Perform Cy3 normalization

Given a PBMExperiment containing Alexa488 probe intensity data and a second PBMExperiment containing Cy3 observed-to-expected ratios, this function performs probe filtering and/or scaling of the Alexa488 data according to the over/under abundance of dsDNA at each probe as quantified from the Cy3 data.

Both the Alexa488 and Cy3 PBMExperiment objects must have a common column in the colData (specified by match_by=) that can be used to match scans across the two objects. While the values of the match_by column may be repeated across Alexa488 scans (e.g. if an array was reused or scanned multiple times), the values must be unique across the Cy3 PBMExperiment object.

cy3Normalize(
  pe,
  cy3pe,
  assay = SummarizedExperiment::assayNames(pe)[1],
  match_by = "id_idx",
  filter = TRUE,
  scale = TRUE,
  verbose = FALSE
)

Arguments

pe	a PBMExperiment object containing Alexa488 intensity data.
cy3pe	a PBMExperiment object containing Cy3 deviation values from using `cy3FitEmpirical` or `cy3FitModel`.
assay	a numeric index or string name specifying the assay to use. (default = `SummarizedExperiment::assayNames(pe)[1]`)
match_by	a string column name in colData of `pe` and `cy3pe` to use for matching scans across the two objects; values of column must be unique for each scan in each object. (default = `"id_idx"`)
filter	a logical value whether to filter "low quality" flagged probes by setting to NA. (default = TRUE)
scale	a logical value whether to perform scaling by ratio of observed vs. expected Cy3 intensity at each probe. (default = TRUE)
verbose	a logical value whether to print verbose output during analysis. (default = FALSE)

Value

Original PBMExperiment object with assay containing Cy3 filtered and/or scaled intensities ("normalized"). If an assay with the same name is already included in the object, it will be overwritten.

Arguments

Value

See also