totData {EasyqpcR} | R Documentation

## Aggregation of qPCR biological replicates and data transformation

### Description

This function aggregates qPCR biological replicates and calculates the main parameters : mean (arithmetic or geometric), the standard deviation and the standard error from your biological replicates of your experience. This function has an algorithm published by Willems et al. (2008) which performs a standardization procedure that can be applied to data sets that display high variation between biological replicates. This enables proper statistical analysis and drawing relevant conclusions. The procedure is not new, it has been used in microarray data analysis and is based on a series of sequential corrections, including log transformation, mean centering, and autoscaling.

### Usage

    totData(data, r, geo = TRUE, logarithm = TRUE, base, transformation = TRUE,
    	nSpl, linear = TRUE, na.rm = na.rm)

### Arguments

`data` | data.frame containing row datas (genes in columns, samples in rows, Cq values).
`r` | numeric, number of qPCR replicates.
`geo` | logical, the function will use the geometrical mean of your biological replicates if TRUE or the arithmetic mean if FALSE.
`logarithm` | logical, the NRQs will be log-transformed.
`base` | numeric, the logarithmic base (2 or 10).
`transformation` | logical, if TRUE, the transformation procedure for highly variable biological replicates (but with the same tendency) will be done.
`nSpl` | numeric, the number of samples.
`linear` | logical, after the transformation procedure done, your raw data will be normalized (anti-log-transformed).
`na.rm` | logical, indicating whether NA values should be stripped before the computation proceeds.

### Details

The standardization procedure used in this function (if TRUE for the transformation argument) is based on the article of Willems et al. (2008). This function perform successively thEerror operations : log-transformation of your raw data, mean of your log-transformed data for each biological replicate, mean centering for each biological replicate, standard deviation of each mean-centered biological replicate, autoscaling of your data, i.e., your mean-centered data for each biological replicate will be divided by the standard deviation of the mean-centered biological replicate and then multiplicated by the mean of the standard deviation of all the biological replicates.

For more information for the way to use this function, please see the vignette.

### Value

`Mean of your qPCR runs` | The geometric (if TRUE for geo) or arithmetic mean of your biological replicates.
`Standard deviations of your qPCR runs` | The standard deviation of your biological replicates.
`Standard errors of your qPCR runs` | The standard error of your biological replicates.
`Transformed data` | If TRUE for transformation, your raw data will be transformed by the algorithm of Willems et al. (2008).
`Reordered transformed data` | The transformed data reordered by rowname.

### Author(s)

Sylvain Le pape <sylvain.le.pape@univ-poitiers.fr>

### References

Erik Willems Luc Leyns, Jo Vandesompele. Standardization of real-time PCR gene expression data from independent biological replicates. Analytical Biochemistry 379 (2008) 127-129 (doi:10.1016/j.ab.2008.04.036). <url:http://www.sciencedirect.com/science/article/pii/S0003269708002649>

### Examples

    data(qPCR_run1,qPCR_run2,qPCR_run3)

    nrmData(data = qPCR_run1 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=c(1, 1, 1, 1), CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)

    nrmData(data = qPCR_run2 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=c(1, 1, 1, 1), CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)

    nrmData(data = qPCR_run3 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=c(1, 1, 1, 1), CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)

    ## Isolate the calibrator NRQ values of the first biological replicate

    a <- nrmData(data = qPCR_run1 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=c(1, 1, 1, 1), CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)[[3]]

    ## Isolate the calibrator NRQ values of the first biological replicate

    b <- nrmData(data = qPCR_run2 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=c(1, 1, 1, 1), CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)[[3]]

    ## Isolate the calibrator NRQ values of the first biological replicate

    c <- nrmData(data = qPCR_run3 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=c(1, 1, 1, 1), CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)[[3]]

    ## Regrouping the calibrator NRQ values of all the biological replicates

    d <- rbind(a, b, c)

    ## Calibration factor calculation

    e <- calData(d)

    ## Attenuation of inter-run variation thanks to the calibration factor for the
    ## first biological replicate

    nrmData(data = qPCR_run1 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=e, CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)

    ## Attenuation of inter-run variation thanks to the calibration factor for the
    ## second biological replicate

    nrmData(data = qPCR_run2 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=e, CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)

    ## Attenuation of inter-run variation thanks to the calibration factor for the
    ## third biological replicate

    nrmData(data = qPCR_run3 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=e, CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)

    ## Isolate the NRQs scaled to control of the first biological replicate

    a1 <- nrmData(data = qPCR_run1 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=e, CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)[1]

    ## Isolate the NRQs scaled to control of the second biological replicate

    b1 <- nrmData(data = qPCR_run2 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=e, CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)[1]

    ## Isolate the NRQs scaled to control of the third biological replicate

    c1 <- nrmData(data = qPCR_run3 , r=3, E=c(2, 2, 2, 2),
    	      Eerror=c(0.02, 0.02, 0.02, 0.02), nSpl=5,
    	      nbRef=2, Refposcol=1:2, nCTL=2,
    	      CF=e, CalPos=5, trace=TRUE, geo=TRUE, na.rm=TRUE)[1]

    ## Data frame transformation

    a2 <- as.data.frame(a1)
    b2 <- as.data.frame(b1)
    c2 <- as.data.frame(c1)

    ## Aggregation of the three biological replicates

    d2 <- rbind(a2, b2, c2)

    totData(data=d2, r=3, geo=TRUE, logarithm=TRUE, base=2,
    	transformation=TRUE, nSpl=5, linear=TRUE,
    	na.rm=TRUE)

[Package EasyqpcR version 1.24.0 Index]