[BioC] help: limma and changing gene results!

[Koen Marien]

Thanks for the clear and fast reply, Jim. Indeed a-(b+c+d) isn't a contrast,
but I think I'm having a different problem. Here is the experiment shortly
explained:
 
I have four populations of cells with three biological replicates for each
population -> a1,a2,a3,b1,b2,b3,c1,c2,c3,d1,d2,d3. I normalized them and
looked at the differentially expressed genes between the 'a' population and
each of those other populations individually: a-b, a-c, a-d. The venn
approach is done with the online web application Venny and only looks at the
common probe set ID's in the three lists (let's call it the 'one-on-one
strategy').
I also looked at the differentially expressed genes when b, c and d values
where put together: a-e with e=b+c+d (let's call it the 'group strategy').
So it's not really the contrasts that are changed.

Now, when looking at the one-on-one strategy list there are only five genes
common in the three groups with a B-value > 2, while in the group strategy
there are 181 probe sets with a B-value > 2.

Relevent code used:
read all the cell files (a,b,c,d)
pd<-data.frame(population=c(rep(1,3),rep(2,8)),replicate=c(seq(1,3),seq(1,8)
)) => group strategy
or
only read the .cel files of two populations (a,b or a,c or a,d)
pd<-data.frame(population=c(rep(1,3),rep(2,3)),replicate=c(seq(1,3),seq(1,3)
)) => one-on-one strategy (repeated three times for each comparison)

group<-factor(eset$population)
design = model.matrix(~0+group)
design
cont.matrix = makeContrasts(eset = (group2 - group1), levels = design)
cont.matrix


Regards

Koen

-----Original Message-----
From: James MacDonald [mailto:jmacdon at med.umich.edu] 
Sent: woensdag 12 mei 2010 4:40
To: Koen Marien
Cc: 'Joseph Skaf'; bioconductor at stat.math.ethz.ch
Subject: Re: [BioC] help: limma and changing gene results!

Hi Koen,

Koen Marien wrote:
> Dear
> 
> 
> Is this also the reason why there is a difference in the (differentially
> expressed) gene lists of a-(b+c+d) and venny(a-b,a-c,a-d)?

I am not familiar with the venny() function, so it's hard to say. But if 
you mean a contrast of a-(b+c+d) versus individual contrasts of a-b, 
a-c, a-d, then no.

In the first place, a-(b+c+d) isn't a contrast, and in most cases 
doesn't make sense. You might mean a-(b+c+d)/3, which is a contrast, and 
tests the difference between the a group and the mean of the other 
three. The denominator will be the same in each case, being based on (in 
simple terms) the average variability of the four groups.

However, if what I am assuming is correct, then the two contrasts are 
quite different, and shouldn't be expected to result in the same gene 
lists. As an example, say the mean of the groups for one gene are:

a = 5
b = 2
c = 5
d = 8

since the denominator will be the same we can ignore that here. So do 
you think there will be a difference in what is called significant when 
we compare

5 - (2+5+8)/3 = 0

and

5 - 2 = 3
5 - 5 = 0
5 - 8 = -3

?

Best,

Jim


> 
> a-(b+c+d): 				putting the b, c and d values in one
> group (b+c+d) and using limma
> venny(a-b,a-c,a-d): 		using limma on the separate groups and
> create a list by looking at the intersection of de venn diagram of the
three
> 
> 					'sublists' a-b, a-c, a-d
> 
> 
> Thanks a lot
> 
> 
> Koen Marien
> student bioscience engineering: cell and gene biotechnology
> University of Ghent, Belgium
> 
> 
> -----Original Message-----
> From: bioconductor-bounces at stat.math.ethz.ch
> [mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of James W.
> MacDonald
> Sent: donderdag 29 april 2010 18:46
> To: Joseph Skaf
> Cc: bioconductor at stat.math.ethz.ch
> Subject: Re: [BioC] help: limma and changing gene results!
> 
> Hi Joseph,
> 
> Joseph Skaf wrote:
>> To whom it may concern,
>>
>> I've been having some problems with consistency in my limma results for
>> genes that are found to have significant differential transcript
> abundance.
>> In a given example, I may have 4 different groups (a, b, c, and d) in an
>> array set of 12.
>>
>> From here, I make a contrast matrix that has contrasts for a-b, a-c, and
>> a-d.  Eventually, I output an eBaye's corrected contrast fit and I use
>> decideTests from there to find out what genes are differentially
> expressed.
>> My misunderstanding is that when I take away an entire group (such as
>> removing all d's) and redo all steps in the limma analysis, I find that I
>> end up with a different set of genes after using decideTests.  I am
> confused
>> here, because I would not think that removing group 'd' from the analysis
>> would have an effect on contrasts a-b and a-c.
>>
>> If anyone could even hint to me a reason as to why this is happening, it
>> would be greatly appreciated.
> 
> It's because of how the denominator for your contrast is computed. The 
> denominator is computed using the intra-group variance for all the 
> groups in your study, not just the two groups being compared in the 
> contrast.
> 
> So if you remove one of the groups, you lose both degrees of freedom as 
> well as the contribution from the intra-group variance of that group. 
> Losing the degrees of freedom will reduce your power to detect 
> differences. Losing the contribution of the intra-group variance will 
> depend on how variable the group d data are compared to groups a-c.
> 
> Best,
> 
> Jim
> 
> 
> 
>> Thanks and regards,
>> Joseph Skaf
>>
>>
>>
> 

-- 
James W. MacDonald, M.S.
Biostatistician
Douglas Lab
5912 Buhl
1241 E. Catherine St.
Ann Arbor MI 48109-5618
734-615-7826
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