Get FlowCode Spectra

Measure spectral unmixing errors per barcode in a sample stained only with FlowCode tag antibodies (flowcode.backbone.fcs). Measures FRET error and returns the calculated correction matrices and thresholds for debarcoding.

Usage

get.flowcode.spectra(
  backbone.rds,
  thresholds.file,
  asp,
  output.dir = "./flowcode_spectra",
  filename = "FlowCode_Spectra.rds",
  figures = TRUE,
  plot.corrections = TRUE,
  verbose = TRUE
)

Arguments

backbone.rds

File name and path to the full (large) rds file produced by running unmix.backbone. For this to work well, all combinations present in the experimental samples should be well represented in the backbone sample used.

thresholds.file

File name and path to the thresholds CSV file produced using the threshold setting app.

asp

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

output.dir

Destination folder for output plots and saved spectra. Default is ./flowcode_spectra.

filename

Logical, whether to create and save plots of the data correction. Default is TRUE.

figures

Logical, controls whether graphs of the process are produced. Default is TRUE.

plot.corrections

Logical, default is FALSE. If TRUE, the FRET unmixing will be applied to flowcode.backbone.fcs and plots of the corrected data (FlowCode channels only) will be produced in output.dir.

verbose

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

Value

A named list with two elements:

1. Thresholds: numeric thresholds for debarcoding

2. FRET: A list of matrices of FRET spectral variations per barcode combo

3. Flowcode.fluors: Character vector of fluorophores linked to the FlowCodes

4. Combos: The valid combinations, specified by the combination file.