[BioC] beadarray: mRNA QC help

[Abhishek Pratap]

Just dropping in a gentle reminder if my last update was missed over
the weekend.

-Abhi

On Fri, Jan 24, 2014 at 3:10 PM, Abhishek Pratap <apratap at sagebase.org> wrote:
> 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