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@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@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

	[[alternative HTML version deleted]]

