expfit {qpcR}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 '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the 'midpoint' method (Peirson et al., 2003) or by subtracting the difference of cpD1 and cpD2 from cpD2 ('ERBCP', unpublished).

Usage

expfit(object, method = c("outlier", "midpoint", "ERBCP"), pval = 0.05, 
n.outl = 3, n.ground = 10, corfact = 1, fix = c("top", "bottom", "middle"), 
nfit = 5, plot = TRUE, ...) 

Arguments

object a 'drc' object.
method one of the three possible methods to be used for defining the position of the fitting window.
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) = b * exp(k * x) + e is fit to the data. Calls outlier and studres for calculation of the studentized residuals and 'outlier' cycles, 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 - corfact * (cpD1-cpD2). The efficiency is calculated a) from the exponential fit with Eff = exp(k) and b) for each cycle within the exponential region from the raw fluorescence values by Eff = frac{F(n)}{F(n-1)}. The inital template fluorescence (F0) is derived from parameter b.

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 as defined by method, fix and nfit.
eff the efficiency calculated from the exponential fit.
eff.cycles the efficiency of all points within the identified region.
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.

Author(s)

Andrej-Nikolai Spiess

References

Standardized determination of real-time PCR efficiency from a single reaction set-up. Tichopad et al., Nucleic Acids Research, 2003, e122.
Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Peirson et al., Nucleic Acids Research, 2003, e73.

Examples

m <- pcrfit(reps, 1, 2, l5())
### using 'outlier' method
expfit(m)
### 'midpoint' method and 7 cycle window
expfit(m, method = "midpoint", nfit = 7)
### 'ERBCP' method with window centered around
### fixpoint
expfit(m, method = "ERBCP", fix = "middle")

[Package qpcR version 1.1-8 Index]