[BioC] Limma

Wed Apr 6 16:52:51 CEST 2011

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."
> >>
> >>
> >>
> >> Universitätsklinikum Schleswig-Holstein Rechtsfähige Anstalt des
> >> öffentlichen Rechts der Christian-Albrechts-Universität zu Kiel und
> >> der Universität zu Lübeck
> >>
> >> Vorstandsmitglieder: Prof. Dr. Jens Scholz (Vorsitzender), Peter
> >> Pansegrau, Christa Meyer Vorsitzende des Aufsichtsrates: Dr. Cordelia
> >> Andreßen
> >> Bankverbindungen: Förde Sparkasse BLZ 210 501 70 Kto.-Nr. 100 206,
> >> Commerzbank AG BLZ 230 800 40 Kto.-Nr. 300 041 200
> >>
> >> Diese E-Mail enthält vertrauliche Informationen und ist nur für die
> >> Personen bestimmt, an welche sie gerichtet ist. Sollten Sie nicht der
> >> bestimmungsgemäße Empfänger sein, bitten wir Sie, uns hiervon
> >> unverzüglich zu unterrichten und die E-Mail zu vernichten.
> >> Wir weisen darauf hin, dass der Gebrauch und die Weiterleitung einer
> >> nicht bestimmungsgemäß empfangenen E-Mail und ihres Inhalts
> >> gesetzlich verboten sind und ggf. Schadensersatzansprüche auslösen
>können.
> >> _______________________________________________
> >> 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
> >
> >
> > ______________________________________________________________________
> > The information in this email is confidential and
> > intend...{{dropped:6}}
> >
> > _______________________________________________
> > 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
>Douglas Lab
>University of Michigan
>Department of Human Genetics
>5912 Buhl
>1241 E. Catherine St.
>Ann Arbor MI 48109-5618
>734-615-7826
>**********************************************************
>Electronic Mail is not secure, may not be read every day, and should not be
>used for urgent or sensitive issues
>
>_______________________________________________
>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