Hi- I am new to analyzing Illumina microarray data. We ran 6 case and 6 control samples (no replicates) on 1 Illumina HT-12 v3 Expression BeadChip. I would like to analyze our data using lumi. I was planning on doing the following: 1) background subtraction in Beadstudio, 2) VST in lumi, and 3) quantile normalization in lumi. I noticed that the normalization methods in lumi are for between chip normalization. Would it still be appropriate to use quantile normalization in my situation since I only have one chip (ie: between array normalization)? Also, will the VST method yield similar results if gene-level data are used instead of probe-level data? Thanks for your time, Christina Markunas cam27@duke.edu [[alternative HTML version deleted]]

HI Christina, I don't bother with beadstudio background subtraction, and have in found that background subtraction in beadstudio or other background subtraction algorithms on illumine chips make little or no difference (in the experiments I have tested it on). But it won't hurt if you do it. Dump unnormalised data from beadstudio, most use the probe profile and not the gene profile. Proceed as you have described. I don't think the main issue is with VST and gene/probe profile; it is if you want to mix together potential splice variants together and call them a "gene" (or perhaps mix crappy probes and good probes together and call the average better). This is why most recommend using the probe profile but the results will probably be very similar either way. Yes you absolutely have to do normalization : treat each of the 12 separate arrays on the chip as being different (which they truly are). Normalization is needed to correct for more than just technical scanning artifacts that *may* be reduced if the arrays are on the same piece of silicon. You will find PLENTY of variation in the arrays on the same chip that normalization will be needed for! Sound like you're on the right track follow the lumi pdf with the default parameters and you should get something quite reasonable. Cheers Paul -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of Christina Markunas Sent: Wednesday, April 01, 2009 11:50 AM To: bioconductor at stat.math.ethz.ch Subject: [BioC] Lumi normalization and VST help Hi- I am new to analyzing Illumina microarray data. We ran 6 case and 6 control samples (no replicates) on 1 Illumina HT-12 v3 Expression BeadChip. I would like to analyze our data using lumi. I was planning on doing the following: 1) background subtraction in Beadstudio, 2) VST in lumi, and 3) quantile normalization in lumi. I noticed that the normalization methods in lumi are for between chip normalization. Would it still be appropriate to use quantile normalization in my situation since I only have one chip (ie: between array normalization)? Also, will the VST method yield similar results if gene-level data are used instead of probe-level data? Thanks for your time, Christina Markunas cam27 at duke.edu [[alternative HTML version deleted]] _______________________________________________ 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