R: Calculation of PCR efficiency by fitting an exponential model
expfit
R Documentation
Calculation of PCR efficiency by fitting an exponential model
Description
An exponential model is fit to a window of defined size on the qPCR raw data. The window is identified either by the second derivative maximum 'cpD2' (default), 'studentized outlier' method as described in Tichopad et al. (2003), the 'midpoint' method (Peirson et al., 2003) or by subtracting the difference of cpD1 and cpD2 from cpD2 ('ERBCP', unpublished).
one of the four possible methods to be used for defining the position of the fitting window.
model
which exponential model to use. expGrowth is default, but the linear-exponential model linexp can also be chosen.
offset
for method = "cpD2", the cycle offset from second derivative maximum.
pval
for method = "outlier", the p-value for the outlier test.
n.outl
for method = "outlier", the number of successive outlier cycles.
n.ground
for method = "midpoint", the number of cycles in the noisy ground phase to calculate the standard deviation from.
corfact
for method = "ERBCP", the correction factor for finding the exponential region. See 'Details'.
fix
for methods "midpoint" and "ERBCP", the orientation of the fitting window based on the identified point. See 'Details'.
nfit
the size of the fitting window.
plot
logical. If TRUE, a graphical display of the curve and the fitted region is shown.
...
other parameters to be passed to the plotting function.
Details
The exponential growth function f(x) = a cdot exp(b cdot x) + c is fit to a subset of the data. Calls efficiency for calculation of the second derivative maximum, takeoff for calculation of the studentized residuals and 'outlier' cycle, and midpoint for calculation of the exponential phase 'midpoint'. For method 'ERBCP' (Exponential Region By Crossing Points), the exponential region is calculated by expR = cpD2 - code{corfact} cdot (cpD1-cpD2). The efficiency is calculated from the exponential fit with E = exp(b) and the inital template fluorescence F_0 = a.
Value
A list with the following components:
point
the point within the exponential region as identified by one of the three methods.
cycles
the cycles of the identified region.
eff
the efficiency calculated from the exponential fit.
AIC
the Akaike Information Criterion of the fit.
resVar
the residual variance of the fit.
RMSE
the root-mean-squared-error of the fit.
init
the initial template fluorescence.
mod
the exponential model of class 'nls'.
Author(s)
Andrej-Nikolai Spiess
References
Standardized determination of real-time PCR efficiency from a single reaction set-up.
Tichopad A, Dilger M, Schwarz G & Pfaffl MW. Nucleic Acids Research (2003), 31:e122.
Comprehensive algorithm for quantitative real-time polymerase chain reaction.
Zhao S & Fernald RD. J Comput Biol (2005), 12:1047-64.
Examples
## Using default SDM method.
m1 <- pcrfit(reps, 1, 2, l5)
expfit(m1)
## Using 'outlier' method.
expfit(m1, method = "outlier")
## Linear exponential model.
expfit(m1, model = "linexp")