Hi James

On Mon, Feb 28, 2011 at 17:42, James W. MacDonald <jmacdon@med.umich.edu>wrote:

> Hi Assa,
>
>
> On 2/28/2011 4:30 AM, Assa Yeroslaviz wrote:
>
>> Hi BioC users,
>>
>> I'm working on a set of affy chips for miRNA. I would like to run some
>> quality control tests but none of the functions I am trying seems to work.
>> I have tried the AffyQCReport, affycoretools and simpleaffy. But every
>> time
>> I am getting the error message that my qcdef file is not there.
>> I downloaded the files from affymetrix and rename the miRNA-1_0_2Xgain.CDF
>> into mirna102xgaincdf.
>>
>
> Not sure what you mean here. Do you in fact mean that you used biocLite()
> to get the correct cdf package for this chip? Which is what you should be
> saying. There is almost never a reason for you to be getting the 'raw' cdf
> from Affy.


Yes you are right. It didn't help much renaming the file, so I tried to do
it according to the instructions in the simpleaffy package.
Unfortunately it is not exactly easy to follow them. It doesn't say much
about how to create a qcdef file.

I download from affymetrix the information I could find and tried to create
a  mirna102xgaincdf.qcdef fie.
This is what I came up with:

array mirna102xgaincdf
alpha1 0.05
alpha2 0.065
spk bioB AFFX-BioB-3_at
spk bioC AFFX-BioC-3_at
spk bioD AFFX-BioDn-3_at
spk creX AFFX-CreX-3_at

I didn't change the alpha values as I don't know what they exactly means.
The spike-ins are the same as in other arrays. My problem begins with the
ratios. There are no house keeping genes on the array, so I don't know what
to take.
This is what Affymetrix has to say about the control probes on these arrays:

Control target content
95 GC-binned background probe sets                     ≤ 94 probes/set
54 BioB, C, D, Cre r2 hybridization control probe sets 11 probes/set
9 BioB, C, D, Cre hybridization control probe sets     20 probes/set
22 oligonucleotide spike-in control probe sets         10 identical
probes/set (i.e., each probe is tiled 10 times)
10 identical probe sets for human 5.8s rRNA(gi555853)  11 probes/set

But the qcdef file can have only spk  for spik-ins and ratio for ratios of
gene pairs. I am not exactly sure, whether it makes sense to add the cre,
bioc,d,b as ratios to the qcdef file, something like that:
AFFX-r2-Ec-c1-bioB-3_at/AFFX-r2-Ec-c1-bioB-5_at
AFFX-r2-Ec-c1-bioC-3_at/AFFX-r2-Ec-c1-bioC-5_at
AFFX-r2-Ec-c1-bioD-3_at/AFFX-r2-Ec-c1-bioD-5_at

I will try this one and will tell what happens

The array has extra probes with the names spike_in
405            spike_in-control-17_st            Unknown

406            spike_in-control-1_st            Unknown

407            spike_in-control-21_st            Unknown

408            spike_in-control-23_st            Unknown

409            spike_in-control-27_st            Unknown

410            spike_in-control-28_st            Unknown

411            spike_in-control-29_st            Unknown
...
426            spike_in-control-8_st            Unknown


Can it be that these suppose to be the spike ins for the array and the cre,
biob-d are instead of the house keeping genes?


>
>  than I did
>>
>>> setQCEnvironment("mirna102xgaincdf", path=getwd())
>>>
>>
>> which apperently works just fine, but:
>>
>>> QCReport(rawData)
>>>
>> Error in setQCEnvironment(cdfn) :
>>   Could not find array definition file ' mirna102xgaincdf.qcdef '.
>> Simpleaffy does not know the QC parameters for this array type.
>> See the package vignette for details about how to specify QC parameters
>> manually.
>>
>
> Right. Which means you should read the package vignette, where they give
> some indication what to do to specify QC parameters. Did you?
>
> This does raise a bit of a problem, as I don't see any actin or GAPDH
> probes on this chip. Perhaps you can get away with just specifying something
> for the spike-in controls. Or maybe not. You will have to try and see what
> happens.


yes I will try it

>
>
>
>  In addition: Warning message:
>> In data.row.names(row.names, rowsi, i) :
>>   some row.names duplicated:
>>
>> 3,4,8,9,10,14,15,16,20,21,22,23,28,29,30,31,35,36,37,38,43,44,45,49,50,51,52,56,57,58,62,63,64,68,69,70,75,76,77,81,82,83,84,88,89,90,94,95,96,97,101,102,103,107,
>>
>> 108,109,113,114,115,120,121,122,126,127,128,132,133,134,135,139,140,141,145,146,147,152,153,154,158,159,160,164,165,166,167,172,173,174,196,197,198,199,200,201,207,208,209,210,211,217,
>>
>> 218,219,220,221,227,228,229,230,231,237,238,239,240,241,245,246,247,253,254,255,256,257,258,264,265,266,267,268,274,275,276,277,278,284,285,286,287,288,294,295,296,297,298,304,305,306,
>>
>> 307,308,314,315,316,317,318,324,325,326,327,328,335,336,337,338,339,345,346,347,348,349,355,356,357,358,359,365,366,367,368,369,375,376,377,378,379,385,386,387,388,389,396,397,398,399,
>>
>> 400,404,405,406,407,413,414,415,416,417,424,425,426,427,428,434,435,436,437,438,444,445,446,447,448,454,455,456,457,458,464,465,466,467,468,474,475,476,477,478,485,486,487,488,489,495,
>>
>> 496,497,498,499,505,506,507,508,509,515,516,517,518,519,525,526,527,528,529
>> [... truncated]
>>
>> I would like to know whether there are some specific qc tools or
>> R-packages
>> for the miRNA from Affymetrix (the older version of the arrays).
>>
>
> I don't know of any miRNA-specific qc packages. But many of the functions
> in simpleaffy, AffyQCReport, and arrayQualityMetrics should work. However,
> you might need to pick and choose what to run rather than simply running the
> main wrapper function, as many of the functions are likely not to work for
> this chip.
>

Unfortunately they ask for a qcdef file.


>
> Best,
>
> Jim
>
>
>
>> I did an MA plot of the arrays and they looks fine to me, but I would
>> still
>> like to sun some more tests.
>>
>> THX,
>> Assa
>>
>>        [[alternative HTML version deleted]]
>>
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>>
>
> --
> James W. MacDonald, M.S.
> Biostatistician
> Douglas Lab
> University of Michigan
> Department of Human Genetics
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