Re: edgeR on ncRNA analysis question

Hi - OK, thanks for the feedback, I will them look carefully at the 
procedure

No, I did not have only WTA, but in the two comparisons the experiment 
samples were different - i.e. these are the same W.T. samples compared with 
two different set of experiment samples, which I did not copy in the output.

Thanks again and keep in touch

Alessandro

-----------------------------------------------------
Alessandro Guffanti - Head, Bioinformatics
Genomnia srl
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Tel. +39-0293305.702 / Fax +39-0293305.777
www.genomnia.com [http://www.genomnia.com/]
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-----Original Message-----
From: Gordon K Smyth <smyth@...>
To: alessandro.guffanti@...
Cc: Bioconductor mailing list <bioconductor@...>, Mark
Robinson 
<mark.robinson@...>
Date: Sun, 1 Dec 2013 13:25:26 +1100 (AUS Eastern Daylight Time)
Subject: edgeR on ncRNA analysis question

It does look like you may have done something wrong.  In fact, the output
doesn't make sense to me.  The CPM and average logCPM values output by
edgeR should be unchanged regardless of the comparison you are testing, so
the two output tables you give cannot be from the same data.  And you seem
to have wildtype samples only??

Normalization of ncRNA reads is very challenging, but there seems a much
more basic problem here.

In the absence of any code leading to the output given, it is impossible
to say more.

Best wishes
Gordon

> Date: Fri, 29 Nov 2013 12:06:40 +0100
> From: alessandro.guffanti@... 
[mailto:alessandro.guffanti%40genomnia.com]
> To: Bioconductor mailing list <bioconductor@... 
[mailto:bioconductor%40r-project.org]>
> Cc: bioinfo@... [mailto:bioinfo%40genomnia.com]
> Subject: [BioC] edgeR on ncRNA analysis question
>
> Der BioC edgeR developers and users:
>
> I am using edgeR for ncRNA transcriptome data analysis - ie mapping RNA 
seq
> results only versus a ncRNA transcript database (bowtie from Color Space
> reads)
>
> There seems to be, unsurprisingly, an high variability on these samples,
> which affects obviously the FDR
>
> However, what surprised us is that the CPM for the same samples in 
different
> comparisons (TMM-normalized) are always very different
>
> As an example:
> *
> **Comparison **A*
>
> Transcript_ID    logFC    logCPM    PValue    FDR    WT_4_CPM    WT_7.CPM  
  WT_10.CPM
> ENST00000456355    1.42    10.91    0.00001    0.03283    2843    2926    
2631
>
>
> *
> **Comparison **B
>
> *
> Transcript_ID    logFC    logCPM    PValue    FDR    WT_4_CPM    WT_7.CPM  
  WT_10.CPM
>
>
> ENST00000456355    0.91    11.11    0.00003    0.00361    190    341    
157
>
>
> Can TMM normalization affect so heavily the CPM values of the same
> samples in different comparisons,
> or do we have something else wrong here ?
>
> Thanks in advance for any feedback on this,
>
> Alessandro G
>
> ---

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