Compute Cy3 scaling factors using emprical reference

PBM arrays are scanned twice, once for Cy3-tagged dUTPs to quantify dsDNA abundance at each probe, and again for the Alexa488-tagged protein. The Cy3 scans can be used to first filter out probes which appear to have poor dsDNA enrichment, and second, to scale Alexa488 intensities to account for differences in dsDNA abundance between probes.

Since probe intensities in the Cy3 scans are independent of the assayed Alexa488-tagged protein, Cy3 scans should be comparable across experiments. Based on this observation, a global Cy3 reference can be constructed by taking the empirical average over a large number of Cy3 scans. Filtering and scaling can then be performed based on deviations from this reference. Given a PBMExperiment of Cy3 scans and an empirical reference generated by cy3GenerateRef, this function returns the residuals from the reference, and the corresponding observed-to-expected ratios for each probe as new assays added to the original Cy3 PBMExperiment object.

The returned PBMExperiment object can be passed to cy3Normalize with a PBMExperiment of Alexa488 scan intensities to filter low quality probes and/or normalize the Alexa488 intensities by the computed ratios.

cy3FitEmpirical(
  pe,
  refpe,
  assay = SummarizedExperiment::assayNames(pe)[1],
  useMean = TRUE,
  standardize = TRUE,
  threshold = 1/2,
  verbose = FALSE
)

Arguments

pe	a PBMExperiment object containing Cy3 intensity data.
refpe	a PBMExperiment object containing Cy3 reference intensities as a single assay named "ref", most likely generated by a call to cy3GenerateRef.
assay	a numeric index or string specifying the Cy3 intensity assay. (default = SummarizedExperiment::assayNames(pe)[1])
useMean	a logical value whether to use the probe-level mean, rather than the probe-level median, from the Cy3 reference (default = TRUE)
standardize	a logical value whether to standardize residuals using MAD (median absolute deviation about the median) intensity computed for PBM Cy3 reference data for probe filtering. (default = TRUE)
threshold	a numeric threshold on the absolute value of the log2 ratio between observed and expected Cy3 intensities. If standardize is specified, the log2 ratios are first scaled before comparing against the threshold. In this case, the threshold is also scaled by the median of the probe-level MAD intensities across all probes. (default = 1/2)
verbose	a logical value whether to print verbose output during analysis. (default = FALSE)

Value

Original Cy3 PBMExperiment object with additional assays corresponding to ratio of observed to expected probe intensities, and whether probes were flagged as low-quality based on abs(log2(ratio)) > threshold.

See also

cy3GenerateRef, cy3Normalize