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

In qpcR: Modelling and Analysis of Real-Time PCR Data

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.

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 + 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.

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)

Example output

Loading required package: MASS
Loading required package: minpack.lm
Loading required package: rgl
Loading required package: robustbase
Loading required package: Matrix
Warning messages:
1: In rgl.init(initValue, onlyNULL) : RGL: unable to open X11 display
2: 'rgl_init' failed, running with rgl.useNULL = TRUE 
3: .onUnload failed in unloadNamespace() for 'rgl', details:
  call: fun(...)
  error: object 'rgl_quit' not found 
Making model for Fluo (l5)
 => Fitting passed...

Calculating delta of first/second derivative maxima...
.
$eff
[1] 1.55

$mr
[1] 0.55

$fcn
[1] 14.87

$fcna
[1] 15.73

$names
[1] "Fluo"

Making model for F1.1 (l5)
 => Fitting passed...

Making model for F1.2 (l5)
 => Fitting passed...

Making model for F1.3 (l5)
 => Fitting passed...

Making model for F1.4 (l5)
 => Fitting passed...

Making model for F2.1 (l5)
 => Fitting passed...

Making model for F2.2 (l5)
 => Fitting passed...

Making model for F2.3 (l5)
 => Fitting passed...

Making model for F2.4 (l5)
 => Fitting passed...

Making model for F3.1 (l5)
 => Fitting passed...

Making model for F3.2 (l5)
 => Fitting passed...

Making model for F3.3 (l5)
 => Fitting passed...

Making model for F3.4 (l5)
 => Fitting passed...

Making model for F4.1 (l5)
 => Fitting passed...

Making model for F4.2 (l5)
 => Fitting passed...

Making model for F4.3 (l5)
 => Fitting passed...

Making model for F4.4 (l5)
 => Fitting passed...

Making model for F5.1 (l5)
 => Fitting passed...

Making model for F5.2 (l5)
 => Fitting passed...

Making model for F5.3 (l5)
 => Fitting passed...

Making model for F5.4 (l5)
 => Fitting passed...

Making model for F6.1 (l5)
 => Fitting passed...

Making model for F6.2 (l5)
 => Fitting passed...

Making model for F6.3 (l5)
 => Fitting passed...

Making model for F6.4 (l5)
 => Fitting passed...

Making model for F7.1 (l5)
 => Fitting passed...

Making model for F7.2 (l5)
 => Fitting passed...

Making model for F7.3 (l5)
 => Fitting passed...

Making model for F7.4 (l5)
 => Fitting passed...

Calculating delta of first/second derivative maxima...
.........10.........20........
$eff
 [1] 1.486 1.492 1.390 1.425 1.494 1.501 1.371 1.417 1.490 1.494 1.371 1.408
[13] 1.490 1.498 1.369 1.406 1.487 1.492 1.357 1.399 1.482 1.491 1.362 1.398
[25] 1.471 1.468 1.294 1.331

$mr
 [1] 0.486 0.492 0.390 0.425 0.494 0.501 0.371 0.417 0.490 0.494 0.371 0.408
[13] 0.490 0.498 0.369 0.406 0.487 0.492 0.357 0.399 0.482 0.491 0.362 0.398
[25] 0.471 0.468 0.294 0.331

$fcn
 [1] 15.18 15.02 14.95 15.43 18.58 18.32 18.25 17.11 21.75 21.58 21.00 20.87
[13] 25.08 24.85 24.98 24.53 28.07 28.00 27.90 27.98 31.68 31.50 31.78 31.68
[25] 39.68 39.39 39.51 39.00

$fcna
 [1] 16.22 16.04 16.31 16.66 19.60 19.32 19.68 18.37 22.78 22.60 22.43 22.16
[13] 26.11 25.86 26.42 25.83 29.11 29.02 29.39 29.31 32.73 32.53 33.25 33.01
[25] 40.77 40.49 41.28 40.60

$names
 [1] "F1.1" "F1.2" "F1.3" "F1.4" "F2.1" "F2.2" "F2.3" "F2.4" "F3.1" "F3.2"
[11] "F3.3" "F3.4" "F4.1" "F4.2" "F4.3" "F4.4" "F5.1" "F5.2" "F5.3" "F5.4"
[21] "F6.1" "F6.2" "F6.3" "F6.4" "F7.1" "F7.2" "F7.3" "F7.4"

qpcR documentation built on May 2, 2019, 5:17 a.m.