#Load in required packages for functions below
require(qpcR)
## Loading required package: qpcR
## Loading required package: MASS
## Loading required package: minpack.lm
## Loading required package: rgl
## Loading required package: robustbase
## Loading required package: Matrix
require(plyr)
## Loading required package: plyr
require(ggplot2)
## Loading required package: ggplot2
require(splitstackshape)
## Loading required package: splitstackshape
## Loading required package: data.table
#Read in raw fluorescence data from 1st Actin replicate
rep1<-read.csv("Actin3rawfluoro.csv", header = T)
#Remove blank first column entitled "X"
rep1$X<-NULL
#Rename columns so that qpcR package and appropriately handle the data
rep1<-rename(rep1, c("Cycle" = "Cycles", "A1" = "H_C_1", "A2" = "N_C_1",
"A3"= "S_C_1", "A4"="H_T_1", "A5"="N_T_1","A6"="S_T_1",
"A7"="NT_C_1","B1" = "H_C_2", "B2" = "N_C_2","B3"= "S_C_2",
"B4"="H_T_2", "B5"="N_T_2", "B6"="S_T_2","B7"="NT_C_2",
"C1" = "H_C_3", "C2" = "N_C_3","C3"= "S_C_3","C4"="H_T_3",
"C5"="N_T_3", "C6"="S_T_3", "C7"="NT_C_3","D1" = "H_C_4",
"D2" = "N_C_4","D3"= "S_C_4", "D4"="H_T_4", "D5"="N_T_4",
"D6"="S_T_4", "D7"="NT_C_4","E1" = "H_C_5", "E2" = "N_C_5",
"E3"= "S_C_5", "E4"="H_T_5", "E5"="N_T_5", "E6"="S_T_5",
"F1" = "H_C_6", "F2" = "N_C_6","F3"= "S_C_6", "F4"="H_T_6",
"F5"="N_T_6", "F6"="S_T_6","G1" = "H_C_7", "G2" = "N_C_7",
"G3"= "S_C_7", "G4"="H_T_7", "G5"="N_T_7", "G6"="S_T_7",
"H1" = "H_C_8", "H2" = "N_C_8","H3"= "S_C_8", "H4"="H_T_8",
"H5"="N_T_8", "H6"="S_T_8"))
#Run data through pcrbatch in qpcR package which analyzes fluorescence and produces efficiency and cycle threshold values
rep1ct<-pcrbatch(rep1, fluo=NULL)
## Making model for H_C_1 (l4)
## => Fitting passed...
##
## Making model for N_C_1 (l4)
## => Fitting passed...
##
## Making model for S_C_1 (l4)
## => Fitting passed...
##
## Making model for H_T_1 (l4)
## => Fitting passed...
##
## Making model for N_T_1 (l4)
## => Fitting passed...
##
## Making model for S_T_1 (l4)
## => Fitting passed...
##
## Making model for NT_C_1 (l4)
## => Fitting passed...
##
## Making model for H_C_2 (l4)
## => Fitting passed...
##
## Making model for N_C_2 (l4)
## => Fitting passed...
##
## Making model for S_C_2 (l4)
## => Fitting passed...
##
## Making model for H_T_2 (l4)
## => Fitting passed...
##
## Making model for N_T_2 (l4)
## => Fitting passed...
##
## Making model for S_T_2 (l4)
## => Fitting passed...
##
## Making model for NT_C_2 (l4)
## => Fitting passed...
##
## Making model for H_C_3 (l4)
## => Fitting passed...
##
## Making model for N_C_3 (l4)
## => Fitting passed...
##
## Making model for S_C_3 (l4)
## => Fitting passed...
##
## Making model for H_T_3 (l4)
## => Fitting passed...
##
## Making model for N_T_3 (l4)
## => Fitting passed...
##
## Making model for S_T_3 (l4)
## => Fitting passed...
##
## Making model for NT_C_3 (l4)
## => Fitting passed...
##
## Making model for H_C_4 (l4)
## => Fitting passed...
##
## Making model for N_C_4 (l4)
## => Fitting passed...
##
## Making model for S_C_4 (l4)
## => Fitting passed...
##
## Making model for H_T_4 (l4)
## => Fitting passed...
##
## Making model for N_T_4 (l4)
## => Fitting passed...
##
## Making model for S_T_4 (l4)
## => Fitting passed...
##
## Making model for NT_C_4 (l4)
## => Fitting passed...
##
## Making model for H_C_5 (l4)
## => Fitting passed...
##
## Making model for N_C_5 (l4)
## => Fitting passed...
##
## Making model for S_C_5 (l4)
## => Fitting passed...
##
## Making model for H_T_5 (l4)
## => Fitting passed...
##
## Making model for N_T_5 (l4)
## => Fitting passed...
##
## Making model for S_T_5 (l4)
## => Fitting passed...
##
## Making model for H_C_6 (l4)
## => Fitting passed...
##
## Making model for N_C_6 (l4)
## => Fitting passed...
##
## Making model for S_C_6 (l4)
## => Fitting passed...
##
## Making model for H_T_6 (l4)
## => Fitting passed...
##
## Making model for N_T_6 (l4)
## => Fitting passed...
##
## Making model for S_T_6 (l4)
## => Fitting passed...
##
## Making model for H_C_7 (l4)
## => Fitting passed...
##
## Making model for N_C_7 (l4)
## => Fitting passed...
##
## Making model for S_C_7 (l4)
## => Fitting passed...
##
## Making model for H_T_7 (l4)
## => Fitting passed...
##
## Making model for N_T_7 (l4)
## => Fitting passed...
##
## Making model for S_T_7 (l4)
## => Fitting passed...
##
## Making model for H_C_8 (l4)
## => Fitting passed...
##
## Making model for N_C_8 (l4)
## => Fitting passed...
##
## Making model for S_C_8 (l4)
## => Fitting passed...
##
## Making model for H_T_8 (l4)
## => Fitting passed...
##
## Making model for N_T_8 (l4)
## => Fitting passed...
##
## Making model for S_T_8 (l4)
## => Fitting passed...
##
## Calculating delta of first/second derivative maxima...
## .........10.........20.........30.........40.........50
## ..
## Analyzing H_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
#pcrbatch creates a file with each sample as an individual column in the dataframe. The problem with this is
#that I want to compare all the Ct (labelled sig.cpD2) and generate expression data for them but these values have to be
#in individual columns. To do this I must transpose the data and set the first row as the column names.
rep1res<-setNames(data.frame(t(rep1ct)),rep1ct[,1])
#Now I must remove the first row as it is a duplicate and will cause errors with future analysis
rep1res<-rep1res[-1,]
#since the sample names are now in the first column the column title is row.names. This makes analys hard based on the ability to call the first column.
#to eliminate this issue, I copied the first column into a new column called "Names"
rep1res$Names<-rownames(rep1res)
#Since each sample name contains information such as Population, Treatment, and Sample Number I want to separate out these factors
#into new columns so that I can run future analysis based on population, treatment, or both. Also note the "drop = F" this is so the original names column remains.
rep1res2<-cSplit_f(rep1res, splitCols=c("Names"), sep="_", drop = F)
#After splitting the names column into three new columns I need to rename them appropriately.
rep1res2<-rename(rep1res2, c("Names_1"="Pop", "Names_2"="Treat", "Names_3"="Sample"))
#I also create a column with the target gene name. This isn't used in this analysis but will be helpful for future work.
rep1res2$Gene<-rep("Actin", length(rep1res2))
#In transposing the data frame, the column entries became factors which cannot be used for equations.
#to fix this, I set the entries for sig.eff (efficiency) and sig.cpD2 (Ct value) to numeric. Be aware, without the as.character function the factors will be transformed inappropriately.
rep1res2$sig.eff<-as.numeric(as.character(rep1res2$sig.eff))
rep1res2$sig.cpD2<-as.numeric(as.character(rep1res2$sig.cpD2))
#Now I plot the Ct values to see how they align without converting them to expression.
ggplot(rep1res2, aes(x=Names,y=sig.cpD2, fill=Pop))+geom_bar(stat="identity")
#Now I want to get expression information from my data set. qpcR has a way of doing this but its complicated and I'm not comfortable using it.
#Luckily there is an equation I can use to do it. The equation is expression = 1/(1+efficiency)^Ctvalue. I tried multiple ways to get this to work in R
#but it doesn't handle the complicated equation easily.
#To work around this, I created a function in R to run the equation and produce an outcome. x = efficiency argument, y=Ctvalue argument
expr<-function(x,y){
newVar<-(1+x)^y
1/newVar
}
#Now I run the data through the function and produce a useful expression value
rep1res2$expression<-expr(rep1res2$sig.eff, rep1res2$sig.cpD2)
#Graphing the expression values is a good way to examine the data quickly for errors that might have occurred.
ggplot(rep1res2, aes(x=Names,y=expression, fill=Pop))+geom_bar(stat="identity")
#Before I'm able to compare the replicates I need to process the raw fluorescence from the second Actin run.
#To do this I perform all the same steps as the previous replicate.
rep2<-read.csv("Actin4rawfluoro.csv", header = T)
rep2$X<-NULL
rep2<-rename(rep2, c("Cycle" = "Cycles", "A1" = "H_C_1", "A2" = "N_C_1",
"A3"= "S_C_1", "A4"="H_T_1", "A5"="N_T_1","A6"="S_T_1",
"A7"="NT_C_1","B1" = "H_C_2", "B2" = "N_C_2","B3"= "S_C_2",
"B4"="H_T_2", "B5"="N_T_2", "B6"="S_T_2","B7"="NT_C_2",
"C1" = "H_C_3", "C2" = "N_C_3","C3"= "S_C_3","C4"="H_T_3",
"C5"="N_T_3", "C6"="S_T_3", "C7"="NT_C_3","D1" = "H_C_4",
"D2" = "N_C_4","D3"= "S_C_4", "D4"="H_T_4", "D5"="N_T_4",
"D6"="S_T_4", "D7"="NT_C_4","E1" = "H_C_5", "E2" = "N_C_5",
"E3"= "S_C_5", "E4"="H_T_5", "E5"="N_T_5", "E6"="S_T_5",
"F1" = "H_C_6", "F2" = "N_C_6","F3"= "S_C_6", "F4"="H_T_6",
"F5"="N_T_6", "F6"="S_T_6","G1" = "H_C_7", "G2" = "N_C_7",
"G3"= "S_C_7", "G4"="H_T_7", "G5"="N_T_7", "G6"="S_T_7",
"H1" = "H_C_8", "H2" = "N_C_8","H3"= "S_C_8", "H4"="H_T_8",
"H5"="N_T_8", "H6"="S_T_8"))
rep2ct<-pcrbatch(rep2, fluo=NULL)
## Making model for H_C_1 (l4)
## => Fitting passed...
##
## Making model for N_C_1 (l4)
## => Fitting passed...
##
## Making model for S_C_1 (l4)
## => Fitting passed...
##
## Making model for H_T_1 (l4)
## => Fitting passed...
##
## Making model for N_T_1 (l4)
## => Fitting passed...
##
## Making model for S_T_1 (l4)
## => Fitting passed...
##
## Making model for NT_C_1 (l4)
## => Fitting passed...
##
## Making model for H_C_2 (l4)
## => Fitting passed...
##
## Making model for N_C_2 (l4)
## => Fitting passed...
##
## Making model for S_C_2 (l4)
## => Fitting passed...
##
## Making model for H_T_2 (l4)
## => Fitting passed...
##
## Making model for N_T_2 (l4)
## => Fitting passed...
##
## Making model for S_T_2 (l4)
## => Fitting passed...
##
## Making model for NT_C_2 (l4)
## => Fitting failed. Tagging name of NT_C_2...
##
## Making model for H_C_3 (l4)
## => Fitting passed...
##
## Making model for N_C_3 (l4)
## => Fitting passed...
##
## Making model for S_C_3 (l4)
## => Fitting passed...
##
## Making model for H_T_3 (l4)
## => Fitting passed...
##
## Making model for N_T_3 (l4)
## => Fitting passed...
##
## Making model for S_T_3 (l4)
## => Fitting passed...
##
## Making model for NT_C_3 (l4)
## => Fitting failed. Tagging name of NT_C_3...
##
## Making model for H_C_4 (l4)
## => Fitting passed...
##
## Making model for N_C_4 (l4)
## => Fitting passed...
##
## Making model for S_C_4 (l4)
## => Fitting passed...
##
## Making model for H_T_4 (l4)
## => Fitting passed...
##
## Making model for N_T_4 (l4)
## => Fitting passed...
##
## Making model for S_T_4 (l4)
## => Fitting passed...
##
## Making model for NT_C_4 (l4)
## => Fitting failed. Tagging name of NT_C_4...
##
## Making model for H_C_5 (l4)
## => Fitting passed...
##
## Making model for N_C_5 (l4)
## => Fitting passed...
##
## Making model for S_C_5 (l4)
## => Fitting passed...
##
## Making model for H_T_5 (l4)
## => Fitting passed...
##
## Making model for N_T_5 (l4)
## => Fitting passed...
##
## Making model for S_T_5 (l4)
## => Fitting passed...
##
## Making model for H_C_6 (l4)
## => Fitting passed...
##
## Making model for N_C_6 (l4)
## => Fitting passed...
##
## Making model for S_C_6 (l4)
## => Fitting passed...
##
## Making model for H_T_6 (l4)
## => Fitting passed...
##
## Making model for N_T_6 (l4)
## => Fitting passed...
##
## Making model for S_T_6 (l4)
## => Fitting passed...
##
## Making model for H_C_7 (l4)
## => Fitting passed...
##
## Making model for N_C_7 (l4)
## => Fitting passed...
##
## Making model for S_C_7 (l4)
## => Fitting passed...
##
## Making model for H_T_7 (l4)
## => Fitting passed...
##
## Making model for N_T_7 (l4)
## => Fitting passed...
##
## Making model for S_T_7 (l4)
## => Fitting passed...
##
## Making model for H_C_8 (l4)
## => Fitting passed...
##
## Making model for N_C_8 (l4)
## => Fitting passed...
##
## Making model for S_C_8 (l4)
## => Fitting passed...
##
## Making model for H_T_8 (l4)
## => Fitting passed...
##
## Making model for N_T_8 (l4)
## => Fitting passed...
##
## Making model for S_T_8 (l4)
## => Fitting passed...
##
## Calculating delta of first/second derivative maxima...
## .........10.........20.........30.........40.........50
## ..
## Analyzing H_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing *NT_C_2* ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing *NT_C_3* ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing *NT_C_4* ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
rep2res<-setNames(data.frame(t(rep2ct)),rep2ct[,1])
rep2res<-rep2res[-1,]
rep2res$Names<-rownames(rep2res)
rep2res2<-cSplit_f(rep2res, splitCols=c("Names"), sep="_", drop = F)
rep2res2<-rename(rep2res2, c("Names_1"="Pop", "Names_2"="Treat", "Names_3"="Sample"))
rep2res2$Gene<-rep("Actin", length(rep2res2))
rep2res2$sig.eff<-as.numeric(as.character(rep2res2$sig.eff))
rep2res2$sig.cpD2<-as.numeric(as.character(rep2res2$sig.cpD2))
ggplot(rep2res2, aes(x=Names,y=sig.cpD2, fill=Pop))+geom_bar(stat="identity")
expr<-function(x,y){
newVar<-(1+x)^y
1/newVar
}
rep2res2$expression<-expr(rep2res2$sig.eff, rep2res2$sig.cpD2)
ggplot(rep2res2, aes(x=Names,y=expression, fill=Pop))+geom_bar(stat="identity")
#Now that I have Ct values, efficiencies and expression values for both replicates I can create a table of the differences between reps.
#To do this I create a data frame with a single formula that creates a column of values generated by subtracting the first run from the second.
repcomp<-as.data.frame(rep1res2$sig.cpD2-rep2res2$sig.cpD2)
#Now I need to add some Names for the samples to use with ggplot.Since the names column contains all the relevant information
#I copy only that column and run the split function on it again as well as the rename function.
repcomp$Names<-rep1res2$Names
repcomp<-cSplit_f(repcomp, splitCols=c("Names"), sep="_", drop = F)
#To better address the difference column in ggplot I need to rename it something simple and short.
repcomp<-rename(repcomp, c("rep1res2$sig.cpD2 - rep2res2$sig.cpD2"="rep.diff", "Names_1"="Pop", "Names_2"="Treat", "Names_3"="Sample"))
#Now I just run the data through ggplot to generate a bar graph exploring the differences between the two replicate in terms of Ct values.
ggplot(repcomp, aes(x=Names, y=rep.diff, fill=Pop))+geom_bar(stat="identity")
#Read in raw fluorescence data from 1st Actin replicate
rep3<-read.csv("Actin1rawfluoro.csv", header = T)
#Remove blank first column entitled "X"
rep3$X<-NULL
#Rename columns so that qpcR package and appropriately handle the data
rep3<-rename(rep3, c("Cycle" = "Cycles", "A1" = "H_C_1", "A2" = "N_C_1",
"A3"= "S_C_1", "A4"="H_T_1", "A5"="N_T_1","A6"="S_T_1",
"A7"="NT_C_1","B1" = "H_C_2", "B2" = "N_C_2","B3"= "S_C_2",
"B4"="H_T_2", "B5"="N_T_2", "B6"="S_T_2","B7"="NT_C_2",
"C1" = "H_C_3", "C2" = "N_C_3","C3"= "S_C_3","C4"="H_T_3",
"C5"="N_T_3", "C6"="S_T_3", "C7"="NT_C_3","D1" = "H_C_4",
"D2" = "N_C_4","D3"= "S_C_4", "D4"="H_T_4", "D5"="N_T_4",
"D6"="S_T_4", "D7"="NT_C_4","E1" = "H_C_5", "E2" = "N_C_5",
"E3"= "S_C_5", "E4"="H_T_5", "E5"="N_T_5", "E6"="S_T_5",
"F1" = "H_C_6", "F2" = "N_C_6","F3"= "S_C_6", "F4"="H_T_6",
"F5"="N_T_6", "F6"="S_T_6","G1" = "H_C_7", "G2" = "N_C_7",
"G3"= "S_C_7", "G4"="H_T_7", "G5"="N_T_7", "G6"="S_T_7",
"H1" = "H_C_8", "H2" = "N_C_8","H3"= "S_C_8", "H4"="H_T_8",
"H5"="N_T_8", "H6"="S_T_8"))
#Run data through pcrbatch in qpcR package which analyzes fluorescence and produces efficiency and cycle threshold values
rep3ct<-pcrbatch(rep3, fluo=NULL)
## Making model for H_C_1 (l4)
## => Fitting passed...
##
## Making model for N_C_1 (l4)
## => Fitting passed...
##
## Making model for S_C_1 (l4)
## => Fitting passed...
##
## Making model for H_T_1 (l4)
## => Fitting passed...
##
## Making model for N_T_1 (l4)
## => Fitting passed...
##
## Making model for S_T_1 (l4)
## => Fitting passed...
##
## Making model for NT_C_1 (l4)
## => Fitting passed...
##
## Making model for H_C_2 (l4)
## => Fitting passed...
##
## Making model for N_C_2 (l4)
## => Fitting passed...
##
## Making model for S_C_2 (l4)
## => Fitting passed...
##
## Making model for H_T_2 (l4)
## => Fitting passed...
##
## Making model for N_T_2 (l4)
## => Fitting passed...
##
## Making model for S_T_2 (l4)
## => Fitting passed...
##
## Making model for NT_C_2 (l4)
## => Fitting passed...
##
## Making model for H_C_3 (l4)
## => Fitting passed...
##
## Making model for N_C_3 (l4)
## => Fitting passed...
##
## Making model for S_C_3 (l4)
## => Fitting passed...
##
## Making model for H_T_3 (l4)
## => Fitting passed...
##
## Making model for N_T_3 (l4)
## => Fitting passed...
##
## Making model for S_T_3 (l4)
## => Fitting passed...
##
## Making model for NT_C_3 (l4)
## => Fitting passed...
##
## Making model for H_C_4 (l4)
## => Fitting passed...
##
## Making model for N_C_4 (l4)
## => Fitting passed...
##
## Making model for S_C_4 (l4)
## => Fitting passed...
##
## Making model for H_T_4 (l4)
## => Fitting passed...
##
## Making model for N_T_4 (l4)
## => Fitting passed...
##
## Making model for S_T_4 (l4)
## => Fitting passed...
##
## Making model for NT_C_4 (l4)
## => Fitting passed...
##
## Making model for H_C_5 (l4)
## => Fitting passed...
##
## Making model for N_C_5 (l4)
## => Fitting passed...
##
## Making model for S_C_5 (l4)
## => Fitting passed...
##
## Making model for H_T_5 (l4)
## => Fitting passed...
##
## Making model for N_T_5 (l4)
## => Fitting passed...
##
## Making model for S_T_5 (l4)
## => Fitting passed...
##
## Making model for H_C_6 (l4)
## => Fitting passed...
##
## Making model for N_C_6 (l4)
## => Fitting passed...
##
## Making model for S_C_6 (l4)
## => Fitting passed...
##
## Making model for H_T_6 (l4)
## => Fitting passed...
##
## Making model for N_T_6 (l4)
## => Fitting passed...
##
## Making model for S_T_6 (l4)
## => Fitting passed...
##
## Making model for H_C_7 (l4)
## => Fitting passed...
##
## Making model for N_C_7 (l4)
## => Fitting passed...
##
## Making model for S_C_7 (l4)
## => Fitting passed...
##
## Making model for H_T_7 (l4)
## => Fitting passed...
##
## Making model for N_T_7 (l4)
## => Fitting passed...
##
## Making model for S_T_7 (l4)
## => Fitting passed...
##
## Making model for H_C_8 (l4)
## => Fitting passed...
##
## Making model for N_C_8 (l4)
## => Fitting passed...
##
## Making model for S_C_8 (l4)
## => Fitting passed...
##
## Making model for H_T_8 (l4)
## => Fitting passed...
##
## Making model for N_T_8 (l4)
## => Fitting passed...
##
## Making model for S_T_8 (l4)
## => Fitting passed...
##
## Calculating delta of first/second derivative maxima...
## .........10.........20.........30.........40.........50
## ..
## Analyzing H_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
#pcrbatch creates a file with each sample as an individual column in the dataframe. The problem with this is
#that I want to compare all the Ct (labelled sig.cpD2) and generate expression data for them but these values have to be
#in individual columns. To do this I must transpose the data and set the first row as the column names.
rep3res<-setNames(data.frame(t(rep3ct)),rep3ct[,1])
#Now I must remove the first row as it is a duplicate and will cause errors with future analysis
rep3res<-rep3res[-1,]
#since the sample names are now in the first column the column title is row.names. This makes analys hard based on the ability to call the first column.
#to eliminate this issue, I copied the first column into a new column called "Names"
rep3res$Names<-rownames(rep3res)
#Since each sample name contains information such as Population, Treatment, and Sample Number I want to separate out these factors
#into new columns so that I can run future analysis based on population, treatment, or both. Also note the "drop = F" this is so the original names column remains.
rep3res2<-cSplit_f(rep3res, splitCols=c("Names"), sep="_", drop = F)
#After splitting the names column into three new columns I need to rename them appropriately.
rep3res2<-rename(rep3res2, c("Names_1"="Pop", "Names_2"="Treat", "Names_3"="Sample"))
#I also create a column with the target gene name. This isn't used in this analysis but will be helpful for future work.
rep3res2$Gene<-rep("Actin", length(rep3res2))
#In transposing the data frame, the column entries became factors which cannot be used for equations.
#to fix this, I set the entries for sig.eff (efficiency) and sig.cpD2 (Ct value) to numeric. Be aware, without the as.character function the factors will be transformed inappropriately.
rep3res2$sig.eff<-as.numeric(as.character(rep3res2$sig.eff))
rep3res2$sig.cpD2<-as.numeric(as.character(rep3res2$sig.cpD2))
#Now I plot the Ct values to see how they align without converting them to expression.
ggplot(rep3res2, aes(x=Names,y=sig.cpD2, fill=Pop))+geom_bar(stat="identity")
#Now I want to get expression information from my data set. qpcR has a way of doing this but its complicated and I'm not comfortable using it.
#Luckily there is an equation I can use to do it. The equation is expression = 1/(1+efficiency)^Ctvalue. I tried multiple ways to get this to work in R
#but it doesn't handle the complicated equation easily.
#To work around this, I created a function in R to run the equation and produce an outcome. x = efficiency argument, y=Ctvalue argument
expr<-function(x,y){
newVar<-(1+x)^y
1/newVar
}
#Now I run the data through the function and produce a useful expression value
rep3res2$expression<-expr(rep3res2$sig.eff, rep3res2$sig.cpD2)
#Graphing the expression values is a good way to examine the data quickly for errors that might have occurred.
ggplot(rep3res2, aes(x=Names,y=expression, fill=Pop))+geom_bar(stat="identity")
#Before I'm able to compare the replicates I need to process the raw fluorescence from the second Actin run.
#To do this I perform all the same steps as the previous replicate.
rep4<-read.csv("Actin2rawfluoro.csv", header = T)
rep4$X<-NULL
rep4<-rename(rep4, c("Cycle" = "Cycles", "A1" = "H_C_1", "A2" = "N_C_1",
"A3"= "S_C_1", "A4"="H_T_1", "A5"="N_T_1","A6"="S_T_1",
"A7"="NT_C_1","B1" = "H_C_2", "B2" = "N_C_2","B3"= "S_C_2",
"B4"="H_T_2", "B5"="N_T_2", "B6"="S_T_2","B7"="NT_C_2",
"C1" = "H_C_3", "C2" = "N_C_3","C3"= "S_C_3","C4"="H_T_3",
"C5"="N_T_3", "C6"="S_T_3", "C7"="NT_C_3","D1" = "H_C_4",
"D2" = "N_C_4","D3"= "S_C_4", "D4"="H_T_4", "D5"="N_T_4",
"D6"="S_T_4", "D7"="NT_C_4","E1" = "H_C_5", "E2" = "N_C_5",
"E3"= "S_C_5", "E4"="H_T_5", "E5"="N_T_5", "E6"="S_T_5",
"F1" = "H_C_6", "F2" = "N_C_6","F3"= "S_C_6", "F4"="H_T_6",
"F5"="N_T_6", "F6"="S_T_6","G1" = "H_C_7", "G2" = "N_C_7",
"G3"= "S_C_7", "G4"="H_T_7", "G5"="N_T_7", "G6"="S_T_7",
"H1" = "H_C_8", "H2" = "N_C_8","H3"= "S_C_8", "H4"="H_T_8",
"H5"="N_T_8", "H6"="S_T_8"))
rep4ct<-pcrbatch(rep4, fluo=NULL)
## Making model for H_C_1 (l4)
## => Fitting passed...
##
## Making model for N_C_1 (l4)
## => Fitting passed...
##
## Making model for S_C_1 (l4)
## => Fitting passed...
##
## Making model for H_T_1 (l4)
## => Fitting passed...
##
## Making model for N_T_1 (l4)
## => Fitting passed...
##
## Making model for S_T_1 (l4)
## => Fitting passed...
##
## Making model for NT_C_1 (l4)
## => Fitting passed...
##
## Making model for H_C_2 (l4)
## => Fitting passed...
##
## Making model for N_C_2 (l4)
## => Fitting passed...
##
## Making model for S_C_2 (l4)
## => Fitting passed...
##
## Making model for H_T_2 (l4)
## => Fitting passed...
##
## Making model for N_T_2 (l4)
## => Fitting passed...
##
## Making model for S_T_2 (l4)
## => Fitting passed...
##
## Making model for NT_C_2 (l4)
## => Fitting passed...
##
## Making model for H_C_3 (l4)
## => Fitting passed...
##
## Making model for N_C_3 (l4)
## => Fitting passed...
##
## Making model for S_C_3 (l4)
## => Fitting passed...
##
## Making model for H_T_3 (l4)
## => Fitting passed...
##
## Making model for N_T_3 (l4)
## => Fitting passed...
##
## Making model for S_T_3 (l4)
## => Fitting passed...
##
## Making model for NT_C_3 (l4)
## => Fitting passed...
##
## Making model for H_C_4 (l4)
## => Fitting passed...
##
## Making model for N_C_4 (l4)
## => Fitting passed...
##
## Making model for S_C_4 (l4)
## => Fitting passed...
##
## Making model for H_T_4 (l4)
## => Fitting passed...
##
## Making model for N_T_4 (l4)
## => Fitting passed...
##
## Making model for S_T_4 (l4)
## => Fitting passed...
##
## Making model for NT_C_4 (l4)
## => Fitting passed...
##
## Making model for H_C_5 (l4)
## => Fitting passed...
##
## Making model for N_C_5 (l4)
## => Fitting passed...
##
## Making model for S_C_5 (l4)
## => Fitting passed...
##
## Making model for H_T_5 (l4)
## => Fitting passed...
##
## Making model for N_T_5 (l4)
## => Fitting passed...
##
## Making model for S_T_5 (l4)
## => Fitting passed...
##
## Making model for H_C_6 (l4)
## => Fitting passed...
##
## Making model for N_C_6 (l4)
## => Fitting passed...
##
## Making model for S_C_6 (l4)
## => Fitting passed...
##
## Making model for H_T_6 (l4)
## => Fitting passed...
##
## Making model for N_T_6 (l4)
## => Fitting passed...
##
## Making model for S_T_6 (l4)
## => Fitting passed...
##
## Making model for H_C_7 (l4)
## => Fitting passed...
##
## Making model for N_C_7 (l4)
## => Fitting passed...
##
## Making model for S_C_7 (l4)
## => Fitting passed...
##
## Making model for H_T_7 (l4)
## => Fitting passed...
##
## Making model for N_T_7 (l4)
## => Fitting passed...
##
## Making model for S_T_7 (l4)
## => Fitting passed...
##
## Making model for H_C_8 (l4)
## => Fitting passed...
##
## Making model for N_C_8 (l4)
## => Fitting passed...
##
## Making model for S_C_8 (l4)
## => Fitting passed...
##
## Making model for H_T_8 (l4)
## => Fitting passed...
##
## Making model for N_T_8 (l4)
## => Fitting passed...
##
## Making model for S_T_8 (l4)
## => Fitting passed...
##
## Calculating delta of first/second derivative maxima...
## .........10.........20.........30.........40.........50
## ..
## Analyzing H_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_1 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_2 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_3 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing NT_C_4 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_5 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_6 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_7 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_C_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing H_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing N_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
##
## Analyzing S_T_8 ...
## Calculating 'eff' and 'ct' from sigmoidal model...
## Using window-of-linearity...
## Fitting exponential model...
## Using linear regression of efficiency (LRE)...
rep4res<-setNames(data.frame(t(rep4ct)),rep4ct[,1])
rep4res<-rep4res[-1,]
rep4res$Names<-rownames(rep4res)
rep4res2<-cSplit_f(rep4res, splitCols=c("Names"), sep="_", drop = F)
rep4res2<-rename(rep4res2, c("Names_1"="Pop", "Names_2"="Treat", "Names_3"="Sample"))
rep4res2$Gene<-rep("Actin", length(rep4res2))
rep4res2$sig.eff<-as.numeric(as.character(rep4res2$sig.eff))
rep4res2$sig.cpD2<-as.numeric(as.character(rep4res2$sig.cpD2))
ggplot(rep4res2, aes(x=Names,y=sig.cpD2, fill=Pop))+geom_bar(stat="identity")
expr<-function(x,y){
newVar<-(1+x)^y
1/newVar
}
rep4res2$expression<-expr(rep4res2$sig.eff, rep4res2$sig.cpD2)
ggplot(rep4res2, aes(x=Names,y=expression, fill=Pop))+geom_bar(stat="identity")
#Now that I have Ct values, efficiencies and expression values for both replicates I can create a table of the differences between reps.
#To do this I create a data frame with a single formula that creates a column of values generated by subtracting the first run from the second.
repcomp<-as.data.frame(rep3res2$sig.cpD2-rep4res2$sig.cpD2)
#Now I need to add some Names for the samples to use with ggplot.Since the names column contains all the relevant information
#I copy only that column and run the split function on it again as well as the rename function.
repcomp$Names<-rep3res2$Names
repcomp<-cSplit_f(repcomp, splitCols=c("Names"), sep="_", drop = F)
#To better address the difference column in ggplot I need to rename it something simple and short.
repcomp<-rename(repcomp, c("rep3res2$sig.cpD2 - rep4res2$sig.cpD2"="rep.diff", "Names_1"="Pop", "Names_2"="Treat", "Names_3"="Sample"))
#Now I just run the data through ggplot to generate a bar graph exploring the differences between the two replicate in terms of Ct values.
ggplot(repcomp, aes(x=Names, y=rep.diff, fill=Pop))+geom_bar(stat="identity")
actstandard<-as.data.frame(cbind(rep1res2$expression,rep1res2$Names,rep1res2$Pop,rep1res2$Treat,rep2res2$expression,rep3res2$expression,rep4res2$expression,rep1res2$sig.cpD2,rep2res2$sig.cpD2,rep3res2$sig.cpD2,rep4res2$sig.cpD2))
actstandard<-rename(actstandard, c(V1="rep1.expr","V2"="name","V3"="pop","V4"="treat"
,"V5"="rep2.expr","V6"="rep3.expr","V7"="rep4.expr",
"V8"="rep1.Ct","V9"="rep2.Ct","V10"="rep3.Ct","V11"="rep4.Ct"))
actstandard$rep1.expr<-as.numeric(as.character(actstandard$rep1.expr))
actstandard$rep2.expr<-as.numeric(as.character(actstandard$rep2.expr))
actstandard$rep3.expr<-as.numeric(as.character(actstandard$rep3.expr))
actstandard$rep4.expr<-as.numeric(as.character(actstandard$rep4.expr))
actstandard$avgexpr<-rowMeans(actstandard[,c("rep1.expr","rep2.expr","rep3.expr","rep4.expr")],na.rm=F)
actstandard<-actstandard[which(actstandard$pop!=c("NT")),]
ggplot(actstandard, aes(x=treat,y=avgexpr, fill=pop))+geom_boxplot()
fit<-aov(avgexpr~pop+treat+pop:treat,data=actstandard)
fit
## Call:
## aov(formula = avgexpr ~ pop + treat + pop:treat, data = actstandard)
##
## Terms:
## pop treat pop:treat Residuals
## Sum of Squares 1.408412e-17 5.411087e-17 9.717390e-18 1.658841e-16
## Deg. of Freedom 2 1 2 42
##
## Residual standard error: 1.987366e-09
## Estimated effects may be unbalanced
TukeyHSD(fit)
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = avgexpr ~ pop + treat + pop:treat, data = actstandard)
##
## $pop
## diff lwr upr p adj
## N-H 1.129511e-09 -5.775478e-10 2.836571e-09 0.2537138
## S-H -3.818664e-11 -1.745246e-09 1.668873e-09 0.9983730
## S-N -1.167698e-09 -2.874757e-09 5.393611e-10 0.2317285
##
## $treat
## diff lwr upr p adj
## T-C -2.123497e-09 -3.281277e-09 -9.657172e-10 0.0006182
##
## $`pop:treat`
## diff lwr upr p adj
## N:C-H:C 2.177609e-09 -7.887820e-10 5.143999e-09 0.2632276
## S:C-H:C 1.906935e-10 -2.775697e-09 3.157084e-09 0.9999612
## H:T-H:C -1.272179e-09 -4.238569e-09 1.694212e-09 0.7938935
## N:T-H:C -1.190764e-09 -4.157155e-09 1.775626e-09 0.8351472
## S:T-H:C -1.539245e-09 -4.505636e-09 1.427145e-09 0.6354210
## S:C-N:C -1.986915e-09 -4.953306e-09 9.794755e-10 0.3597559
## H:T-N:C -3.449787e-09 -6.416178e-09 -4.833967e-10 0.0143596
## N:T-N:C -3.368373e-09 -6.334763e-09 -4.019824e-10 0.0178899
## S:T-N:C -3.716854e-09 -6.683245e-09 -7.504635e-10 0.0068191
## H:T-S:C -1.462872e-09 -4.429263e-09 1.503518e-09 0.6833126
## N:T-S:C -1.381458e-09 -4.347848e-09 1.584933e-09 0.7324909
## S:T-S:C -1.729939e-09 -4.696330e-09 1.236452e-09 0.5135711
## N:T-H:T 8.141429e-11 -2.884976e-09 3.047805e-09 0.9999994
## S:T-H:T -2.670668e-10 -3.233457e-09 2.699324e-09 0.9997952
## S:T-N:T -3.484811e-10 -3.314872e-09 2.617909e-09 0.9992493
