Get Fluorophore Variants

Assesses variation in the spectral signature of a single-stained flow cytometry control sample. Uses SOM-based clustering on the brightest positive events in the file.

get.fluor.variants(
  fluor,
  file.name,
  control.dir,
  spectra,
  af.spectra,
  spectral.channel,
  universal.negative,
  control.type,
  raw.thresholds,
  unmixed.thresholds,
  flow.channel,
  som.dim,
  n.cells,
  asp,
  verbose,
  output.dir,
  sim.threshold,
  figures
)

Arguments

fluor

The name of the fluorophore.

file.name

A named vector of file names for the samples.

control.dir

The directory containing the control files.

spectra

A matrix containing the spectral data. Fluorophores in rows, 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.

spectral.channel

A vector of spectral channels.

universal.negative

A named vector of unstained negative samples, with names corresponding to the fluorophores.

control.type

Character, either "beads" or "cells". Determines the type of control sample being used and the subsequent processing steps.

raw.thresholds

A named vector of numerical values corresponding to the threshold for positivity in each raw detector channel. Determined by the 99.5th percentile on the unstained sample, typically.

unmixed.thresholds

A named vector of numerical values corresponding to the threshold for positivity in each unmixed channel. Determined by the 99.5th percentile on the unstained sample, typically after single-cell AF unmixing.

flow.channel

A named vector of peak raw channels, one per fluorophore.

som.dim

Numeric, default 10. Number of x and y dimensions to use in the SOM for clustering the spectral variation.

n.cells

Numeric, default 2000. Number of cells to use for defining the variation in spectra. Up to n.cells cells will be selected as positive events in the peak channel for each fluorophore, above the pos.quantile in the unstained sample.

asp

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

verbose

Logical, default is TRUE. Set to FALSE to suppress messages.

output.dir

File path to whether the figures and .rds data file will be saved. Default is NULL, in which case asp$variant.dir will be used.

sim.threshold

Numeric, default 0.98. Threshold for cosine similarity- based exclusion of spectral variants. Any variant less than sim.threshold by cosine.similarity from the optimized spectrum for that fluorophore (from spectra) will be excluded from output. This helps to exclude autofluorescence contamination.

figures

Logical, controls whether the variation in spectra for each fluorophore is plotted in output.dir. Default is TRUE.

Value

A matrix with the flow expression data.