[BioC] beadarray: mRNA QC help

[Abhishek Pratap]

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' )
>>
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>
>
>
>
> --
> 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
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