qpcR (version 1.3-7.1)

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 additionally an 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

## on single curve
## using baseline shifting
m1 <- pcrfit(reps, 1, 2, l5)
maxRatio(m1, baseshift = 0.3)     

## on a 'modlist'
## using 'baseline shifting' 
ml1 <- modlist(reps, model = l5) 
maxRatio(ml1, baseshift = 0.5)