maxRatio {qpcR} | R Documentation |
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 |
smooth |
logical. If |
plot |
Should diagnostic plots be displayed? |
... |
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.
Value
A list with the following components:
eff |
the maximum efficiency. Equals to |
mr |
the maximum ratio. |
fcn |
the cycle number at |
fcna |
an adjusted |
names |
the names of the runs as taken from the original dataframe. |
Note
This function has been approved by the original author (Eric Shain).
Author(s)
Andrej-Nikolai Spiess
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.
## Not run:
ml1 <- modlist(reps, model = l5)
maxRatio(ml1, baseshift = 0.5)
## End(Not run)