[BioC] edgeR contrast

[James W. MacDonald]

Hi Julia,

This appears to be a ChIP-Seq experiment, in which case I wouldn't analyze
it this way. Instead, I would use something like MACS to call peaks, using
the IgG fractions as the 'input' fraction. In other words, the IgG fraction
is used to help distinguish real IP regions from those regions that have
high sequencing depth due to technical factors. You would then use edgeR to
compare IP.treat versus IP.control. This is not a trivial analysis, and you
should look at this paper
(*http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066778/
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066778/>) *for more
information on how you should normalize your counts. But maybe I completely
misunderstand the experiment.

In that case, back to your question. contrasts 3 and 4 aren't valid
contrasts, so you should just ignore those results. Contrast 1 is an
interaction contrast, and is testing for genes that have different amounts
of IP binding between treatment and control, after adjusting for
non-specific binding. If this isn't ChIP-Seq, but instead is some
transcript binding experiment, then this is likely the contrast you want.

Best,

Jim




On Tue, Sep 2, 2014 at 6:42 AM, Julia [guest] <guest at bioconductor.org>
wrote:

> I try different contrasts with edgeR to get a feeling for my data and also
> to find out the best contrast for my question. I would like to know what
> genes are enriched in IP.treat compared to IP.control, both adjusted for
> unspecific IgG binding.
> So it seems like contrast 1 is the best:
> (IP.treat-IgG.treat)-(IP.control-IgG.control), however it seems like
> IgG.control is added to IP.treat as – and – is +. I then tried contrast
> 3 and 4, and get totally different results with genes only FC>1.
> My question: Is it allowed to have more levels -1 than +1 or how can it be
> explained that contrast3 and 4 look very similar but totally different than
> contrast1 (and IP)?
>
>
> Levels           IP contrast1 contrast2 contrast3 contrast4 contrast5
>
>   IP.treat        1         1         1         1         1         0
>
>   IP.control     -1        -1        -1        -1        -1         0
>
>   IgG.treat       0        -1         0        -1        -1         1
>
>   IgG.control     0         1         0        -1         0        -1
>
>
>
>
>
> Thanks for any help.
>
> Julia
>
>  -- output of sessionInfo():
>
> .
>
> --
> Sent via the guest posting facility at bioconductor.org.
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor at r-project.org
> https://stat.ethz.ch/mailman/listinfo/bioconductor
> Search the archives:
> http://news.gmane.org/gmane.science.biology.informatics.conductor
>



-- 
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099

	[[alternative HTML version deleted]]