R/AllClasses.R

In HowardChao/RNASeqWorkflow: RNASeqR: an R package for automated two-group RNA-Seq analysis workflow

#' @title RNASeqR
#'
#' @description  An S4 class for checking and storing RNA-Seq workflow
#'  parameters of this package.
#'
#'
#' @slot os.type 'linux' or 'osx'. The operating system type.
#' @slot python.variable A list storing python environment.
#'    \code{(check.answer, python.version)}
#' @slot python.2to3 Logical value whether \code{2to3} command is available
#'    on the workstation.
#' @slot path.prefix Path prefix of 'gene_data/', 'RNASeq_bin/',
#'    'RNASeq_results/', 'Rscript/' and 'Rscript_out/' directories.
#' @slot input.path.prefix Path prefix of 'input_files/' directory,
#' @slot genome.name Variable of genome name defined in this RNA-Seq workflow
#'   (ex. \code{genome.name}.fa, \code{genome.name}.gtf).
#' @slot sample.pattern  Regular expression of paired-end fastq.gz files under
#'   'input_files/raw_fastq.gz'. Expression not includes \code{_[1,2].fastq.gz}.
#' @slot independent.variable Independent variable for the biological.
#' experiment design of two-group RNA-Seq workflow.
#' @slot case.group Group name of the case group.
#' @slot control.group Group name of the control group.
#' @slot indices.optional Logical value whether 'indices/' is exit in
#'   'input_files/'.
#' @slot fastq.gz.type Specify the fastq.gz file type. 'PE' represents 
#'   paired-end and 'SE' represents single-end.
#'
#' @name RNASeqRParam-class
#'
#' @rdname RNASeqRParam-class
#'
#' @exportClass RNASeqRParam
#' @author Kuan-Hao Chao
#' @examples
#' data(yeast)
#' "@@"(yeast, os.type)
#' "@@"(yeast, python.variable)
#' "@@"(yeast, python.2to3)
#' "@@"(yeast, path.prefix)
#' "@@"(yeast, input.path.prefix)
#' "@@"(yeast, genome.name)
#' "@@"(yeast, sample.pattern)
#' "@@"(yeast, independent.variable)
#' "@@"(yeast, case.group)
#' "@@"(yeast, control.group)
#' "@@"(yeast, indices.optional)
#' "@@"(yeast, fastq.gz.type)
setClass("RNASeqRParam",
         representation(
           os.type              = "character",
           python.variable      = "list",
           python.2to3          = "logical",
           path.prefix          = "character",
           input.path.prefix    = "character",
           genome.name          = "character",
           sample.pattern       = "character",
           independent.variable = "character",
           case.group           = "character",
           control.group        = "character",
           indices.optional     = "logical",
           fastq.gz.type        = "character"
         )
)



#' @title RNASeqRParam
#' @description  Constructor function for RNASeqRParam objects
#'
#' @name RNASeqRParam-constructor
#'
#' @param path.prefix Path prefix of 'gene_data/', 'RNASeq_bin/',
#'   'RNASeq_results/', 'Rscript/' and 'Rscript_out/' directories.
#' @param input.path.prefix Path prefix of 'input_files/' directory.
#' @param genome.name variable of genome name defined in this RNA-Seq workflow
#'   (ex. \code{genome.name}.fa, \code{genome.name}.gtf).
#' @param sample.pattern  Regular expression of paired-end fastq.gz files under
#'   'input_files/raw_fastq.gz'. Expression not includes \code{_[1,2].fastq.gz}.
#' @param independent.variable Independent variable for the biological
#'   experiment design of two-group RNA-Seq workflow.
#' @param case.group Group name of the case group.
#' @param control.group Group name of the control group.
#' @param fastq.gz.type Specify the fastq.gz file type. 'PE' represents 
#'   paired-end and 'SE' represents single-end.
#'
#' @return an object of class \code{RNASeqRParam}
#'
#' @author kuan-hao Chao
#'
#' @rdname RNASeqRParam-constructor
#'
#' @export
#' @author Kuan-Hao Chao
#' @examples
#' input_files.path <- system.file("extdata/", package = "RNASeqRData")
#' rnaseq_result.path <- tempdir(check = TRUE)
#' exp <- RNASeqRParam(path.prefix          = rnaseq_result.path,
#'                     input.path.prefix    = input_files.path,
#'                     genome.name          = "Saccharomyces_cerevisiae_XV_Ensembl",
#'                     sample.pattern       = "SRR[0-9]*_XV",
#'                     independent.variable = "state",
#'                     case.group           = "60mins_ID20_amphotericin_B",
#'                     control.group        = "60mins_ID20_control",
#'                     fastq.gz.type        = "PE")
RNASeqRParam <- function(path.prefix          = NA,
                         input.path.prefix    = NA,
                         genome.name          = NA,
                         sample.pattern       = NA,
                         independent.variable = NA,
                         case.group           = NA,
                         control.group        = NA,
                         fastq.gz.type        = NA) {
  # check input parameters
  CheckInputParamNa(path.prefix,
                    input.path.prefix,
                    genome.name,
                    sample.pattern,
                    independent.variable,
                    case.group,
                    control.group,
                    fastq.gz.type)
  if (fastq.gz.type != 'PE' && fastq.gz.type !='SE') {
    stop("'fastq.gz.type' must be 'PE' or 'SE'. Please check your input again.")
  }
  # 1. check operating system
  characters.os.type <- CheckOperatingSystem()
  # 2. check python version
  python.version.list <- CheckPython()
  bool.python.avail <- python.version.list$check.answer
  two.to.three.result <- Check2to3()
  # 3. check validity of path.prefix
  bool.prefix.path <- CheckPrefixPath(path.prefix = path.prefix)
  if (bool.prefix.path){
    # add '/' to the path.prefix
    if (substr(path.prefix, nchar(path.prefix), nchar(path.prefix)) != "/") {
      path.prefix <- paste0(path.prefix, "/")
    }
  }
  # 4. check input.path.prefix
  bool.input.path.prefix <- CheckInputPrefixPath(input.path.prefix)
  if (bool.input.path.prefix){
    # add '/' to the path.prefix
    if (substr(input.path.prefix,
               nchar(input.path.prefix),
               nchar(input.path.prefix)) != "/") {
      input.path.prefix <- paste0(input.path.prefix, "/")
    }
  }
  # 5. check 'phenodata'
  bool.phenodata <- CheckPhenodata(input.path.prefix,
                                   genome.name,
                                   sample.pattern,
                                   independent.variable)
  # check sample.pattern is valid file name !!
  # 6. check sample.pattern that can't have '.fastq.gz'
  fast.gz.extend <- tools::file_ext(sample.pattern)
  if (fast.gz.extend == "gz" || fast.gz.extend == "fastq") {
    message("(\u2718) : 'sample.pattern' can't include file ",
            "extension(.gz or .fastq or any) or any '.'\n\n")
    stop("'sample.pattern' with extension error.")
  }
  # 6. check 'input_files/' necessary files with 'genome.name', 'sample.pattern'
  input.dir.files.list <- CheckInputDirFiles(input.path.prefix,
                                             genome.name,
                                             sample.pattern,
                                             fastq.gz.type)
  bool.input.dir.files <- input.dir.files.list$check.answer
  # This determine whether to run 'CreateHisat2Index'
  bool.input.dir.indices <- input.dir.files.list$optional.indices.bool
  # below still need to fix
  # 7. check 'case.group' and 'control.group'
  bool.control.control.group <- CheckCaseControlGroup(input.path.prefix,
                                                      independent.variable,
                                                      case.group, control.group)

  if ( (characters.os.type == "linux" || characters.os.type == "osx") &&
      bool.python.avail && bool.prefix.path && bool.input.path.prefix &&
      bool.input.dir.files && bool.phenodata && bool.control.control.group) {
    message("\n**************************************\n")
    message("************** Success! **************\n")
    message("**************************************\n")
    new("RNASeqRParam",
        os.type              = characters.os.type,
        python.variable      = python.version.list,
        python.2to3          = two.to.three.result,
        path.prefix          = path.prefix,
        input.path.prefix    = input.path.prefix,
        genome.name          = genome.name,
        sample.pattern       = sample.pattern,
        independent.variable = independent.variable,
        case.group           = case.group,
        control.group        = control.group,
        indices.optional     = bool.input.dir.indices,
        fastq.gz.type        = fastq.gz.type)
  }
}

# inner function : check whether input values are NA
CheckInputParamNa <- function(path.prefix, input.path.prefix, genome.name,
                              sample.pattern, independent.variable,
                              case.group, control.group, fastq.gz.type) {
  message("************** Checking input parameters ************\n")
  if (is.na(path.prefix) || is.na(input.path.prefix) ||
      is.na(genome.name) || is.na(sample.pattern) ||
      is.na(independent.variable) ||
      is.na(case.group) || is.na(control.group) || is.na(fastq.gz.type)) {
    if (is.na(path.prefix)) {
      message("(\u2718) : 'path.prefix' is missing.\n\n")
    }
    if (is.na(input.path.prefix)) {
      message("(\u2718) : 'input.path.prefix' is missing.\n\n")
    }
    if (is.na(genome.name)) {
      message("(\u2718) : 'genome.name' is missing.\n\n")
    }
    if (is.na(sample.pattern)) {
      message("(\u2718) : 'sample.pattern' is missing.\n\n")
    }
    if (is.na(independent.variable)) {
      message("(\u2718) : 'independent.variable' is missing.\n\n")
    }
    if (is.na(case.group)) {
      message("(\u2718) : 'case.group' is missing.\n\n")
    }
    if (is.na(control.group)) {
      message("(\u2718) : 'control.group' is missing.\n\n")
    }
    if (is.na(fastq.gz.type)) {
      message("(\u2718) : 'fastq.gz.type' is missing.\n\n")
    }
    stop("Input parameters ERROR")
  } else {
    message("(\u2714) : Input parameters are all not NA !! \n\n")
  }
}

# inner function : get operating system
CheckOperatingSystem <- function(print = TRUE){
  if (print) {
    message("************** Checking operating system type ************\n")
  }
  sysinf <- Sys.info()
  if (!is.null(sysinf)){
    os <- sysinf["sysname"]
    if (os == "Darwin")
      os <- "osx"
  } else {
    ## mystery machine
    os <- .Platform$OS.type
    if (grepl("^darwin", R.version$os))
      os <- "osx"
    if (grepl("linux-gnu", R.version$os))
      os <- "linux"
  }
  os <- tolower(os)
  if (os != "linux" && os != "osx") {
    message("(\u2718) : Your system operating system is ", os, "\n")
    message("       This program supports only 'linux' and 'osx'\n\n")
    stop("Operating system ERROR")
  }
  if (print) {
    message("(\u2714) : Your system operating system is '", os, "'\n\n")
  }
  return(os)
}

# inner fucntion : check python version
CheckPython <- function() {
  message("************** Checking python version ************\n")
  # have to check python !!!
  if (reticulate::py_available(initialize = TRUE)){
    message("(\u2714) : Python is available on your device!\n")
    python.version <- as.numeric(reticulate::py_config()$version)
    message("       \u25CF Python version : ",
            reticulate::py_config()$version, "\n\n")
    if (python.version >= 3) {
      return.value <- list("check.answer" = TRUE, "python.version" = 3)
    } else if (python.version < 3 && python.version >= 2 ){
      return.value <- list("check.answer" = TRUE, "python.version" = 2)
    }
    return(return.value)
  } else {
    message("(\u2718) : Python is not available on this device.",
            "Please install python.",
            "(It's fine for python2 and python3)\n\n")
    stop("Python unavaliable ERROR")
  }
}

# inner function : check 2to3 availability
Check2to3 <- function() {
  message("************** Checking 2to3 command ************\n")
  a <- system2("2to3", "--help", stdout = FALSE)
  if (a == 0) {
    message("(\u2714) : 2to3 command is available on your device!\n\n")
    return(TRUE)
  } else {
    message("(\u2718) : 2to3 command is not available on your device!\n\n")
    return(FALSE)
  }
}

# inner function : check prefix.path
CheckPrefixPath <- function(path.prefix) {
  # Check the prefix exist
  if (isTRUE(dir.exists(path.prefix))){
    message("************** Setting prefix path ************\n")
    if (substr(path.prefix, nchar(path.prefix), nchar(path.prefix)) != "/") {
      path.prefix <- paste0(path.prefix, "/")
    }
    message("(\u270e) : The following files will be installed under '",
            path.prefix, "'\n\n")
    return(TRUE)
  } else if (is.na(path.prefix)) {
    message("(\u2718) : Please give value to prefix path.\n\n")
    stop("'prefix.path' NA ERROR")
  } else {
    message("(\u2718) : Prefix path '",
            path.prefix,"' is invalid. Please try another one.\n\n")
    stop("'prefix.path' invalid ERROR")
  }
}

# inner function : check prefix.path
CheckInputPrefixPath <- function(input.path.prefix) {
  # Check the prefix exist
  if (isTRUE(dir.exists(input.path.prefix))){
    if (substr(input.path.prefix,
               nchar(input.path.prefix),
               nchar(input.path.prefix)) != "/") {
      input.path.prefix <- paste0(input.path.prefix, "/")
    }
    path.prefix.input_files <- paste0(input.path.prefix, "input_files/")
    if (isTRUE(dir.exists(path.prefix.input_files))){
      message("************** Setting input prefix path ************\n")
      message("(\u270e) : 'input_files' is found under '",
              input.path.prefix,
              "' directory.\n")
      message("       \u25CF Validity of 'input_files/' will be checked.\n\n")
      return(TRUE)
    } else {
      message("(\u2718) : 'input_files/' is not found under '",
              input.path.prefix,
              "'. Please make sure the spelling is correct ",
              "or try another input.path.prefix.\n\n")
      stop("'input.prefix' path invalid ERROR")
    }
  } else if (is.na(input.path.prefix)) {
    message("(\u2718) : Please give value to input prefix path.\n\n")
    stop("'input.prefix' path NA ERROR")
  } else {
    message("(\u2718) : Input prefix path '", input.path.prefix,
            "' is invalid. Please try another one.\n\n")
    stop("'input.prefix' path invalid ERROR")
  }
}

# inner function : check input.path.prefix
CheckInputDirFiles <- function(input.path.prefix, genome.name, sample.pattern,
                               fastq.gz.type) {
  message("************** Checking hierarchy of '",
          input.path.prefix, "input_files/' ************\n")
  # only check whether exist
  gtf.file <- file.exists(paste0(input.path.prefix,
                                 "input_files/",
                                 genome.name, ".gtf"))
  # only check whether exist
  fa.file <- file.exists(paste0(input.path.prefix,
                                "input_files/",
                                genome.name, ".fa"))
  # check exist and rules
  raw.fastq.dir <- dir.exists(paste0(input.path.prefix,
                                     "input_files/raw_fastq.gz/"))
  # check exist and rules
  phenodata.file <- file.exists(paste0(input.path.prefix,
                                       "input_files/phenodata.csv"))
  # check exist and rules ( this is optional)
  ht2.dir <- dir.exists(paste0(input.path.prefix,
                               "input_files/indices/"))
  optional.indices.bool <- FALSE
  # check whether sample pattern matches the file names~
  if (raw.fastq.dir) {
    if (fastq.gz.type == 'SE') {
      raw.fastq <- list.files(path = paste0(input.path.prefix,
                                            "input_files/raw_fastq.gz/"),
                              pattern = sample.pattern, all.files = FALSE,
                              full.names = FALSE,
                              recursive = FALSE,
                              ignore.case = FALSE)
      extract.fastq.gz.sample.names <- unique(gsub("_1.fastq.gz", "",
                                                   raw.fastq))
      check.fastq.gz.1 <- vapply(extract.fastq.gz.sample.names,
                                 function(x) paste0(x, "_1.fastq.gz"),
                                 USE.NAMES = FALSE,
                                 FUN.VALUE = "a")
      # checking the valid file naming of '.fastq.gz'
      for ( i in seq_along(check.fastq.gz.1)) {
        bool.check.1 <- check.fastq.gz.1[i] %in% raw.fastq
        if (!bool.check.1) {
          message("(\u2718) : ",
                  gsub("_1.fastq.gz", "",
                       check.fastq.gz.1[i]),
                  " should not be found in 'input_files/raw_fastq.gz/\n")
          stop("Single-end file ERROR")
        }
      }
      if (sample.pattern == "") {
        message("(\u2718) : The value of 'sample.pattern' is \"\" !!\n")
        stop("'sample.pattern' ERROR")
      }
      raw.fastq.number <- length(raw.fastq)
    } else if (fastq.gz.type == 'PE') {
      raw.fastq <- list.files(path = paste0(input.path.prefix,
                                            "input_files/raw_fastq.gz/"),
                              pattern = sample.pattern, all.files = FALSE,
                              full.names = FALSE,
                              recursive = FALSE,
                              ignore.case = FALSE)
      extract.fastq.gz.sample.names <- unique(gsub("_[1-2]*.fastq.gz", "",
                                                   raw.fastq))
      check.fastq.gz.1 <- vapply(extract.fastq.gz.sample.names,
                                 function(x) paste0(x, "_1.fastq.gz"),
                                 USE.NAMES = FALSE,
                                 FUN.VALUE = "a")
      check.fastq.gz.2 <- vapply(extract.fastq.gz.sample.names,
                                 function(x) paste0(x, "_2.fastq.gz"),
                                 USE.NAMES = FALSE,
                                 FUN.VALUE = "a")
      # checking the valid file naming of '.fastq.gz'
      for ( i in seq_along(check.fastq.gz.1)) {
        bool.check.1 <- check.fastq.gz.1[i] %in% raw.fastq
        if (!bool.check.1) {
          message("(\u2718) : ",
                  check.fastq.gz.1[i],
                  " is not found in 'input_files/raw_fastq.gz/\n")
          stop("Pair-end file ERROR")
        }
      }
      for ( i in seq_along(check.fastq.gz.2)) {
        bool.check.2 <- check.fastq.gz.2[i] %in% raw.fastq
        if (!bool.check.2) {
          message("(\u2718) : ",
                  check.fastq.gz.2[i],
                  " is not found in 'input_files/raw_fastq.gz/\n")
          stop("Pair-end file ERROR")
        }
      }
      if (sample.pattern == "") {
        message("(\u2718) : The value of 'sample.pattern' is \"\" !!\n")
        stop("'sample.pattern' ERROR")
      }
      raw.fastq.number <- length(raw.fastq) 
    }
  }
  if (isTRUE(ht2.dir)) {
    ht2.files <- list.files(path = paste0(input.path.prefix,
                                          "input_files/indices/"),
                            pattern = paste0("^", genome.name,
                                             "_tran.[0-9]*.ht2$"),
                            all.files = FALSE,
                            full.names = FALSE,
                            recursive = FALSE,
                            ignore.case = FALSE)
    ht2.files.number <- length(ht2.files)
  }
  if (!isTRUE(gtf.file)) {
    grab.gtf.file <- Sys.glob(file.path(path = paste0(input.path.prefix,
                                                      "input_files"), "*.gtf"))
    message("(\u2718) : '", genome.name,
            ".gtf (user input)' and '", grab.gtf.file,
            " (find in directory)' ", " are mismatched.\n")
  } else {
    message("(\u2714) : '", genome.name,
            ".gtf'", " is in 'input_files'\n")
  }
  if (!isTRUE(fa.file)) {
    grab.fa.file <- Sys.glob(file.path(path = paste0(input.path.prefix,
                                                     "input_files"), "*.fa"))
    message("(\u2718) : '", genome.name, ".fa (user input)' and '",
            grab.fa.file, " (find in directory)' are mismatched.\n")
  } else {
    message("(\u2714) : '",
            genome.name, ".fa' is in 'input_files'\n")
  }
  if (!isTRUE(raw.fastq.dir)) {
    message("(\u2718) : 'raw_fastq.gz/' is missing.\n")
  } else {
    message("(\u2714) : 'raw_fastq.gz/' is in 'input_files'\n")
  }
  if (isTRUE(raw.fastq.dir) && raw.fastq.number == 0) {
    message("(\u2718) : Sample pattern \"",
            sample.pattern, "\" is not found in 'raw_fastq.gz/'.\n")
  } else if (isTRUE(raw.fastq.dir) && raw.fastq.number >= 0) {
    for (i in raw.fastq) {
      message("(\u2714) : 'raw_fastq.gz/", i,
              "' is in 'input_files/'\n")
    }
  }
  if (!isTRUE(phenodata.file)) {
    message("(\u2718) : '", "phenodata.csv is missing.\n")
  } else {
    message("(\u2714) : '", "phenodata.csv is in 'input_files'\n")
  }
  if (!isTRUE(ht2.dir)) {
    ## not exist
    message("(\u26A0) : 'indices/' is optional.",
            " You can download the corresponding 'XXX.ht2' from",
            " 'https://ccb.jhu.edu/software/hisat2/index.shtml'",
            " to speed up the process.\n")
  } else {
    ## exist
    message("(\u2714) : 'indices/' is in 'input_files'\n")
  }
  if (isTRUE(ht2.dir) && ht2.files.number == 0) {
    message("(\u26A0) : 'indices/' directory has been created but there",
            " are no samples in 'indices/' or files' names",
            paste0("\"^", genome.name, "_tran.[0-9]*.ht2$\" "),
            "in 'indices/' are not found.\n      ",
            "No files will be copied.\n      ",
            "(1). Check whether files name",
            paste0("'", genome.name, "_tran.[0-9]*.ht2'"),
            "matches the files in 'indices' directory.\n      ",
            "(2). If you don't have",
            paste0("'", genome.name, "_tran.[0-9].ht2'"),
            "files, remove 'indices' directory\n\n")
    stop("'input_files/indices/' ERROR")
  } else if (isTRUE(ht2.dir) && ht2.files.number >= 0) {
    optional.indices.bool <- TRUE
    for (i in ht2.files) {
      message("(\u2714) : 'indices/", i, "' is in 'input_files/'\n")
    }
  }
  if (gtf.file && raw.fastq.dir && raw.fastq.dir &&
      raw.fastq.number != 0 && phenodata.file) {
    message("\n(\u2714) : '", input.path.prefix,
                   "input_files/", "' is valid !\n")
    if (ht2.dir && (ht2.files.number != 0) ) {
      message("(\u2714) : optional directory 'indices/' is valid !\n")
      optional.indices.bool <- TRUE
    }
    message("\n")
    return.value <- list("check.answer" = TRUE,
                         "optional.indices.bool" = optional.indices.bool)
    return(return.value)
  } else {
    stop("'input_files/' checking ERROR")
  }
}

# inner function : check validity of phenodata
CheckPhenodata <- function(input.path.prefix,
                           genome.name,
                           sample.pattern,
                           independent.variable) {
  # have to sort the column !! and sort them in the correct order
  message("************** Checking phenodata  ************\n")
  pheno_data <- read.csv(paste0(input.path.prefix, "input_files/phenodata.csv"))
  # Covert all column to character
  pheno_data <- data.frame(lapply(pheno_data, as.character),
                           stringsAsFactors = FALSE)
  # Checl 'ids' is in the 'phenodata.csv'
  if (!("ids" %in% colnames(pheno_data))) {
    message("(\u2718) : 'ids' can't find in the ",
            "column of phenodata.csv.\n\n")
    stop("'ids' invalid ERROR")
  }
  # "id" : must be distinct, same as input_files raw reads name !
  message("\u25B6 Checking whether \"raw_fastq.gz files\" ",
          "matches \"'ids' of phenodata.csv\" \n")
  raw.fastq <- list.files(path = paste0(input.path.prefix,
                                        "input_files/raw_fastq.gz/"),
                          pattern = sample.pattern,
                          all.files = FALSE,
                          full.names = FALSE,
                          recursive = FALSE,
                          ignore.case = FALSE)
  extract.fastq.gz.sample.names <- unique(gsub("_[1-2]*.fastq.gz", "",
                                               raw.fastq))
  bool.length <- length(extract.fastq.gz.sample.names) == length(pheno_data$ids)
  bool.identical <- identical(sort(extract.fastq.gz.sample.names),
                              sort(pheno_data$ids))
  if (!bool.length || !bool.identical) {
    message("(\u2718) : 'ids' column doesn't match the smaple_id in ",
            "'input_files/raw_fastq.gz'. Please check the file and ",
            "'input_files/raw_fastq.gz' directory.\n" )
    stop("'ids' mismatch ERROR")
  }
  ids.list <- paste(sort(extract.fastq.gz.sample.names), collapse = ", ")
  message("(\u2714) : Column 'ids' of phenodata.csv is valid. \n")
  message("      \u25CF sample ids are : \"", ids.list, "\"\n")
  # Check again independent.variable in the list
  if (!(independent.variable %in% colnames(pheno_data))) {
    message("(\u2718) : 'independent.variable' : '",
            independent.variable,
            "' can't find in the column of phenodata.csv.\n\n")
    stop("'independent.variable' invalid ERROR")
  }
  message("(\u2714) : 'independent.variable' : '",
          independent.variable,
          "' is in the column of phenodata.csv. \n\n")
  message("\u25B6 Checking whether '",
          independent.variable,
          "' is a two-group 'independent.variable' ...\n")
  length.independent.variable <- length(table(pheno_data[independent.variable]))
  if (!(length.independent.variable == 2)) {
    message("(\u2718) : 'independent.variable' : '",
            independent.variable, "' is a ",
            length.independent.variable,
            "-group 'independent.variable'. Not 2-group.\n")
    message("          groups that found : ",
            paste0(names(table(pheno_data[independent.variable])),
                   collapse = ", "), "\n")
    stop("'independent.variable' none-two-group ERROR")
  }
  message("(\u2714) : Column 'independent.variable' : '",
          independent.variable, "' of phenodata.csv is valid. \n")
  message("      \u25CF 'independent.variable' : '",
          independent.variable, "'\n\n")
  return(TRUE)
}

# inner function
CheckCaseControlGroup <- function(input.path.prefix,
                                  independent.variable,
                                  case.group,
                                  control.group) {
  message("************** Checking 'case.group' ",
          "& 'control.group' ************\n")
  pheno_data <- read.csv(paste0(input.path.prefix, "input_files/phenodata.csv"))
  # Covert all column to character
  pheno_data <- data.frame(lapply(pheno_data, as.character),
                           stringsAsFactors = FALSE)
  cont.in <- case.group %in%
    as.character(data.frame(table(pheno_data[independent.variable]))$Var1)
  exp.in <- control.group %in%
    as.character(data.frame(table(pheno_data[independent.variable]))$Var1)
  # Check 'case.group' is on group of 'independent.variable'
  if (!cont.in) {
    message("(\u2718) : 'case.group' : '", case.group,
            "' is not a group of in 'independent.variable'.\n\n")
    stop("'case.group' invalid ERROR")
  }
  if (!exp.in) {
    message("(\u2718) : 'control.group' : '", control.group,
            "' is not a group of in 'independent.variable'.\n\n")
    stop("'control.group' invalid ERROR")
  }
  if (exp.in && cont.in) {
    message("(\u2714) :    'case.group' : '",
            case.group,
            "' is a group of in 'independent.variable'.\n")
    message("(\u2714) : 'control.group' : '",
            control.group,
            "' is a group of in 'independent.variable'.\n\n")
    if (control.group == case.group) {
      message("(\u2718) : 'case.group' and 'control.group' are same.\n\n")
      stop("'case.group' &'control.group' same ERROR")
    }
  return(TRUE)
  }
}