bead_normalize: Bead-based normalization

In prybakowska/CytoQP: Cytometry data quality control and cleaninig

Bead-based normalization

Description

Performs bead-based normalization using beads spiked in the sample. It is based on functions from CATALYST package.

Usage

bead_normalize(
  files,
  cores = 1,
  markers_to_keep = NULL,
  non_mass_channel = NULL,
  beads = "dvs",
  norm_to_ref = NULL,
  remove_beads = TRUE,
  to_plot = TRUE,
  out_dir = NULL,
  k = 80,
  ...
)

Arguments

files	Character vector or list with the paths of the raw files.
cores	Number of cores to be used.Works only for not-Widows users.
markers_to_keep	Character vector, marker names to be kept after the normalization, can be full marker name e.g. "CD45" or "CD". If NULL (default) all markers will be normalized and kept in flowframe. Selection of the markers will reduce file volume and speedup the analysis. Non-mass channels like Time, Event_length, Gaussian parameters and in addition palladium barcoding channels are kept if non_mass_ch set to NULL.
non_mass_channel	Character vector, non-mass channels to keep for further analysis. Can be full channel name like Eu151Di or 151. By default "Time" and "event_length" will be always kept in the flow frame.
beads	Character, as in CATALYST::normCytof, "dvs" (for bead masses 140, 151, 153 ,165, 175) or "beta" (for bead masses 139, 141, 159, 169, 175) or a numeric vector of masses. Default is set to "dvs".
norm_to_ref	flow frame, created by baseline_file function to which input data will be normalized, default is set to NULL.
remove_beads	Logical, as in CATALYST::normCytof if beads should be removed from fcs files. Default set to TRUE. Note, should be set to FALSE if none of the channels is beads-specific.
to_plot	Logical if to plot bead gate and bead normalization lines for each file.Defaults is set to TRUE.
out_dir	Character, pathway to where the bead normalized fcs files and plots should be saved, for plots only if argument to_plot = TRUE, default is set to file.path(getwd(), BeadNorm).
k	The same as in CATALYST::normCytof, integer width of the median window used for bead smoothing (affects visualizations only).
...	Additional arguments to pass to normCytof.

Value

Save bead-normalized fcs files and plots to out_dir.

Examples

# set input directory (pathway to the files that are going to be normalized)
raw_data_dir <- file.path(dir, "RawFiles")

# set a directory where bead-normalized fcs files and plots will be saved
bead_norm_dir <- file.path(dir, "BeadNorm")

# define full pathway to the files that you want to normalize
files <- list.files(raw_data_dir,
                    pattern = ".FCS$",
                    full.names = TRUE)

# create baseline file to which all the files will be normalized
set.seed(2)
ref_sample <- baseline_file(fcs_files = files,
                            beads = "dvs",
                            out_dir = bead_norm_dir)

# Normalize files
bead_normalize(files, cores = 1,
               out_dir = bead_norm_dir,
               non_mass_channel = NULL,
               norm_to_ref = ref_sample,
               to_plot = TRUE,
               remove_beads = TRUE,
               k = 80,
               markers_to_keep = c("CD", "HLA", "IgD", "TCR", "Ir",
                                 "Viability","IL", "IFNa",
                                   "TNF", "TGF", "MIP", "MCP", "Granz"))

prybakowska/CytoQP documentation built on June 28, 2022, 12:36 a.m.