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Define Gate by Landmarks

Source: R/define_gate_landmarks.R
define.gate.landmarks.Rd

Performs gating on scatter parameters using events selected on the basis of high fluorescence intensity in the expected peak channel in the single-stained control samples. This approach allows for identification of cells fairly reliably by "landmarks", especially when using well-defined, abundant populations such a T cells (e.g., CD3, CD4, CD8 single-stained samples), monocytes (CD14-stained sample) or neutrophils (CD66, CD33).

Usage

define.gate.landmarks(
  control.file,
  control.dir,
  asp,
  gate.name,
  gating.params = NULL,
  n.cells = 2000,
  percentile = 70,
  grid.n = 100,
  bandwidth.factor = 1,
  fsc.channel = NULL,
  ssc.channel = NULL,
  fsc.lims = NULL,
  ssc.lims = NULL,
  output.dir = "./figure_gate",
  filename = "landmark_gate_definition_",
  color.palette = "plasma",
  boundary.color = "black",
  points.to.plot = 1e+05,
  width = 5,
  height = 5,
  verbose = TRUE,
  control.table = NULL,
  check = TRUE
)

Arguments

control.file

File path and name for the CSV file defining the single- color control file names, fluorophores they represent, marker names, peak channels, and gating requirements.

control.dir

File path to the single-stained control FCS files.

asp

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

gate.name

Character, name for the gate. Useful for distinguishing gates when you have multiple types. Must match one string (name) in the gate.name column of the control.file.

gating.params

Previously saved gating parameters. Load in the .rds file using readRDS and pass the result here if you wish to replicate a previous run. This is essentially just an updated version of asp containing any modifications based on information passed as arguments to define.gate.landmarks.

n.cells

The number of cells to use for defining the gate boundary. This many cells will be selected from the peak channel (brightest first) in the single-color controls. For example, if you set 200 and marked files such as CD3-PE.fcs and CD19-FITC.fcs as gate.define=TRUE in the control file, the brightest 200 events in the YG1 channel from the CD3-PE file and the brightest 200 events in the B1 channel for the CD19-FITC file would be used to define the gate.

percentile

Numeric 1 - 100, default 70. The percentile cutoff for density in the scatter to use for defining the gate. For example, a value of 50 would take the 50% of cells closest to the density peak, more or less. Smaller numbers will define a tighter gate.

grid.n

Numeric, default 100. The binning grid for the kernel density estimation. If n.cells is very low, you may wish to lower this number to compress the search space.

bandwidth.factor

Numeric, default 1. A multiplier for the bandwidth for the kernel density estimation. Larger numbers will smooth the density, reducing discrimination between peaks in the density (such as between live and dead cells).

fsc.channel

Channel to use for Forward Scatter. Default NULL will use the asp$default.scatter.parameter[1], which is appropriate for your machine.

ssc.channel

Channel to use for Side Scatter. Default NULL will use the asp$default.scatter.parameter[2], which is appropriate for your machine. For machines with multiple side scatter measurements, you can change this.

fsc.lims

Numeric vector. Limits for searching and plotting the FSC. The default NULL uses cc(asp$scatter.data.min.x, asp$scatter.data.max.x).

ssc.lims

Numeric vector. Limits for searching and plotting the SSC. The default NULL uses cc(asp$scatter.data.min.y, asp$scatter.data.max.y).

output.dir

File path where you want to save the results. Default is ./figure_gate.

filename

Character, name for the output files. Default is gate_definition.

color.palette

Optional character string defining the viridis color palette to be used for the fluorophore traces. Default is plasma. Use rainbow to be similar to FlowJo or SpectroFlo. Other options are the viridis color options: magma, inferno, plasma, viridis, cividis, rocket, mako and turbo.

boundary.color

Color for the gate boundary line on the plot. Default is black.

points.to.plot

Numeric, default 1e5. Maximum number of points to show on the plot. More points will take longer, but you really shouldn't have even close to this number when defining the gate with landmarks.

width

Numeric, default 5. Width of the saved plot.

height

Numeric, default 5. Height of the saved plot.

verbose

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

control.table

Dataframe or table of the control file, read in via define.flow.control() and cleaned up by that function.

check

Logical, default is TRUE. Set to FALSE to skip consistency checks on gate defining columns in the control table: large.gate, is.viability and sample.type.

Value

A set of points describing the gate boundary.

References

Laniewski, Nathan. flowstate. https://github.com/nlaniewski/flowstate

See also