efficiency {qpcR} | R Documentation |
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.
efficiency(object, plot = TRUE, type = "cpD2", thresh = NULL, shift = 0, amount = NULL)
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. |
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.
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. |
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.
Andrej-Nikolai Spiess
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.
## 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)