CombineTechReps: Combine technical replicates of Cq and amplification...

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

Description

Combine the technical replicates of Cq values and amplification efficiencies on the basis of mean, median and geometric mean and compute standard deviation of Cq and efficiency within the replicates in object of class "RTqPCRBatch".

Usage

## S4 method for signature 'RTqPCRBatch'
CombineTechReps(RTqPCRBatch, calc = "Mean",
  cRepCq = TRUE, ...)

Arguments

RTqPCRBatch	Object of class RTqPCRBatch. It can be output of CqValues or ReplaceValue or NonDetects or ReplaceAboveCutOff or ReplaceNAs functions.
calc	Which methods to be used out of Mean, Median and Geometric mean.
cRepCq	if TRUE, then combine replicates in Cq. If FALSE, then combine replicates in amplification efficiencies.
...	Other parameters to be passed to downstream methods.

Details

See the vignettes in RTqPCR package.

Value

Object of class "RTqPCRBatch" with new slots.

Author(s)

Navneet Phogat, Matthias Kohl, [e-mail]

Examples

## Read in the raw fluorescent data

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

## Read in the sample information data

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

Cqeffs.LC480 <- CqValues(merge.LC480, PCRtype = "LC480", Effmethod = "sigfit", baseline = "none")
Cqeffs.LC480 #To see the overview of data
exprs(Cqeffs.LC480)[1:5]  ##to visualise the first five CqValues
effs(Cqeffs.LC480)[1:5]  ##to visualise the first five amplification efficiencies
exprs(Cqeffs.LC480)  ##to visualise all Cq values
effs(Cqeffs.LC480)   ##to visualise all amplification efficiencies

##  Implement the functions ReplaceNAs, ReplaceAboveCutOff, ReplaceValue and NonDetects as per requirement.

## Combine technical replicates - In this example, we are combining the replicates directly from 
## the output of CqValues function

##  replicates can be combined through following three methods

## 1) based on mean (default method is for Cq values and is based on mean)

## i) combine Cq values and compute the standard deviation of Cq values within replicates

Cqreps <- CombineTechReps(Cqeffs.LC480)
Cqreps   ## to visualise the overview of the rsulting data


ii) combine amplification efficiencies and compute the standard deviation of amplification efficiencies
within replicates

effsreps <- CombineTechReps(Cqeffs.LC480, cRepCq = FALSE)

## 2) Based on median

## i) Combine Cq values and compute standard deviation of Cq values within replicates

Cqreps2 <- CombineTechReps(Cqeffs.LC480, calc = "Median")

ii) Combine amplification efficiencies and compute their standard deviation within replicates

effsreps2 <- CombineTechReps(Cqeffs.LC480, calc = "Median", cRepCq = FALSE)

## 3) Based on Geometric Mean

## i) combine Cq values and compute their standard deviation within replicates

Cqreps3 <- CombineTechReps(Cqeffs.LC480, calc = "Geom")

ii) combine amplification efficiencies and compute their standard deviation within replicates

effsreps <- CombineTehcReps(Cqeffs.LC480, calc = "Geom", cRepCq = FALSE)

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