Unmix FlowCode

Unmix FlowCode samples, correcting FRET errors and debarcoding the data.

Usage

unmix.flowcode(
  raw.data,
  spectra,
  af.spectra,
  spectra.variants,
  flowcode.spectra,
  asp,
  thresholds = NULL,
  weights = NULL,
  cell.weighting = FALSE,
  cell.weight.regularize = TRUE,
  k = 1,
  parallel = TRUE,
  threads = if (parallel) 0 else 1,
  verbose = TRUE
)

Arguments

raw.data

Expression data from raw FCS files. Cells in rows and detectors in columns. Columns should be fluorescent data only and must match the columns in spectra.

spectra

Spectral signatures of fluorophores, normalized between 0 and 1, with fluorophores in rows and detectors in columns.

af.spectra

Spectral signatures of autofluorescences, normalized between 0 and 1, with fluorophores in rows and detectors in columns. Prepare using get.af.spectra.

spectra.variants

Named list (names are fluorophores) carrying matrices of spectral signature variations for each fluorophore. Prepare using get.spectral.variants(). Default is NULL.

flowcode.spectra

Structured output from get.flowcode.spectra(), which details the combination-level spectral unmixing errors due to FRET-like artefacts.

asp

The AutoSpectral parameter list. Prepare using get.autospectral.param.

thresholds

Optional named numeric vector of positivity thresholds for the FlowCode fluorophores. Overrides the thresholds provided by flowcode.spectra. Default is NULL, which is unused.

weights

Optional numeric vector of weights (one per fluorescent detector). Default is NULL, in which case weighting will be done by channel means (Poisson variance).

cell.weighting

Logical, whether to use cell-specific weighting for a more Poisson-like unmixing. Default is FALSE.

cell.weight.regularize

Logical, whether to regularize cell-specific weights towards the bulk mean weighting set by weights. 50:50 averaging. Default is TRUE. Only active if cell.weighting=TRUE.

k

Numeric, controls the number of variants tested for each fluorophore, autofluorescence and FRET spectrum. Default is 10, which will be good, 1 is fastest. Values up to 10 provide additional benefit in unmixing quality.

parallel

Logical, whether to use parallel processing for the per-cell unmixing. Default is FALSE.

threads

Numeric. Number of threads to use for parallel processing. Defaults to 1 for sequential processing, or 0 (all cores) if parallel=TRUE.

verbose

Logical, whether to send messages to the console. Default is TRUE.

Value

Unmixed data with cells in rows and fluorophores in columns.