This function estimates a smoothed surface from positive control spots on
an RPPA slide. The surface is used to perform spatial corrections (i.e.,
because of uneven hybridization) on the array.
It is used before
RPPAFit, one slide at a time.
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object of class
object of class
object of class
character string specifying fit measure to smooth
numeric scalar used to identify the background cutoff with value in range [0..1]
numeric scalar used as smoothing model argument.
numeric scalar used as model parameter with value in range [0..2]
logical scalar. If
The observed spot intensities are assumed to be a combination of true signal, background noise, and hybridization effects according to the following model:
Y_rc = Y * H_rc + B_rc
where Y_rc is the observed intensity, Y is the true signal, H_rc is the effect of hybridization, and B_rc is the background noise. The subscripts "r" and "c" refer to the physical row and column of the spot on the array. Background noise is estimated locally by the array software. The hybridization effect is estimated fitting a generalized additive model (GAM) to positive control spots printed uniformly across the array.
The estimated surface is used to scale the intensities on the array. Each intensity is adjusted by the amount that is needed to make the positive control surface flat at the value of the median of the surface. This is done by dividing each spot by the estimated surface value and then multiplying by the median of the surface.
Positive control spots that are expressed below the cutoff for the noise region are excluded from the computation of the surface.
Sometimes, positive control spots are printed in a dilution series to avoid saturation problems with these spots. When this happens, the observed intensities are adjusted by the positive control surface that has the most similar expression level.
design argument must have already been augmented with
slide design information.
cutoff argument passed to
quantile is percentile
of the background estimates used to define the noise region of slide.
k argument passed to
s sets upper limit on
degrees of freedom associated with smoothing.
gamma argument passed to
gam provides a constant
multiplier used to inflate model degrees of freedom in the
GCV or UBRE/AIC score.
rppa with an additional measurement column
named after the
measure with an
Adj. prefix. For example,
if the measure was
Mean.Net, the name of the adjusted column
Neeley ES, Baggerly KA, Kornblau SM.
Surface Adjustment of Reverse Phase Protein Arrays Using Positive Control Spots
Cancer Informatics (2012) 11: 77-86.
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extdata.dir <- system.file("extdata", package="SuperCurveSampleData") txtdir <- file.path(extdata.dir, "rppaSingleSubgridData") designfile <- file.path(txtdir, "slidedesign.tsv") rppa <- RPPA("Waldo.txt", path=txtdir, software="microvigene", alt.layout="superslide") designparams <- RPPADesignParams(grouping="blockSample", designfile=designfile) design <- RPPADesignFromParams(rppa, designparams) rppa.adj <- spatialAdjustment(rppa, design) colnames(rppa.adj)