Dear Ina, It is my understanding that the intensities stored in the txt files have undergone 'local' background correction. This is a standard part of Illumina's image processing (i.e. not optional if you are going to use the intensities stored in the txt files by specifying useImages=FALSE). This should not be confused with the 'global' background correction step carried out in BeadStudio which makes use of the negative control probes. This occurs after the data has been summarised and is optional. We have found that the Illumina's default image processing steps (which includes local background correction) perform pretty well on spike-in data (see Dunning et al, BMC Bioinformatics 2008, 9:85 http://www.ncbi.nlm.nih.gov/pubmed/18254947), so I'd expect that the analysis you propose (with useImages=FALSE, and backgroundMethod="normexp") should give good results. In principle, running your analysis with useImages=FALSE, backgroundMethod="normexp" should get you to the same place as if you used useImages=TRUE and backgroundMethod="normexp". Note that the values stored in the txt files are subject to rounding, so in practice the intensities will be off by a little bit. I hope this clarifies things for you. Best wishes, Matt > Matt, > when I work with the Grn values from the .txt files (i.e. I read the data > into beadarray with useImages=FALSE), everything looks good. Only when I read > from the .tif files (useImages=TRUE), then I have a problem. What I am unclear > about it whether the Grn values in the .txt files are foreground intensities > or already background corrected. I posed this question to our Illumina > representative but his answer is not clear to me (see below). > "The value "Grn" is in fact the foreground signal intensity of that > particular bead. There has not been a global background subtraction > applied at this step. There is a local pixel level correction that is > done to generate this bead level intensity value, but this image > pre-processing cannot be avoided." > > So I am wondering whether I can simply work with the Grn values from the .txt > files and still perform background subtraction, using useImages=FALSE and > backgroundMethod=normexp? I.e., should this produce the same result as setting > useImages=TRUE and backgroundMethod=normexp? > > Thanks, Ina