qpcR (version 1.4-1)

maxRatio: The maxRatio method as in Shain et al. (2008)

Description

The maximum ratio (MR) is determined along the cubic spline interpolated curve of \(\frac{F_n}{F_{n-1}}\) and the corresponding cycle numbers FCN and its adjusted version FCNA are then calculated for MR.

Usage

maxRatio(x, method = c("spline", "sigfit"), baseshift = NULL, 
         smooth = TRUE, plot = TRUE, ...)

Arguments

x

an object of class 'pcrfit' (single run) or 'modlist' (multiple runs).

method

the parameters are either calculated from the cubic spline interpolation (default) or a sigmoidal fit.

baseshift

numerical. Shift value in case of type = "spline". See 'Details'.

smooth

logical. If TRUE and type = "spline", invokes a 5-point convolution filter (filter). See 'Details'.

plot

Should diagnostic plots be displayed?

...

other parameters to be passed to eff or plot.

Value

A list with the following components:

eff

the maximum efficiency. Equals to mr + 1.

mr

the maximum ratio.

fcn

the cycle number at mr.

fcna

an adjusted fcn, as described in Shain et al.

names

the names of the runs as taken from the original dataframe.

Details

In a first step, the raw fluorescence data can be smoothed by a 5-point convolution filter. This is optional but feasible for many qPCR setups with significant noise in the baseline region, and therefore set to TRUE as default. If baseshift is a numeric value, this is added to each response value \(F_n = F_n + baseshift\) (baseline shifting). Finally, a cubic spline is fit with a resolution of 0.01 cycles and the maximum ratio (efficiency) is calculated by \(MR = max(\frac{F_n}{F_{n-1}}-1)\). \(FCN\) is then calculated as the cycle number at \(MR\) and from this the adjusted \(FCNA = FCN -log_2(MR)\). Sometimes problems are encountered in which, due to high noise in the background region, randomly high efficiency ratios are calculated. This must be resolved by tweaking the baseshift value.

References

A new method for robust quantitative and qualitative analysis of real-time PCR. Shain EB & Clemens JM. Nucleic Acids Research (2008), 36: e91.

Examples

# NOT RUN {
## On single curve using baseline shifting.
m1 <- pcrfit(reps, 1, 2, l5)
maxRatio(m1, baseshift = 0.3)     

## On a 'modlist' using baseline shifting.
# }
# NOT RUN {
ml1 <- modlist(reps, model = l5) 
maxRatio(ml1, baseshift = 0.5)
# }