[BioC] assist in constructing a design matrix & contrast matrix for Illumina microarray

[Steve Lianoglou]

Howdy,

On Nov 11, 2009, at 10:43 AM, James W. MacDonald wrote:

> Hi Maria,
>
> Maria Dolores Serafica wrote:
>>   Dear Bioconductor Staff,
>>   I sent this email on Oct 27 & would like to know if it was  
>> received and if
>>   someone can help.
>>   I am analysing Illumina Beadchips (HumanWG6) for 42 SAMPLES.
>>   Experimental design: (factorial) time course, samples grown on 4
>>   media; have zero control sampled at onset only[ one way multigroup
>>   analysis]; 2-3 reps per treatment.
>>   I used the lumi script by Pan Du et al (2009) for preprocessing
>>   and normalisation . This part  was okay. The samples were subsetted
>>   and are R objects.
>>   SAMPLE 1 REFERENCE SAMPLE  DAY 0 (triplicate)
>>   SAMPLE 2 MEDIA A DAY 7 (triplicate)
>>   SAMPLE 3 MEDIA A DAY 14 (triplicate)
>>   SAMPLE 4 MEDIA A Day 30 (triplicate)
>>   SAMPLE 5 MEDIA A DAY60  (singleton)
>>   SAMPLE 6 MEDIA A DAY90 (replicates)
>>   SAMPLE 7 MEDIA B DAY 7 (triplicate)
>>   SAMPLE 8 MEDIA B DAY 14 (triplicate)
>>   SAMPLE 9 MEDIA B DAY 30 (triplicate)
>>   SAMPLE 10 MEDIA B DAY60 (singleton
>>   SAMPLE 11 MEDIA B DAY 90 (TRIPLICATE)
>>   SAMPLE 12 MEDIA C DAY 7 (REPLICATES)
>>   SAMPLE 13 MEDIA C DAY 14 (REPLICATES)
>>   SAMPLE 14 MEDIA C DAY 30 (REPLICATES)
>>   SAMPLE 15 MEDIA D DAY 7 (REPLICATES)
>>   SAMPLE 16 MEDIA D DAY 14 (REPLCATES)
>>   SAMPLE 17 MEDIA D DAY 30 (REPLICATES)
>>   SAMPLE 18 MEDIA D (DAY 90 (REPLICATES
>>   This is a differentiation experiment.
>>    I tried the lumi script using the modified limma (PanDu et al  
>> 2009) based
>>   on the Barnes subset data but the script does not apply to my  
>> experimental
>>   set up.  Can someone assist me in making a design matrix and a  
>> contrast
>>   matrix.
>>   I am new to R and would like to use limma for identifying  
>> differentially
>>   expressed genes.  I have read the limma users guide(Gordon  
>> Smyth), LAB4
>>   limma 2005, James MacDonald's BioC 2009.
>>   The contrasts (comparisons) I am looking for:
>>   a. differential gene expression of each sample WITH the reference
>>   b. early differentiation genes as affected by media, time and both
>>   c. late differentiation genes as affected by media or both.
>
> It's good that you have done some homework and are trying to learn  
> how to do the analysis, but in the end you are asking somebody to do  
> your work for you (for free), and it's not likely that you will get  
> many people who want to do that.

Or, at the very least, you can show us how you're trying to do what  
you're doing, and someone will likely comment on what's going awry.

To be honest, I have no idea what reference Pan Du (2009) is, and  
don't really have any desire to add that to my reading list, atm.

So, why don't you start with an easy task. Specifically, by reading  
the limma user guide, it *should* be pretty straight forward to  
achieve (a) in your goals: finding differential expression of a  
perterbation-sample vs. its reference is in there.

To be even more specific:

You should be able to find differential expression between your SAMPLE  
1 (reference triplicate) vs. SAMPLE 2 (media day 7 triplicate). I'm  
guessing your code will look very similar to section 8.2.

I recall at first I wasn't very comfortable using the  
limma::modelMatrix function, and I I would create my own design matrix  
by hand. Once you have that (with appropriate column names), building  
the contrast matrix should be easy rather straightforward.

So .. have fun with that. I'd imagine that might take a while (the  
limma guide is *big*), so I guess we'll get your follow up questions  
sometime tomorrow :-)

And, like I said, the more specific you are about what's troubling you  
for this specific task (getting differential expression between  
sample2 and sample1, for example), the greater the likelihood you'll  
get an informative answer.

Good luck,

-steve

--
Steve Lianoglou
Graduate Student: Computational Systems Biology
   |  Memorial Sloan-Kettering Cancer Center
   |  Weill Medical College of Cornell University
Contact Info: http://cbio.mskcc.org/~lianos/contact