R/scruff.R
In 87875172/Scuff: Single Cell RNA-Seq UMI Filtering Facilitator (scruff)
Defines functions
scruff
Documented in
scruff
#' Run scruff pipeline
#'
#' Run the \code{scruff} pipeline. This function performs all
#' \code{demultiplex}, \code{alignRsubread}, and \code{countUMI} functions.
#' Write demultiplex statistics, alignment statistics, and UMI filtered count
#' matrix in output directories. Return a SingleCellExperiment object
#' containing the count matrix, cell and gene annotations, and all QC metrics.
#'
#' @param project The project name. Default is
#' \code{paste0("project_", Sys.Date())}.
#' @param experiment A character vector of experiment names. Represents the
#' group label for each FASTQ file, e.g. "patient1, patient2, ...". The number
#' of cells in a experiment equals the length of cell barcodes \code{bc}. The
#' length of \code{experiment} equals the number of FASTQ files to be
#' processed.
#' @param lane A character or character vector of flow cell lane numbers. If
#' FASTQ files from multiple lanes are concatenated, any placeholder would be
#' sufficient, e.g. "L001".
#' @param read1Path A character vector of file paths to the read1 FASTQ files.
#' These are the read files with UMI and cell barcode information.
#' @param read2Path A character vector of file paths to the read2 FASTQ files.
#' These read files contain genomic sequences.
#' @param bc A vector of pre-determined cell barcodes. For example, see
#' \code{?barcodeExample}.
#' @param index Path to the \code{Rsubread} index of the reference genome. For
#' generation of Rsubread indices, please refer to \code{buildindex} function
#' in \code{Rsubread} package.
#' @param reference Path to the reference GTF file. The TxDb object of the GTF
#' file will be generated and saved in the current working directory with
#' ".sqlite" suffix.
#' @param bcStart Integer or vector of integers containing the cell barcode
#' start positions (inclusive, one-based numbering).
#' @param bcStop Integer or vector of integers containing the cell barcode
#' stop positions (inclusive, one-based numbering).
#' @param bcEdit Maximally allowed Hamming distance for barcode correction.
#' Barcodes with mismatches equal or fewer than this will be assigned a
#' corrected barcode if the inferred barcode matches uniquely in the provided
#' predetermined barcode list. Default is 0, meaning no cell barcode
#' correction is performed.
#' @param umiStart Integer or vector of integers containing the start positions
#' (inclusive, one-based numbering) of UMI sequences.
#' @param umiStop Integer or vector of integers containing the stop positions
#' (inclusive, one-based numbering) of UMI sequences.
#' @param umiEdit Maximally allowed Hamming distance for UMI correction. For
#' read alignments in each gene, by comparing to a more abundant UMI with more
#' reads, UMIs having fewer reads and with mismatches equal or fewer than
#' \code{umiEdit} will be assigned a corrected UMI (the UMI with more reads).
#' Default is 0, meaning no UMI correction is performed. Doing UMI correction
#' will decrease the number of transcripts per gene.
#' @param keep Read trimming. Read length or number of nucleotides to keep for
#' read 2 (the read that contains transcript sequence information). Longer
#' reads will be clipped at 3' end. Shorter reads will not be affected. This
#' number should be determined based on the sequencing kit that was used in
#' library preparation step.
#' @param cellPerWell Number of cells per well. Can be an integer (e.g. 1)
#' indicating the number of cells in each well or an vector with length equal
#' to the total number of cells in the input alignment files specifying the
#' number of cells in each file. Default is 1.
#' @param unique Argument passed to \code{align} function in \code{Rsubread}
#' package. Boolean indicating if only uniquely mapped reads should be
#' reported. A uniquely mapped read has one single mapping location that has
#' less mis-matched bases than any other candidate locations. If set to
#' \strong{FALSE}, multi-mapping reads will be reported in addition to
#' uniquely mapped reads. Number of alignments reported for each multi-mapping
#' read is determined by the nBestLocations parameter. Default is
#' \strong{FALSE}.
#' @param nBestLocations Argument passed to \code{align} function in
#' \code{Rsubread} package. Numeric value specifying the maximal number of
#' equally-best mapping locations that will be reported for a multi-mapping
#' read. 1 by default. The allowed value is between 1 to 16 (inclusive). In
#' the mapping output, "NH" tag is used to indicate how many alignments are
#' reported for the read and "HI" tag is used for numbering the alignments
#' reported for the same read. This argument is only applicable when unique
#' option is set to \strong{FALSE}.
#' @param minQual Minimally acceptable Phred quality score for cell barcode and
#' UMI sequences. Phread quality scores are calculated for each nucleotide in
#' these tags. Tags with at least one nucleotide with score lower than this
#' will be filtered out. Default is \strong{10}.
#' @param yieldReads The number of reads to yield when drawing successive
#' subsets from a fastq file, providing the number of successive records to be
#' returned on each yield. This parameter is passed to the \code{n} argument
#' of the \code{FastqStreamer} function in \emph{ShortRead} package. Default
#' is \strong{1e06}.
#' @param alignmentFileFormat File format of sequence alignment results.
#' \strong{"BAM"} or \strong{"SAM"}. Default is \strong{"BAM"}.
#' @param demultiplexOutDir Output folder path for demultiplex results.
#' Demultiplexed cell specifc FASTQ files will be stored in folders in this
#' path, respectively. \strong{Make sure the folder is empty.} Default is
#' \code{"./Demultiplex"}.
#' @param alignmentOutDir Output directory for alignment results. Sequence
#' alignment maps will be stored in folders in this directory, respectively.
#' \strong{Make sure the folder is empty.} Default is \code{"./Alignment"}.
#' @param countUmiOutDir Output directory for UMI counting results. UMI
#' filtered count matrix will be stored in this directory. Default is
#' \code{"./Count"}.
#' @param demultiplexSummaryPrefix Prefix for demultiplex summary filename.
#' Default is \code{"demultiplex"}.
#' @param alignmentSummaryPrefix Prefix for alignment summary filename. Default
#' is \code{"alignment"}.
#' @param countPrefix Prefix for UMI filtered count matrix filename. Default is
#' \code{"countUMI"}.
#' @param logfilePrefix Prefix for log file. Default is current date and time
#' in the format of \code{format(Sys.time(), "\%Y\%m\%d_\%H\%M\%S")}.
#' @param overwrite Boolean indicating whether to overwrite the output
#' directory. Default is \strong{FALSE}.
#' @param verbose Boolean indicating whether to print log messages. Useful for
#' debugging. Default to \strong{FALSE}.
#' @param cores Number of cores to use for parallelization. Default is
#' \code{max(1, parallelly::availableCores() - 2)}, i.e. the number of
#' available cores minus 2.
#' @param threads \strong{Do not change}. Number of threads/CPUs used for
#' mapping for each core. Refer to \code{align} function in \code{Rsubread}
#' for details. Default is \strong{1}. It should not be changed in most cases.
#' @param ... Additional arguments passed to the \code{align} function in
#' \code{Rsubread} package.
#' @return A \code{SingleCellExperiment} object.
#' @examples
#' \dontrun{
#' # prepare required files
#'
#' data(barcodeExample, package = "scruff")
#' fastqs <- list.files(system.file("extdata", package = "scruff"),
#' pattern = "\\.fastq\\.gz", full.names = TRUE)
#' fasta <- system.file("extdata", "GRCm38_MT.fa", package = "scruff")
#' gtf <- system.file("extdata", "GRCm38_MT.gtf", package = "scruff")
#'
#' library(Rsubread)
#' # Specify the basename for Rsubread index
#' indexBase <- "GRCm38_MT"
#' # Create index files for GRCm38_MT.
#' buildindex(basename = indexBase, reference = fasta, indexSplit = FALSE)
#'
#' # run scruff pipeline
#' sce <- scruff(project = "example",
#' experiment = c("1h1"),
#' lane = c("L001"),
#' read1Path = c(fastqs[1]),
#' read2Path = c(fastqs[2]),
#' bc = barcodeExample,
#' index = indexBase,
#' reference = gtf,
#' bcStart = 1,
#' bcStop = 8,
#' umiStart = 9,
#' umiStop = 12,
#' keep = 75,
#' cellPerWell = c(rep(1, 46), 0, 0),
#' overwrite = TRUE,
#' verbose = TRUE)
#' }
#'
#' # or use the built-in SingleCellExperiment object generated using
#' # example dataset (see ?sceExample)
#' data(sceExample, package = "scruff")
#' @export
scruff <- function(project = paste0("project_", Sys.Date()),
experiment,
lane,
read1Path,
read2Path,
bc,
index,
reference,
bcStart,
bcStop,
bcEdit = 0,
umiStart,
umiStop,
umiEdit = 0,
keep,
cellPerWell = 1,
unique = FALSE,
nBestLocations = 1,
minQual = 10,
yieldReads = 1e06,
alignmentFileFormat = "BAM",
demultiplexOutDir = "./Demultiplex",
alignmentOutDir = "./Alignment",
countUmiOutDir = "./Count",
demultiplexSummaryPrefix = "demultiplex",
alignmentSummaryPrefix = "alignment",
countPrefix = "countUMI",
logfilePrefix = format(Sys.time(), "%Y%m%d_%H%M%S"),
overwrite = FALSE,
verbose = FALSE,
cores = max(1, parallelly::availableCores() - 2),
threads = 1,
...) {
# run pipeline
message(Sys.time(), " Start running scruff ...")
print(match.call(expand.dots = TRUE))
de <- demultiplex(
project = project,
experiment = experiment,
lane = lane,
read1Path = read1Path,
read2Path = read2Path,
bc = bc,
bcStart = bcStart,
bcStop = bcStop,
bcEdit = bcEdit,
umiStart = umiStart,
umiStop = umiStop,
keep = keep,
minQual = minQual,
yieldReads = yieldReads,
outDir = demultiplexOutDir,
summaryPrefix = demultiplexSummaryPrefix,
overwrite = overwrite,
verbose = verbose,
cores = cores,
logfilePrefix = logfilePrefix
)
al <- alignRsubread(
sce = de,
index = index,
unique = unique,
nBestLocations = nBestLocations,
format = alignmentFileFormat,
outDir = alignmentOutDir,
cores = cores,
threads = threads,
summaryPrefix = alignmentSummaryPrefix,
overwrite = overwrite,
verbose = verbose,
logfilePrefix = logfilePrefix,
...
)
co <- countUMI(
sce = al,
reference = reference,
umiEdit = umiEdit,
format = alignmentFileFormat,
outDir = countUmiOutDir,
cellPerWell = cellPerWell,
cores = cores,
outputPrefix = countPrefix,
verbose = verbose,
logfilePrefix = logfilePrefix
)
message(Sys.time(), " Finished running scruff ...")
return(co)
}
87875172/Scuff documentation built on March 28, 2024, 2:33 a.m.
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Defines functions scruff
Documented in scruff
#' Run scruff pipeline
#'
#' Run the \code{scruff} pipeline. This function performs all
#' \code{demultiplex}, \code{alignRsubread}, and \code{countUMI} functions.
#' Write demultiplex statistics, alignment statistics, and UMI filtered count
#' matrix in output directories. Return a SingleCellExperiment object
#' containing the count matrix, cell and gene annotations, and all QC metrics.
#'
#' @param project The project name. Default is
#' \code{paste0("project_", Sys.Date())}.
#' @param experiment A character vector of experiment names. Represents the
#' group label for each FASTQ file, e.g. "patient1, patient2, ...". The number
#' of cells in a experiment equals the length of cell barcodes \code{bc}. The
#' length of \code{experiment} equals the number of FASTQ files to be
#' processed.
#' @param lane A character or character vector of flow cell lane numbers. If
#' FASTQ files from multiple lanes are concatenated, any placeholder would be
#' sufficient, e.g. "L001".
#' @param read1Path A character vector of file paths to the read1 FASTQ files.
#' These are the read files with UMI and cell barcode information.
#' @param read2Path A character vector of file paths to the read2 FASTQ files.
#' These read files contain genomic sequences.
#' @param bc A vector of pre-determined cell barcodes. For example, see
#' \code{?barcodeExample}.
#' @param index Path to the \code{Rsubread} index of the reference genome. For
#' generation of Rsubread indices, please refer to \code{buildindex} function
#' in \code{Rsubread} package.
#' @param reference Path to the reference GTF file. The TxDb object of the GTF
#' file will be generated and saved in the current working directory with
#' ".sqlite" suffix.
#' @param bcStart Integer or vector of integers containing the cell barcode
#' start positions (inclusive, one-based numbering).
#' @param bcStop Integer or vector of integers containing the cell barcode
#' stop positions (inclusive, one-based numbering).
#' @param bcEdit Maximally allowed Hamming distance for barcode correction.
#' Barcodes with mismatches equal or fewer than this will be assigned a
#' corrected barcode if the inferred barcode matches uniquely in the provided
#' predetermined barcode list. Default is 0, meaning no cell barcode
#' correction is performed.
#' @param umiStart Integer or vector of integers containing the start positions
#' (inclusive, one-based numbering) of UMI sequences.
#' @param umiStop Integer or vector of integers containing the stop positions
#' (inclusive, one-based numbering) of UMI sequences.
#' @param umiEdit Maximally allowed Hamming distance for UMI correction. For
#' read alignments in each gene, by comparing to a more abundant UMI with more
#' reads, UMIs having fewer reads and with mismatches equal or fewer than
#' \code{umiEdit} will be assigned a corrected UMI (the UMI with more reads).
#' Default is 0, meaning no UMI correction is performed. Doing UMI correction
#' will decrease the number of transcripts per gene.
#' @param keep Read trimming. Read length or number of nucleotides to keep for
#' read 2 (the read that contains transcript sequence information). Longer
#' reads will be clipped at 3' end. Shorter reads will not be affected. This
#' number should be determined based on the sequencing kit that was used in
#' library preparation step.
#' @param cellPerWell Number of cells per well. Can be an integer (e.g. 1)
#' indicating the number of cells in each well or an vector with length equal
#' to the total number of cells in the input alignment files specifying the
#' number of cells in each file. Default is 1.
#' @param unique Argument passed to \code{align} function in \code{Rsubread}
#' package. Boolean indicating if only uniquely mapped reads should be
#' reported. A uniquely mapped read has one single mapping location that has
#' less mis-matched bases than any other candidate locations. If set to
#' \strong{FALSE}, multi-mapping reads will be reported in addition to
#' uniquely mapped reads. Number of alignments reported for each multi-mapping
#' read is determined by the nBestLocations parameter. Default is
#' \strong{FALSE}.
#' @param nBestLocations Argument passed to \code{align} function in
#' \code{Rsubread} package. Numeric value specifying the maximal number of
#' equally-best mapping locations that will be reported for a multi-mapping
#' read. 1 by default. The allowed value is between 1 to 16 (inclusive). In
#' the mapping output, "NH" tag is used to indicate how many alignments are
#' reported for the read and "HI" tag is used for numbering the alignments
#' reported for the same read. This argument is only applicable when unique
#' option is set to \strong{FALSE}.
#' @param minQual Minimally acceptable Phred quality score for cell barcode and
#' UMI sequences. Phread quality scores are calculated for each nucleotide in
#' these tags. Tags with at least one nucleotide with score lower than this
#' will be filtered out. Default is \strong{10}.
#' @param yieldReads The number of reads to yield when drawing successive
#' subsets from a fastq file, providing the number of successive records to be
#' returned on each yield. This parameter is passed to the \code{n} argument
#' of the \code{FastqStreamer} function in \emph{ShortRead} package. Default
#' is \strong{1e06}.
#' @param alignmentFileFormat File format of sequence alignment results.
#' \strong{"BAM"} or \strong{"SAM"}. Default is \strong{"BAM"}.
#' @param demultiplexOutDir Output folder path for demultiplex results.
#' Demultiplexed cell specifc FASTQ files will be stored in folders in this
#' path, respectively. \strong{Make sure the folder is empty.} Default is
#' \code{"./Demultiplex"}.
#' @param alignmentOutDir Output directory for alignment results. Sequence
#' alignment maps will be stored in folders in this directory, respectively.
#' \strong{Make sure the folder is empty.} Default is \code{"./Alignment"}.
#' @param countUmiOutDir Output directory for UMI counting results. UMI
#' filtered count matrix will be stored in this directory. Default is
#' \code{"./Count"}.
#' @param demultiplexSummaryPrefix Prefix for demultiplex summary filename.
#' Default is \code{"demultiplex"}.
#' @param alignmentSummaryPrefix Prefix for alignment summary filename. Default
#' is \code{"alignment"}.
#' @param countPrefix Prefix for UMI filtered count matrix filename. Default is
#' \code{"countUMI"}.
#' @param logfilePrefix Prefix for log file. Default is current date and time
#' in the format of \code{format(Sys.time(), "\%Y\%m\%d_\%H\%M\%S")}.
#' @param overwrite Boolean indicating whether to overwrite the output
#' directory. Default is \strong{FALSE}.
#' @param verbose Boolean indicating whether to print log messages. Useful for
#' debugging. Default to \strong{FALSE}.
#' @param cores Number of cores to use for parallelization. Default is
#' \code{max(1, parallelly::availableCores() - 2)}, i.e. the number of
#' available cores minus 2.
#' @param threads \strong{Do not change}. Number of threads/CPUs used for
#' mapping for each core. Refer to \code{align} function in \code{Rsubread}
#' for details. Default is \strong{1}. It should not be changed in most cases.
#' @param ... Additional arguments passed to the \code{align} function in
#' \code{Rsubread} package.
#' @return A \code{SingleCellExperiment} object.
#' @examples
#' \dontrun{
#' # prepare required files
#'
#' data(barcodeExample, package = "scruff")
#' fastqs <- list.files(system.file("extdata", package = "scruff"),
#' pattern = "\\.fastq\\.gz", full.names = TRUE)
#' fasta <- system.file("extdata", "GRCm38_MT.fa", package = "scruff")
#' gtf <- system.file("extdata", "GRCm38_MT.gtf", package = "scruff")
#'
#' library(Rsubread)
#' # Specify the basename for Rsubread index
#' indexBase <- "GRCm38_MT"
#' # Create index files for GRCm38_MT.
#' buildindex(basename = indexBase, reference = fasta, indexSplit = FALSE)
#'
#' # run scruff pipeline
#' sce <- scruff(project = "example",
#' experiment = c("1h1"),
#' lane = c("L001"),
#' read1Path = c(fastqs[1]),
#' read2Path = c(fastqs[2]),
#' bc = barcodeExample,
#' index = indexBase,
#' reference = gtf,
#' bcStart = 1,
#' bcStop = 8,
#' umiStart = 9,
#' umiStop = 12,
#' keep = 75,
#' cellPerWell = c(rep(1, 46), 0, 0),
#' overwrite = TRUE,
#' verbose = TRUE)
#' }
#'
#' # or use the built-in SingleCellExperiment object generated using
#' # example dataset (see ?sceExample)
#' data(sceExample, package = "scruff")
#' @export
scruff <- function(project = paste0("project_", Sys.Date()),
experiment,
lane,
read1Path,
read2Path,
bc,
index,
reference,
bcStart,
bcStop,
bcEdit = 0,
umiStart,
umiStop,
umiEdit = 0,
keep,
cellPerWell = 1,
unique = FALSE,
nBestLocations = 1,
minQual = 10,
yieldReads = 1e06,
alignmentFileFormat = "BAM",
demultiplexOutDir = "./Demultiplex",
alignmentOutDir = "./Alignment",
countUmiOutDir = "./Count",
demultiplexSummaryPrefix = "demultiplex",
alignmentSummaryPrefix = "alignment",
countPrefix = "countUMI",
logfilePrefix = format(Sys.time(), "%Y%m%d_%H%M%S"),
overwrite = FALSE,
verbose = FALSE,
cores = max(1, parallelly::availableCores() - 2),
threads = 1,
...) {
# run pipeline
message(Sys.time(), " Start running scruff ...")
print(match.call(expand.dots = TRUE))
de <- demultiplex(
project = project,
experiment = experiment,
lane = lane,
read1Path = read1Path,
read2Path = read2Path,
bc = bc,
bcStart = bcStart,
bcStop = bcStop,
bcEdit = bcEdit,
umiStart = umiStart,
umiStop = umiStop,
keep = keep,
minQual = minQual,
yieldReads = yieldReads,
outDir = demultiplexOutDir,
summaryPrefix = demultiplexSummaryPrefix,
overwrite = overwrite,
verbose = verbose,
cores = cores,
logfilePrefix = logfilePrefix
)
al <- alignRsubread(
sce = de,
index = index,
unique = unique,
nBestLocations = nBestLocations,
format = alignmentFileFormat,
outDir = alignmentOutDir,
cores = cores,
threads = threads,
summaryPrefix = alignmentSummaryPrefix,
overwrite = overwrite,
verbose = verbose,
logfilePrefix = logfilePrefix,
...
)
co <- countUMI(
sce = al,
reference = reference,
umiEdit = umiEdit,
format = alignmentFileFormat,
outDir = countUmiOutDir,
cellPerWell = cellPerWell,
cores = cores,
outputPrefix = countPrefix,
verbose = verbose,
logfilePrefix = logfilePrefix
)
message(Sys.time(), " Finished running scruff ...")
return(co)
}
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