TXT file reporting the percentage of events removed in each FCS
#'   file.
#' @author Gianni Monaco, Chen Hao
#' @examples
#' ## a sample dataset as flowSet object
#' data(Bcells)
#' ## quality control on a flowFrame object
#' resQC <- flow_auto_qc(Bcells[[1]], html_report = FALSE, mini_report = FALSE, fcs_QC = FALSE, folder_results = FALSE)
#' @import flowCore
#' @import ggplot2
#' @import plyr
#' @import knitr
#' @import reshape2
#' @import rmarkdown
#' @importFrom changepoint cpt.meanvar
#' @importFrom scales pretty_breaks
#' @importFrom graphics hist legend lines
#' @importFrom methods as is new
#' @importFrom stats convolve frequency is.ts mad median na.omit qt runif ts tsp
#' @importFrom utils write.table
#' @export
flow_auto_qc <- function(fcsfiles, remove_from = "all", output = 1,
     timeCh = NULL, second_fractionFR = 0.1, alphaFR = 0.01, decompFR = TRUE,
     ChExcludeFS = c("FSC", "SSC"), outlier_binsFS = FALSE, pen_valueFS = 500,
     max_cptFS = 3, ChExcludeFM = c("FSC", "SSC"), sideFM = "both", neg_valuesFM = 1,
     html_report = "_QC", mini_report = "QCmini", fcs_QC = "_QC", fcs_highQ = FALSE,
     fcs_lowQ = FALSE, folder_results = "resultsQC") {

    ## load the data
  if( is.character(fcsfiles) ){
    FileType <- toupper(strsplit(basename(fcsfiles[1]), split="\\.")[[1]][-1])
    if(length(FileType) == 0){
      warning("It was not possible to retrieve the file extension. The data will be processed as FCS.", call. =FALSE)
      FileType <- "FCS"
    else if(FileType == "LMD"){
      set <- read.flowSet(files = fcsfiles, dataset = 2)
      set <- read.flowSet(files = fcsfiles) 
    names <- fcsfiles
  }else if(is(fcsfiles, "flowSet")){
    FileType <- "FCS"
    set <- fcsfiles
    names <- flowCore::sampleNames(fcsfiles)
  }else if(is(fcsfiles,"flowFrame")){
    FileType <- "FCS"
    set <- as(fcsfiles,"flowSet")
    names <- identifier(fcsfiles)
   stop("As first argument, use a flowSet or a character vector with the path of the FCS files")

  N_cell_set <- flow_set_qc(set)
  area.color <- rep("red", length(set))

  if (missing(timeCh) || is.null(timeCh)) {
    timeCh <- findTimeChannel(set[[1]])
  if (is.null(timeCh)) {
    warning("Impossible to retrieve the time channel automatically. The quality control can only be performed on signal acquisition and dynamic range.", call. =FALSE)

  # in some cases, especially if the FCS file has been modified, there
  # could be more than one slots for the Timestep parameter. the first in
  # numerical order should be the original value.
  word <- which(grepl("TIMESTEP", names(keyword(set[[1]])),
                      ignore.case = TRUE))
  timestep <- as.numeric(keyword(set[[1]])[[word[1]]])
  if( !length(timestep) ){
    if(FileType == "LMD"){
      timestep <- 0.0009765625 # this timestep corresponds to 1/1024
      warning("The TIMESTEP keyword was not found and hence it was set to 0.01. Graphs labels indicating time might not be correct", call. =FALSE)
      timestep <- 0.01
  if( second_fractionFR == "timestep" ){
      second_fractionFR <- timestep
  }else if( second_fractionFR < timestep ){
      stop("The argument second_fractionFR must be greater or equal to timestep.", call. =FALSE)

  if(folder_results != FALSE){
      folder_results <- strip.sep(folder_results)
      dir.create(folder_results, showWarnings = FALSE)
      folder_results <- paste0(folder_results, .Platform$file.sep)
  } else { folder_results <- ""}

  out <- list()

  for (i in 1:length(set)) {
    filename_ext <- flowCore::identifier(set[[i]])
    filename <- sub("^([^.]*).*", "\\1", filename_ext)

    if (html_report != FALSE) {
        reportfile <- paste0(filename, html_report, ".html")
    if (mini_report != FALSE) {
        minireport <-  paste0(folder_results, mini_report, ".txt")
            write.table(t(c("Name file", "n. of events", "% anomalies", "analysis from",
                "% anomalies flow Rate",  "% anomalies Signal",  "% anomalies Margins")),
                minireport, sep="\t", row.names = FALSE, quote = FALSE, col.names = FALSE)
    if (fcs_QC != FALSE) {
        QC.fcs.file <- paste0(folder_results, filename, fcs_QC, ".fcs")
    if (fcs_highQ != FALSE) {
        good.fcs.file <- paste0(folder_results, filename, fcs_highQ, ".fcs")
    if (fcs_lowQ != FALSE) {
        bad.fcs.file <- paste0(folder_results,filename, fcs_lowQ, ".fcs")
    cat(paste0("Quality control for the file: ", filename, "\n"))
    # select different color for the analyzed FCS in the set plot
    area <- area.color
    area[i] <- "blue"

    # check the time channel of the file
    if (!is.null(timeCh)) {
        if (length(unique(exprs(set[[i]])[, timeCh])) == 1){
            cat("The time channel contains a single value. It cannot be used to recreate the flow rate. \n")
            warning(paste0("The time channel in ", filename_ext, " contains a single value. It cannot be used to recreate the flow rate. \n"), call. =FALSE)
            TimeChCheck <- "single_value"
            TimeChCheck <- NULL
        TimeChCheck <- "NoTime"

    # get the size of the bins
    FSbinSize <- min(max(1, ceiling(nrow(set[[1]])/100)), 500)
    # order events in the FCS file if a proper Time channel is present
    if (is.null(TimeChCheck)) {
      ordFCS <- ord_fcs_time(set[[i]], timeCh)
      ordFCS <- set[[i]]

    origin_cellIDs <- 1:nrow(ordFCS)

    ### Describe here the arguments for the functions of the flow Rate and Flow Signal
    FR_bin_arg <- list( second_fraction = second_fractionFR, timeCh = timeCh,
                  timestep = timestep)
    FR_QC_arg <- list( alpha = alphaFR, use_decomp = decompFR)
    FS_bin_arg <- list( binSize = FSbinSize, timeCh = timeCh, timestep = timestep, TimeChCheck = TimeChCheck)
    FS_QC_arg <- list( ChannelExclude = ChExcludeFS, pen_valueFS, max_cptFS, outlier_binsFS )
    FM_QC_arg <- list( ChannelExclude = ChExcludeFM, side= sideFM, neg_values = neg_valuesFM)

    #### The actual analysis is performed here
      if (is.null(TimeChCheck)) {
        FlowRateData <- do.call(flow_rate_bin, c(ordFCS, FR_bin_arg ))
        FlowRateQC <- do.call(flow_rate_check, c(ordFCS, list(FlowRateData), FR_QC_arg ))
        FlowRateQC$goodCellIDs <- origin_cellIDs
        FlowRateQC$res_fr_QC$badPerc <- 0
      FlowSignalData <- do.call(flow_signal_bin, c(ordFCS,FS_bin_arg))
      FlowSignalQC <- do.call(flow_signal_check, c(ordFCS,list(FlowSignalData),FS_QC_arg))
      FlowMarginQC <- do.call(flow_margin_check, c(ordFCS, FM_QC_arg))

      ####selection of good cells
      if(remove_from == "all"){
       goodCellIDs <- intersect(FlowRateQC$goodCellIDs, intersect(FlowSignalQC$goodCellIDs, FlowMarginQC$goodCellIDs))
       analysis <- "Flow Rate, Flow Signal and Flow Margin"
      }else if(remove_from == "FR_FS"){
        goodCellIDs <- intersect(FlowRateQC$goodCellIDs, FlowSignalQC$goodCellIDs)
        analysis <- "Flow Rate and Flow Signal"
      }else if(remove_from == "FR_FM"){
        goodCellIDs <- intersect(FlowRateQC$goodCellIDs, FlowMarginQC$goodCellIDs)
        analysis <- "Flow Rate and Flow Margin"
      }else if(remove_from == "FS_FM"){
        goodCellIDs <- intersect(FlowSignalQC$goodCellIDs, FlowMarginQC$goodCellIDs)
        analysis <- "Flow Signal and Flow Margin"
      }else if(remove_from == "FR"){
        goodCellIDs <- FlowRateQC$goodCellIDs
        analysis <- "Flow Rate"
      }else if(remove_from == "FS"){
        goodCellIDs <- FlowSignalQC$goodCellIDs
        analysis <- "Flow Signal"
      }else if(remove_from == "FM"){
        goodCellIDs <- FlowMarginQC$goodCellIDs
        analysis <- "Flow Margin"

      badCellIDs <- setdiff(origin_cellIDs, goodCellIDs)
      totalBadPerc <- round(length(badCellIDs)/length(origin_cellIDs), 4)
      sub_exprs <- exprs(ordFCS)
      params <- parameters(ordFCS)
      keyval <- keyword(ordFCS)
      if (fcs_highQ != FALSE || output == 1) {
          goodfcs <- flowFrame(exprs = sub_exprs[goodCellIDs, ], parameters = params, description = keyval)
         if (fcs_highQ != FALSE) {suppressWarnings(write.FCS(goodfcs, good.fcs.file)) }
      if (fcs_QC != FALSE || output == 2 ){
          QCvector <- FlowSignalData$cellBinID[,"binID"]
          if(length(QCvector) > 9000) QCvector <- runif(length(QCvector), min=1, max=9000)
          QCvector[badCellIDs] <- runif(length(badCellIDs), min=10000, max=20000)
          newFCS <- addQC(QCvector, remove_from, sub_exprs, params, keyval)
          if (fcs_QC != FALSE){ suppressWarnings(write.FCS(newFCS, QC.fcs.file)) }
    if (length(badCellIDs) > 0 && fcs_lowQ != FALSE) {
      badfcs <- flowFrame(exprs = sub_exprs[badCellIDs, ],parameters = params,description = keyval)
      suppressWarnings(write.FCS(badfcs, bad.fcs.file))
    if (mini_report != FALSE) {
        write.table(t(c(filename, as.integer(dim(set[[i]])[1]),
        totalBadPerc * 100, analysis, FlowRateQC$res_fr_QC$badPerc * 100,
        FlowSignalQC$Perc_bad_cells$badPerc_tot * 100,
        FlowMarginQC$badPerc * 100)), minireport, sep="\t",
        append=TRUE, row.names = FALSE, quote = FALSE, col.names = FALSE)
     if (html_report != FALSE) {
       h_FS_graph <- round(0.4 * (ncol(ordFCS)),1)
       if (!is.null(ChExcludeFS)){
           ChannelExcludedFS <- as.character(grep(paste(ChExcludeFS, collapse="|"),
               ordFCS@parameters$name, value = TRUE))
       if (!is.null(ChExcludeFM)){
         ChannelExcludedFM <- as.character(grep(paste(ChExcludeFM, collapse="|"),
                                                ordFCS@parameters$name, value = TRUE))
       template_path <- system.file("rmd","autoQC_report.Rmd", package='flowAI')
       new_template <- paste0(folder_results, filename, "_template.Rmd")
       file.copy(template_path, new_template)
       # apparently the render function does not work well if there is not a space character in the name of the template
       if(folder_results != FALSE){  
          rmarkdown::render(new_template, html_document(), output_dir = folder_results, output_file = reportfile, quiet = TRUE )
          rmarkdown::render(new_template, html_document(), output_file = reportfile, quiet = TRUE )

      if(output == 1){
          out <- c(out, goodfcs)
      }else if ( output == 2){
          out <- c(out, newFCS)
      }else if( output == 3 ){
          out[[i]] <- badCellIDs
          names(out)[i] <- filename
  if( output == 1 || output == 2){
      if(length(out) == 1){ return( out[[1]] )
        OutSet <- as(out, "flowSet")
        flowCore::sampleNames(OutSet) <- names
        pData(OutSet) <- pData(set)
        return( OutSet ) }
  if( output == 3 ){ return(out) }

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flowAI documentation built on Nov. 8, 2020, 6:44 p.m.