Gordon K Smyth | 1 Dec 03:25 2013
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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@...
> To: Bioconductor mailing list <bioconductor@...>
> Cc: bioinfo@...
> 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
>
> ---
>
> R version 3.0.2 (2013-09-25)
> Platform: x86_64-w64-mingw32/x64 (64-bit)
>
> locale:
> [1] LC_COLLATE=English_United States.1252
> [2] LC_CTYPE=English_United States.1252
> [3] LC_MONETARY=English_United States.1252
> [4] LC_NUMERIC=C
> [5] LC_TIME=English_United States.1252
>
> attached base packages:
> [1] stats     graphics  grDevices utils     datasets  methods base
>
> other attached packages:
> [1] edgeR_3.4.0  limma_3.18.3
>
>
> -- 
> Alessandro Guffanti
> Head, Bioinformatics
> *Genomnia srl*
> Via Nerviano, 31/B -- 20020 Lainate (MI)
> Tel. +39-0293305.702 / Fax +39-0293305.777
> www.genomnia.com <http://www.genomnia.com>
> alessandro.guffanti@... <mailto:alessandro.guffanti@...>

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