CqEffs: Compute Cq values and amplification efficiencies

In NPhogat/RTqPCR: A work flow for analysis and normalization of pre-processing RT-qPCR data

Description

Compute Cq values from an object of Class "RTqPCRBatch" and produce an object of Class "RTqPCRBatch" as a result.

Usage

## S4 method for signature 'RTqPCRBatch'
CqEffs(object, PCRtype, Effmethod = "expfit",
  group = NULL, model = l5, check = "uni2", checkPAR = parKOD(),
  remove = "none", exclude = NULL, type = "cpD2", labels = NULL,
  norm = FALSE, baselinemethod = "mean", basecyc = 1:8, basefac = 1,
  smooth = NULL, smoothPAR = list(span = 0.1), factor = 1, opt = FALSE,
  optPAR = list(sig.level = 0.05, crit = "ftest"), plot = FALSE,
  verbose = FALSE, ...)

Arguments

object	An object of Class "RTqPCRBatch".
PCRtype	The type of RT-qPCRs ("LC480" or "Mx3005P") for which Cq values and amplification efficiencies are to be computed.
Effmethod	A character vector defining the methods for computing amplification efficiency.
group	A vector containing the grouping for possible replicates.
model	The model to be used for all runs. Default model is l5.
check	The method for kinetic outlier detection in KOD. Method "uni2" is set as default which is a test on sigmoidal structure.
checkPAR	Parameters to be supplied to the check method. See parKOD.
remove	Indicates which runs to be removed. Either none of them, those which failed to fit or from the outlier methods.
exclude	Indicates samples to be excluded from calculation, either "" for samples with missing column names or a regular expression defining columns (samples); see Examples in modlist
type	The point on the amplification curve which is used for efficiency estimation; see efficiency.
labels	A vector containing labels which define replicate groups. See more details in pcrbatch and ratiobatch.
norm	a logical Value which determines whether the raw data should be normalized within [0, 1] before model fitting or not.
baselinemethod	Type of baseline subtraction. More details in efficiency.
basecyc	Cycles to be considered for the baseline subtraction.
basefac	A factor when using averaged baseline cycles, such as 0.95.
smooth	The curve smoothing method. See more details in pcrbatch.
smoothPAR	parameters to be supplied to smoothing method in smooth.
factor	A multiplication factor for the fluorescence response values.
opt	A logical value which determines whether model selection should be applied to each model or not.
optPAR	Parameters to be supplied for model selection in mselect.
plot	A logical value. If TRUE, the single runs are plotted from the internal modlist for diagnostics.
verbose	A logical value. If TRUE, fitting and tagging results will be displayed in the console.
...	Other parameters to be passed to the downstream methods.

Value

Object of Class "RTqPCRBatch".

Author(s)

Navneet Phogat, Matthias Kohl, [e-mail]

Examples

## 1) To compute the CqValues and amplification efficiencies for LC480 light cycler

## Read in the raw fluorescent data of LC480 light cycler

LC480.example <- file.path(path, "LC480_Example.txt") 
cycData.LC480 <- read.RTqPCR(LC480.example, PCRtype = "LC480")

## Read in the sample information data of LC480 light cycler

SampleInfoLC480 <- file.path(path, "LC480_example_SampleInfo.txt")
samInfoLC480 <- read.RTqPCRSampleInfo(SampleInfoLC480, PCRtype = "LC480")

## Merge the fluorescence and sample information data through merge function

merge.LC480<-merge(cycData.LC480,samInfo.LC480) 

## Compute the Cq values and amplification efficiencies

## i) sigmoidal model (sigfit - default method)
## There are two ways two call this method. One is by defining the Effmethod as "sigfit".
## The second is calling CqValues function directly, without defining the Effmethod. As, it
## is a default method, so there is no need to define the Effmethod as "sigfit".

Cqeffs.LC4801 <- CqValues(merge.LC480, PCRtype = "LC480", Effmethod = "sigfit", baseline = "none")

## without mentioning Effmethod (as a default method)
Cqeffs.LC4801 <- CqValues(merge.LC480, PCRtype = "LC480", baseline = "none")

Cqeffs.LC4801 #To see the overview of data
exprs(Cqeffs.LC4801)[1:5]  ##to visualise the first five CqValues
effs(Cqeffs.LC4801)[1:5]  ##to visualise the first five amplification efficiencies
exprs(Cqeffs.LC4801)  ##to visualise all Cq values
effs(Cqeffs.LC4801)   ##to visualise all amplification efficiencies

## ii)  fit exponential model (expfit)

Cqeffs.LC4802 <- CqValues(merge.LC480, PCRtype = "LC480", Effmethod = "expfit", baseline = "none")
Cqeffs.LC4802 
exprs(Cqeffs.LC4802)[1:5]  
effs(Cqeffs.LC4802)[1:5]  
exprs(Cqeffs.LC4802)  
effs(Cqeffs.LC4802)   

## iii) window of linearity (sliwin)

Cqeffs.LC4803 <- CqValues(merge.LC480, PCRtype = "LC480", Effmethod = "sliwin", baseline = "none")
Cqeffs.LC4803 
exprs(Cqeffs.LC4803)[1:5]  
effs(Cqeffs.LC4803)[1:5]  
exprs(Cqeffs.LC4803)  
effs(Cqeffs.LC4803)

## iv) linear regression of efficiency (LRE)

Cqeffs.LC4804 <- CqValues(merge.LC480, PCRtype = "LC480", Effmethod = "expfit", baseline = "none")
Cqeffs.LC4804 
exprs(Cqeffs.LC4804)[1:5]  
effs(Cqeffs.LC4804)[1:5]  
exprs(Cqeffs.LC4804)  
effs(Cqeffs.LC4804)
## 2) To compute the Cq values and amplification efficiencies for the Mx3005P RT-qPCR

## Read in the raw fluorescent data of Mx3005P RT-qPCR

Mx3005P.example <- file.path(path, "Mx3005P_Example.txt") 
cycData.Mx <- read.RTqPCR(Mx3005P.example, PCRtype = "Mx3005P")

## Read in the sample information data of Mx3005P RT-qPCR

SampleInfoMx <- file.path(path, "Mx3005P_example_SampleInfo.txt")
samInfoMx <- read.RTqPCRSampleInfo(SampleInfoMx, PCRtype = "Mx3005P")

## Merge the fluorescence and sample information data

merge.Mx<-merge(cycData.Mx,samInfoMx)

## To compute the Cq values and amplification efficiencies

## i) SIgmoidal model (sigfit - default method)

Cqeffs.Mx<- CqValues(merge.Mx, PCRtype = "Mx3005P", Effmethod = "sigfit", baseline = "none")
Cqeffs.Mx #To see the overview of data
exprs(Cqeffs.Mx)[1:5]  ##to visualise the first five CqValues
effs(Cqeffs.Mx)[1:5]  ##to visualise the first five amplification efficiencies
exprs(Cqeffs.Mx)  ##to visualise all Cq values
effs(Cqeffs.Mx)   ##to visualise all amplification efficiencies

## ii) fit exponential model (expfit)

Cqeffs.Mx<- CqValues(merge.Mx, PCRtype = "Mx3005P", Effmethod = "expfit", baseline = "none")
Cqeffs.Mx #To see the overview of data
exprs(Cqeffs.Mx)[1:5]  
effs(Cqeffs.Mx)[1:5]  
exprs(Cqeffs.Mx)  
effs(Cqeffs.Mx)

## iii) Window of linearity (sliwin)

Cqeffs.Mx<- CqValues(merge.Mx, PCRtype = "Mx3005P", Effmethod = "sliwin", baseline = "none")
Cqeffs.Mx
exprs(Cqeffs.Mx)[1:5]
effs(Cqeffs.Mx)[1:5]  
exprs(Cqeffs.Mx)  
effs(Cqeffs.Mx)

## iv) linear regression of efficiency

Cqeffs.Mx<- CqValues(merge.Mx, PCRtype = "Mx3005P", Effmethod = "LRE", baseline = "none")
Cqeffs.Mx 
exprs(Cqeffs.Mx)[1:5]  
effs(Cqeffs.Mx)[1:5]  
exprs(Cqeffs.Mx)  
effs(Cqeffs.Mx)

NPhogat/RTqPCR documentation built on July 12, 2020, 12:56 p.m.