countUMI: Count the number of UMIs for each gene and output count...

In compbiomed/scuff: Single Cell RNA-Seq UMI Filtering Facilitator (scruff)

Count the number of UMIs for each gene and output count matrix

Description

Count unique UMI:gene pairs for single cell RNA-sequencing alignment files. Write resulting count matrix to output directory. Columns are samples (cells) and rows are gene IDs. The input sequence alignment files must be generated using FASTQ files generated by the demultiplex function in scruff package. Return a SingleCellExperiment object containing the count matrix, cell and gene annotations, and all QC metrics.

Usage

countUMI(
  sce,
  reference,
  umiEdit = 0,
  format = "BAM",
  outDir = "./Count",
  cellPerWell = 1,
  cores = max(1, parallelly::availableCores() - 2),
  outputPrefix = "countUMI",
  verbose = FALSE,
  logfilePrefix = format(Sys.time(), "%Y%m%d_%H%M%S")
)

Arguments

sce	A SingleCellExperiment object of which the colData slot contains the alignment_path column with paths to input cell-specific sequence alignment files (BAM or SAM format).
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.
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 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.
format	Format of input sequence alignment files. "BAM" or "SAM". Default is "BAM".
outDir	Output directory for UMI counting results. UMI corrected count matrix will be stored in this directory. Default is "./Count".
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.
cores	Number of cores used for parallelization. Default is max(1, parallelly::availableCores() - 2), i.e. the number of available cores minus 2.
outputPrefix	Prefix for expression table filename. Default is "countUMI".
verbose	Print log messages. Useful for debugging. Default to FALSE.
logfilePrefix	Prefix for log file. Default is current date and time in the format of format(Sys.time(), "%Y%m%d_%H%M%S").

Value

A SingleCellExperiment object.

Examples

## Not run: 
data(barcodeExample, package = "scruff")
# The SingleCellExperiment object returned by alignRsubread function and the
# alignment BAM files are required for running countUMI function
# First demultiplex example FASTQ files
fastqs <- list.files(system.file("extdata", package = "scruff"),
    pattern = "\\.fastq\\.gz", full.names = TRUE)

de <- demultiplex(
    project = "example",
    experiment = c("1h1"),
    lane = c("L001"),
    read1Path = c(fastqs[1]),
    read2Path = c(fastqs[2]),
    barcodeExample,
    bcStart = 1,
    bcStop = 8,
    umiStart = 9,
    umiStop = 12,
    keep = 75,
    overwrite = TRUE)

# Alignment
library(Rsubread)
# Create index files for GRCm38_MT.
fasta <- system.file("extdata", "GRCm38_MT.fa", package = "scruff")
# Specify the basename for Rsubread index
indexBase <- "GRCm38_MT"
buildindex(basename = indexBase, reference = fasta, indexSplit = FALSE)

al <- alignRsubread(de, indexBase, overwrite = TRUE)

# Counting
gtf <- system.file("extdata", "GRCm38_MT.gtf", package = "scruff")
sce = countUMI(al, gtf, cellPerWell=c(rep(1, 46), 0, 0))

## End(Not run)

# or use the built-in SingleCellExperiment object generated using
# example dataset (see ?sceExample)
data(sceExample, package = "scruff")

compbiomed/scuff documentation built on March 28, 2024, 10:54 a.m.