normCytof {CATALYST} | R Documentation

## Bead-based normalization

### Description

an implementation of Finck et al.'s normalization of mass cytometry data using bead standards with automated bead gating.

### Usage

    normCytof(
      x,
      beads,
      k = 500,
      trim = 5,
      remove_beads = TRUE,
      norm_to = NULL,
      assays = c("counts", "exprs"),
      overwrite = TRUE,
      transform = TRUE,
      cofactor = NULL,
      plot = TRUE,
      verbose = TRUE
    )

### Arguments

`x` | a `SingleCellExperiment`.
`beads` | `"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.
`k` | integer width of the median window used for bead smoothing (affects visualizations only!).
`trim` | a single non-negative numeric. A median+/-`trim`*mad rule is applied to preliminary bead populations to remove bead-bead doublets and low signal beads prior to estimating normalization factors.
`remove_beads` | logical. If TRUE, bead events will be removed from the input `SingleCellExperiment` and returned as a separate object?
`norm_to` | a `flowFrame` or character string specifying an FCS file from which to compute baseline bead intensities, and to which the input data should be normalized to.
`assays` | lnegth 2 character string specifying which assay data to use; both should be in `assayNames(x)` and correspond to count- and expression-like data, respectively.
`overwrite` | logical; should the specified `assays` (both, when `transform = TRUE`) be overwritten with the normalized data? If `FALSE`, normalized counts (and expressions, if `transform = TRUE`) will be stored in assay(s) `normcounts/exprs`, respectively.
`transform` | logical; should normalized counts be arcsinh-transformed with the specified `cofactor`(s)?
`cofactor` | numeric cofactor(s) to use for optional arcsinh-transformation when `transform = TRUE`; single value or a vector with channels as names. If NULL, `normCytof` will try and access the cofactor(s) stored in `int_metadata(x)`, thus re-using the same transformation applied previsouly.
`plot` | logical; should bead vs. DNA scatters and smoothed bead intensities before vs. after normalization be included in the output?
`verbose` | logical; should extra information on progress be reported?

### Value

a list of the following `SingleCellExperiment`...

- `data`: The filtered input SCE (when `remove_beads = TRUE`); otherwise, `colData` columns `is_bead` and `remove` indicate whether an event as been identified as a bead or doublet. If `overwrite = FALSE`, assays `normcounts/exprs` are added; otherwise, the specified `counts/exprs` assays are overwritten.

- `beads`, `removed`: SCEs containing subsets of events identified as beads and that were removed, respectively. The latter includes bead-cell and cell-cell doublets)

...and `ggplot` objects:

- `scatter`: scatter plot of DNA vs. bead intensities with indication of the applied gates

- `lines`: running-median smoothed bead intensities before and after normalization

### Author(s)

Helena L Crowell helena.crowell@uzh.ch

### References

Finck, R. et al. (2013). Normalization of mass cytometry data with bead standards. Cytometry A 83A, 483-494.

### Examples

    data(raw_data)
    sce <- prepData(raw_data)

    # apply normalization & write normalized data to separate assays
    res <- normCytof(sce, beads = "dvs", k = 80, overwrite = FALSE)

    ncol(res$beads)   # no. of bead events
    ncol(res$removed) # no. of events removed

    res$scatter # plot DNA vs. bead intensities including applied gates
    res$lines   # plot smoothed bead intensities before vs. after normalization

    # filtered SCE now additionally includes
    # normalized count & expression data
    assayNames(res$data)

[Package CATALYST version 1.14.1 Index]