[BioC] Limma

Wed Apr 6 17:16:43 CEST 2011

Dear Jenny,
Thank you,
I agree that "absence" does not mean that the transcript is not expressed
because if the probe sequence used to target that transcript  is not
performing well so non expression is the result of the technical problem
rather than the abundance of the transcript in the sample analyzed. We can
also have other factors such image acquisition conditions, etc.

But for my analyses, as I have a large sample size (n > 1500), if a
transcript is absent in all RNA samples, we cannot expect that this is just
an artifact.
What I am doing is  calculating detection calls and then I have three
situation: 1) probes present in all samples, 2) probes absent in all
samples, and 3) probes absent in a subset of samples. In order to not lose
information, I am not filtering probes of category (3)

And I also examine the concordance of detection call for all probes
(spliced variants or technical replicate probes) tagging the same gene, so
if I have 3 probes  
for the same gene, all absent I consider this as confidence to say that the
gene is not expressed.

In addition, I am using other gene expression datasets (for the same cell
type), generated using different platforms to check whether the probe absent
in dataset A is also absent in dataset B, C..

Now for the reviewer, he cannot understand how we say that a gene is not
expressed and on the other hand, we use the intensity for this  gene to
calculate a fold change. But in signal processing, we can have a quantity of
signal even it corresponds to noise and not to the real signal.

Best wishes,
Seraya

-----Original Message-----
From: Jenny Drnevich [mailto:drnevich at illinois.edu] 
Sent: Mittwoch, 6. April 2011 16:53
To: Seraya Maouche; 'James W. MacDonald'; 'Wei Shi'
Cc: Bioconductor at r-project.org
Subject: Re: [BioC] Limma

Hi Seraya,

I think your explanation for the reviewer is on track. Despite the fact that
some microarray platforms allow the calculation of a metric called "present"
or "absent", they really cannot detect whether a gene is truly expressed or
not. 
Instead, it's just a metric saying if the signal was above some noise
threshold. Now, the signal measured will always be non-zero, so we can use
these numbers to calculate fold-change regardless of the "present" or
"absent" call. You are right to throw out only those genes that are called
"absent" in all samples, but for the rest of the genes, the "present/absent"
metric is not good enough to categorize "off" versus "on" genes, so we just
use the numbers measured, calculate fold-changes and do statistical tests.
Hopefully the reviewer will be satisfied with an explanation of this sort.

Good luck,
Jenny

At 09:39 AM 4/6/2011, Seraya Maouche wrote:
>Dear  Jim, dear Wei,
>Thanks for your help, it is not a two color analysis, it is Illumina.
>
>Best wishes
>seraya
>
>-----Original Message-----
>From: James W. MacDonald [mailto:jmacdon at med.umich.edu]
>Sent: Mittwoch, 6. April 2011 15:55
>To: Wei Shi
>Cc: Seraya Maouche; Bioconductor at r-project.org
>Subject: Re: [BioC] Limma
>
>Hi Wei,
>
>I think you misunderstood the OP. Seraya _didn't_ remove genes that 
>were only present in one condition. The problem is that the reviewer 
>didn't like ratios with a zero in the denominator, which is a fair
complaint.
>
>I don't do two color analyses, so don't know what the consensus is for 
>handling logratios where the denominator is really close to zero. Since 
>you guys do this stuff all the time, perhaps you have some pointers?
>
>Best,
>
>Jim
>
>
>
>On 4/5/2011 6:43 PM, Wei Shi wrote:
> > Hi Seraya:
> >
> >       Genes which are present in one condition but not in the other 
> > should
>NOT be removed from your analysis. Only those gene which are absent in 
>both conditions should be filtered out to improve the power to detect 
>differentially expressed genes. It is very likely that a lot of genes 
>of biological interest were not included in your analysis results due 
>to the removal of genes which are present in one condition but not in the
other.
>Have a look at the case study for processing Illumina BeadChip data in 
>limma user guide about the probe filtering.
> >
> >       Hope this helps.
> >
> > Cheers,
> > Wei
> >
> > On Apr 6, 2011, at 2:05 AM, Seraya Maouche wrote:
> >
> >> Dear Prof Gordon, dear Bioconductor members,
> >>
> >> I have performed gene expression analysis using Limma (Illumina 
> >> human
> >> ref8) comparing two types of cells (referred below as cond1 and cond2).
> >> Based on detection call, I filtered out transcripts which are 
> >> absent in both types of cells. Transcripts which were expressed 
> >> only in one cell type were included in the analysis.
> >>
> >> I have received the comment below from a reviewer who seems not 
> >> agree to calculate fold change for genes expressed only in one
condition.
> >> Would it be possible to have your opinion about this.
> >>
> >> Thank you in advance for your time, S Maouche
> >>
> >> "There is a little conceptual difficulty related to the cond1/cond2 
> >> comparisons for genes that are considered not detected. If a gene 
> >> product is absent (0) in one cell then no fold change can be 
> >> computed (table 2). I don’t know how to circumvent this difficult 
> >> except by saying that the “noise” is considered to reflect low 
> >> expression. The terms “not detected” and “not expressed” are often 
> >> used interchangeably while this is not the same. Detection is based 
> >> on the definition adopted and in many places of the manuscript it 
> >> should be used in place of expression."
> >>
> >>
> >>
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>--
>James W. MacDonald, M.S.
>Biostatistician
>Douglas Lab
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