Hi Levi Thanks for the quick feedback. I am still following up on few of the things that you recommended. I checked with the collab and these were not FFPE specimens. Also I did a quick signal to noise ratio calculation (95% / 5% intensity quantiles ). I am attaching the plot. May be that indicates something for the Illumina bead array platform that I am not aware of. To me it looks skewed. Cheers! -Abhi On Thu, Jan 23, 2014 at 4:26 PM, Levi Waldron <levi.waldron at="" hunter.cuny.edu=""> wrote: > Dear Abhi, > > it looks to me like your chips had low hybridization, based both on the low > percentage of probes detected and on the low interquartile range of > intensities. Fig. 2 from PMID23136189 (admittedly my publication) shows the > range of IQRs for samples within several published studies using Illumina > BeadArrays for FFPE tissues. The normal IQR is around 2, which corresponds > to around 50-60% present at p<0.05. Your typical IQR is around 1, and I > can't directly read percent present but it looks around 20-30%. I would be > concerned, and do some additional checks: 1) if you have technical > replicates, check them by MA plot and sample-wise correlations, 2) MA plots > against the median pseudochip, 3) check for detection and high intensity of > the positive control probes (labelled CY3_HYB and HOUSEKEEPING on Illumina > chips I've looked at), and 4) run the ffpe::sortedIqrPlot() function to see > whether IQR or percent present are related to correlation to median > pseudochip in your experiment. Are you using FFPE specimens? > > Sincerely, > Levi > > > > On Thu, Jan 23, 2014 at 6:38 PM, Abhishek Pratap <apratap at="" sagebase.org=""> > wrote: >> >> Hi All >> >> I am trying to analyze some mRNA illumina bead level data through bead >> array. Based on the detection p-values(plot attached): if I read it >> right significant # probes >> have no expression which concerns me but I could have easily missed >> some important step. >> >> I am attaching the two box plots 1.) intensity and 2.) probe level >> detection p-values + code >> >> Let me know your opinion. >> >> Thanks! >> -Abhi >> >> beadlevel_data <- readIllumina(dir=dataDir, useImages=F, >> illuminaAnnotation="Humanv4") >> >> #intensity boxplot >> boxplot(beadlevel_data, transFun = logGreenChannelTransform, col = >> "green", >> ylab = expression(log[2](intensity)), las=2, outline=FALSE, >> main= "Array intensities") >> >> #summarize : detection p-val boxplot >> BSData <- summarize(beadlevel_data) >> det = calculateDetection(BSData) >> boxplot(det, main='dset1_mRNA_probe_detection_pvals (Null: there is no >> expression) ', ylab='p-value(detection score)',xaxt='n', >> xlab='individual samples/array' ) >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at r-project.org >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > > -- > Levi Waldron > Assistant Professor of Biostatistics > City University of New York School of Public Health, Hunter College > 2180 3rd Ave Rm 538 > New York NY 10035-4003 > phone: 212-396-7747 -------------- next part -------------- A non-text attachment was scrubbed... Name: dset1_mRNA_array_singal_to_noise_ratio.png Type: image/png Size: 46883 bytes Desc: not available URL: <https: stat.ethz.ch="" pipermail="" bioconductor="" attachments="" 20140124="" f5a225b1="" attachment.png="">