Hi everyone, I am confused by the histogram of normalized Agilent microarray data. It is human single color array, containing around 700 microarrays and 43K probes. After normalization, I plotted the express value of all probes in single microarray, one example is attached. I expected to see a more or less symmetric distribution, however, the values seems truncated. In the beginning I thought it may relate to offset value, but I have tried different value 16, 1, 0, still got similar distribution. Any explanation or suggestions? Here are codes for normalization: library(limma) targets <- readTargets("targets.txt") x <- read.maimages(targets, source="agilent",green.only=TRUE) y.bg <- backgroundCorrect(x, method="normexp") y.bgn <- normalizeBetweenArraysy.bg, method="quantile") g.ex <- avereps(y.bgn, ID=y.bgn$genes$ProbeName) da.norm <- g.ex$E Here are R session: R version 3.0.2 (2013-09-25) Platform: x86_64-apple-darwin10.8.0 (64-bit) locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] graphics grDevices utils datasets stats methods base other attached packages: [1] ggplot2_0.9.3.1 reshape2_1.2.2 plyr_1.8 loaded via a namespace (and not attached): [1] colorspace_1.2-4 dichromat_2.0-0 digest_0.6.3 grid_3.0.2 gtable_0.1.2 labeling_0.2 [7] MASS_7.3-29 munsell_0.4.2 proto_0.3-10 RColorBrewer_1.0-5 scales_0.2.3 stringr_0.6.2 Best, chunxuan -------------- next part -------------- A non-text attachment was scrubbed... Name: Rplot.pdf Type: application/pdf Size: 83343 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20131110="" 0db4eae9="" attachment.pdf="">