Dear community,
I am having trouble importing data to HTqPCR. The data was acquired on a (relatively new) Roche LightCycler 480 and is stored as a text file (values separated by tabs). I compared my file to the example data included in the package and realized that it differs. At the end of this post, I am providing an example of the structure of my data.
Because of the different formats, I chose to import the data as "plain" (in this example, I am trying to import the first row of data, attached at the end of this post). My full data (which is only a test, hence the low number of measurements) consists of 84 measurements in a 96 wells plate, where each row contains 6 samples tested for Gene 1, then again the same 6 samples with Gene 2 and so on until Gene 14) :
test <- list(feature=4, position=2, Ct=5, flag=20, type=9)
path<-("qpcr")
raw <- readCtData("Test.txt", path = path, n.features = 2, format="plain", column.info=test, n.data =6, na.value = 40L, header=TRUE)
I am receiving the following warning
Warning messages:
1: In FUN(newX[, i], ...) : NAs introduced by coercion
2: In readCtData("Test.txt", path = path, n.features = 2, format = "plain", :
One or more samples contain NAs. Consider replacing these with e.g. Ct=40 now.
This happens also if I change n.features = 12 and n.data =1 (and all other kinds of combinations I could come up with, which make no sense) as well as when providing sample names in a separate list and adding samples = samples and/or sep="\t" to the options for import. Also using the actual names of the columns did not help. The option na.value=40 seems to be ignored, sometimes with, sometimes without warnings.
Using format="LightCycler" I get
Error in `[.data.frame`(sample, , column.info[["Ct"]]) :
undefined columns selected
which is not a big surprise given that my textfile looks different from the example.
I can see that there is a problem in the imported data using
plotCtOverview(raw)
Which shows me funny bars no matter how I try to adjust the n.features and n.data. Consulting the manual & reference guide, e.g. for changing the Ct layout, did not help, unfortunately.
I would be very happy about your input, it feels as if I am missing a simple solution, but all attempts of troubleshooting in the past days did not lead to anything.
Thanks!
Example Data:
Color Position Sample Name Gene Name Cq Concentration Call Excluded Sample Type Standard Cq Mean Cq Error Concentration Mean Concentration Error Replicate Group Dye Edited Call Slope EPF Failure Notes Sample Prep Notes Number
255;132;197;238 A1 1 Gene1 25.36 - Positive Unchecked Unknown - 25.36 0.00 - - SYBR Green I 3.49 6.81 None 1
255;132;197;238 A2 2 Gene1 29.01 - Positive Unchecked Unknown - 29.01 0.00 - - SYBR Green I 3.00 6.17 None 2
255;132;197;238 A3 3 Gene1 32.07 - Positive Unchecked Unknown - 32.07 0.00 - - SYBR Green I 2.20 5.25 None 3
255;132;197;238 A4 4 Gene1 36.34 - Positive Unchecked Unknown - 36.34 0.00 - - SYBR Green I 3.50 3.37 None 4
255;132;197;238 A5 5 Gene1 39.56 - Positive Unchecked Unknown - 39.56 0.00 - - SYBR Green I 0.32 0.67 None 5
255;132;197;238 A6 6 Gene1 - - Negative Unchecked Negative control - - - - - SYBR Green I 0.00 0.00 None 6
255;95;103;174 A7 1 Gene2 10.55 - Positive Unchecked Unknown - 10.55 0.00 - - SYBR Green I 3.96 7.39 None 7
255;205;18;55 A8 2 Gene2 13.65 - Positive Unchecked Unknown - 13.65 0.00 - - SYBR Green I 3.71 7.21 None 8
255;34;176;146 A9 3 Gene2 17.33 - Positive Unchecked Unknown - 17.33 0.00 - - SYBR Green I 3.72 7.21 None 9
255;47;73;113 A10 4 Gene2 21.12 - Positive Unchecked Unknown - 21.12 0.00 - - SYBR Green I 2.72 6.55 None 10
255;234;79;79 A11 5 Gene2 24.81 - Positive Unchecked Unknown - 24.81 0.00 - - SYBR Green I 3.44 6.93 None 11
255;66;41;24 A12 6 Gene2 35.55 - Positive Unchecked Negative control - 35.55 0.00 - - SYBR Green I 3.81 4.15 Neg Ctrl is positive 12
