Hi, Just adding to Steve's comments that the limma userguide in the latest release ( Oct. 28) has a case study for processing Illumina BeadChip data now. Cheers, Wei Steve Lianoglou wrote: > 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 > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor