Question: Problems importing (new) LightCycler data to HTqPCR
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gravatar for H.H.
4.6 years ago by
H.H.0
H.H.0 wrote:

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

 

 

dataimport htqpcr • 991 views
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