efficiency {qpcR}R Documentation

Calculation of qPCR efficiency and several other important qPCR parameters

Description

This function calculates the PCR efficiency of a model of class 'pcrfit' and 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.

Usage

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

Arguments

object an object of class 'pcrfit'.
plot logical. If TRUE, a graph is displayed. If FALSE, values are printed out.
type the method of efficiency estimation. See 'Details'.
thresh an (optional) numeric value for a fluorescence threshold border. Overrides type.
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, as to be defined in type:

"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)),
"CQ" taken from the 20% value of the fluorescence at "cpD2" as developed by Corbett Research (comparative quantification),
"Cy0" the intersection of a tangent on the first derivative maximum with the abscissa 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 as defined in type or
a numeric value taken from the fluorescence threshold output of the PCR software as defined in thresh.

The initial fluorescence F(0) for relative or absolute quantification is either calculated by setting x = 0 in the sigmoidal model of object giving init1 or by calculating an exponential model down (init2) with 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.

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.
Rsq.ad the adjusted 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.
cpR the PCR cycle within the exponential region calculated as under 'Details'.
cpT the PCR cycle corresponding to the fluorescence threshold as defined in thresh.
Cy0 the PCR threshold cycle 'Cy0' according to Guescini et al. See 'Details'.
cpCQ the PCR cycle corresponding to the 20% fluorescence value at 'cpD2'.
fluo the raw fluorescence value at the point defined by type or thresh.
init1 the initial template fluorescence from the sigmoidal model, calculated as under 'Details'.
init2 the initial template fluorescence from an exponential model, 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

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)
 
## selection of best model
## using one cycle 'downstream'
## of second derivative max
m2 <- mselect(m1)
efficiency(m2, type = "cpD2", shift = -1)    

## using "maxE" method, with calculation of PCR efficiency
## 2 cycles 'upstream' from the cycle of max efficiency
efficiency(m2, type = "maxE", shift = 2)

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

## using threshold cycle (i.e. 15.32) 
## from PCR software
efficiency(m2, type = 15.32)

## using Cy0 method from
## Guescini et al. (2008),
## add Cy0 tangent
efficiency(m2, type = "Cy0")
Cy0(m2, add = TRUE)

## using a defined fluorescence
## threshold value from PCR software
efficiency(m2, thresh = 1)
 
## 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 1.2-4 Index]