efficiency {qpcR}R Documentation

Calculation of qPCR efficiency and several other important qPCR parameters

Description

This function calculates the PCR efficiency of a fitted 'drc' or 'mchoice' model and additionally several other important values for qPCR quantification like the first and second derivatives and the corresponding maxima thereof (i.e. threshold cycles). These values can be subsequently used for the calculation of PCR kinetics, fold induction etc. All values are included in a graphical output of the fit. Additionally, several measures of goodness-of-fit are calculated, i.e. the Akaike Information Criterion (AIC), the residual variance and the R-square value. For relative and absolute quantification, the initial fluorescence F(0) is calculated and can be used for optical calibration if the template amount is defined (see 'Details').

Usage

  efficiency(object, plot = TRUE, type = "cpD2", shift = 0, amount = NULL)

Arguments

object an object of class 'drc' or 'mchoice' model.
plot logical. If TRUE, a graph is displayed. If FALSE, values are printed out.
type the method of efficiency estimation. See 'Details'.
shift a user defined shift in cycles from the values defined by type. See Examples.
amount the template amount or molecule number for quantitative calibration.

Details

The efficiency is always calculated from the efficiency curve (in blue), which is calculated according to E = frac{F(n)}{F(n-1)} from the fitted curve, but taken from different points at the curve:

"cpD2" taken from the maximum of the second derivative curve,
"cpD1" taken from the maximum of the first derivative curve,
"maxE" taken from the maximum of the efficiency curve,
"expR" taken from the exponential region by expR = cpD2-(cpD1-cpD2)),
"Cy0" as calculated according to Guescini et al. or
a numeric value (taken from the threshold cycle output of the PCR software, i.e. 15.24).

The initial fluorescence F(0) for relative or absolute quantification is calculated by F(0) = frac{F(n)}{E(n)^{Cyc}}, with F(n) = raw fluorescence at the cycle number defined by type, E(n) = PCR efficiency at the cycle number defined by type and Cyc = the cycle number defined by type. If a template amount is defined, a conversion factor cf = frac{amount}{F(0)} is given. The different measures for goodness-of-fit give an overview for the validity of the efficiency estimation. First and second derivatives are calculated from the fitted function and the maxima of the derivatives curve and the efficiency curve are obtained. This function calls the first derivatives from the 'drc' library and the internal functions deriv2.b and deriv2.l for the calculation of second derivatives.

Value

A list with the following components:

eff the PCR efficiency.
resVar the residual variance.
AICc the bias-corrected Akaike Information Criterion.
AIC the Akaike Information Criterion.
Rsq the R-square value.
cpD1 the first derivative maximum (point of inflection in 'l4' or 'b4' models, can be used for defining the threshold cycle).
cpD2 the second derivative maximum (turning point of cpD1, more often used for defining the threshold cycle).
cpE the PCR cycle with the highest efficiency, if "maxE" method is chosen.
cpR the PCR cycle within the exponential region calculated as under 'Details', if "expR" method is chosen.
Cy0 the PCR threshold cycle 'Cy0' according to Guescini et al. See 'Details'.
fluo the raw fluorescence value at the point defined by type.
init the initial template fluorescence, i.e. F(0), calculated as under 'Details'.
cf the conversion factor between raw fluorescence and template amount, if the latter is defined.

Note

Three parameter models ('b3' or 'l3') do not work very well in calculating the PCR efficiency. It is advisable not to take too many cycles of the plateau phase prior to fitting the model as this has a strong effect on the validity of the efficiency estimates.

Author(s)

Andrej-Nikolai Spiess & Christian Ritz

References

Weihong Liu and David A. Saint (2002) Validation of a quantitative method for real time PCR kinetics, BBRC, 294, 347 - 353. A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition.
Guescini M et al, BMC Bioinformatics, 2008, 9: 326.

Examples

## Fitting initial model
m1 <-  pcrfit(reps, 1, 2, l4())
efficiency(m1)
 
## of best model
#uses type="cpD2"
m2 <- mchoice(m1)
efficiency(m2) 

## using "maxE" method, with calculation of PCR efficiency
## one cycle 'upstream' of the cycle of maximum efficiency
efficiency(m2, type = "maxE", shift = 1)

##using the exponential region
efficiency(m2, type = "expR")

## using threshold value (15.32) from PCR software
efficiency(m2, type = 15.32)
 
## using the first 30 cycles and a template amount
## (optical calibration)
m3 <-  pcrfit(reps[1:30, ], 1, 2, l5())
efficiency(m3, amount = 1E3)    

[Package qpcR version 1.1-8 Index]