[BioC] Fwd: help

[Heidi Dvinge]

Hello Rakesh,

>
> sir,
>   i am new on R language and dealing with light-cycler qpcr data using
> HtqPCR package.everything goes all right till fold change.
>
> when i use t-test then following error occured.
>
>>  qDE.ttest <- ttestCtData(sr.norm[, 1:2], groups = files$Treatment[1:2],
> calibrator = "Control")
> Error in t.test.default(x[, g1], x[, g2], alternative = alternative,
> paired
> = paired,  :
>   data are essentially constant
>
This error isn't actually from HTqPCR, but from the underlying t.test
function. Have you checked whether these samples are indeed different?
E.g. plot(getCt(sr.norm[, 1:2])). Does the sample apply to all your
samples, e.g. qDE.ttest <- ttestCtData(sr.norm[, c(1,3)], groups =
files$Treatment[c(1,3)], calibrator = "Control")

It sounds like you may have a case where for example all replicates of a
given gene in both your two samples has the Ct value 40, i.e. is
undetected. Have you replaced such values with NA during your workflow?

You can check the variation in your samples using e.g. plotCtVariation, or
plotCtCor to look at the correlation between samples. In general, the
HTqPCR plotting functions are quite useful for figuring our how your data
'behaves'. The vignette gives examples of how to use most of these plots,
and they're all listed in figure 1.

> when i select 4 samples then
>
> qDE.ttest <- ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4],
> calibrator = "Control")
> Error in ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4],
> calibrator = "Control") :
>   Two factor levels required for 'groups'
>

In this case you input 4 different groups (Control, 30min, 2hr, 4hr) into
a 2-sided t-test. This never going to work; exactly 2 different groups are
required. You will need to go back and figure out why the 2-sample
approach you tried above doesn't work, i.e. where/why some of your samples
are too similar to each other.

HTH
\Heidi
>
> my parent file look like this
>
> File    Treatment
> control.txt    Control
> 30min.txt    30min
> 2hr.txt    2hr
> 4hr.txt    4hr
> 8hr.txt    8hr
> 12hr.txt    12hr
> 16hr.txt    16hr
> 24hr.txt    24hr
> 48hr.txt    48hr
>
> every sample have 26 features(13 replicate)
>
> so how i perform t- test on these data.
> please sir help me out.
>
>
> thanking you
>
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>
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