qpcR (version 1.2-1)

expfit: 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the 'midpoint' method (Peirson et al., 2003) or by subtracting the difference of cpD1 and cpD2 from cpD2 ('ERBCP', unpublished).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 = 1:5, corfact = 1, 
       fix = c("top", "bottom", "middle"), nfit = 5, plot = TRUE, ...)

Arguments

object

an object of class 'pcrfit'.

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.

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 efficiencies 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 of class 'nls'.

Details

The exponential growth function $f(x) = b * exp(k * x) + e$ is fit to the data. Calls outlier for the 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 $expR = cpD2 - \code{corfact} * (cpD1-cpD2)$. 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.

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

## using 'outlier' method
m1 <- pcrfit(reps, 1, 2, l5)
expfit(m1)

## 'midpoint' method and 7 cycle window
expfit(m1, method = "midpoint", nfit = 7)

## 'ERBCP' method with window centered around
## fixpoint
expfit(m1, method = "ERBCP", fix = "middle")