[BioC] Analysis of many Flagged spots

[J.delasHeras at ed.ac.uk]

Quoting Davide Valentini <Davide.Valentini at ki.se>:

>
>
> Hi to all,
>
> I've to deal with a dataset that has a huge amount of flagged as "bad"
> spots. My data are from peptide microarrays and the proportion of flags
> is around the 90% in each slide. Luckily I have a good set of samples
> (35 cases and 35 controls), but I'm not an expert with this kind of
> problem. Should I treat the flagged data as missing values and so try to
> impute new values instead the flagged "bad" spots ? I know the KNN
> imputation or the SVD imputation.
> What is normally done with the spots flagged as "bad" (-100, following
> GenePix criteria + additional criteria) in cDNA experiments ?
>
> Sorry if the question looks banal, but as I said I'm not an expert on
> this field. Any help is useful, also links regarding flagged data
> analysis...
>
> Thanks in advance,
>
> Davide


Hi Davide,

when looking at the actual images, are the high number of "bad" spots  
indicating artifacts on the slides (dirt, scratches, high and uneven  
background...) or just a reflection of your probes lighting up just a  
small proportion of the spots?
Genepix flags as "Not Found" spots where there's no signal, but if the  
background is a little high and uneven, it sometimes will try hard to  
find a spot, and find a group of pixels to call a spot, which then  
often fails other quality criteria (shape-related) and teh spot is  
flagged as "Bad" rather than "Not Found". I think you should look at  
the scanned images to get a very good idea of what's going on.

Regarding what to do with flagged spots... I tend to disregard GenePix  
flags. Ocassionally there'll be some dust or a bubble affecting the  
signal on a few spots in a given array. I just proceed, relying on the  
fact that I have replicates and that I hope I won't get two specs of  
dust on two arrays on teh exact same place. If a given array concerns  
me, and I need to use it in my analysis, I may then create some  
weights (like the Genepix flags) to exclude particular spots from a  
given slide. I do my analyses using the Limma package, which allows  
you to use weights at different levels.
I find it useful to remove all spots that have negligible signal in  
both channels (2-colour data) on ALL arrays. In my latest experiments  
that amounts to up to 30% of the spots. I remove them completely. If  
you have a large % of spots in your arrays that won't light up with  
your samples, it may be a good idea to identify them and remove them  
from the analysis... If you end up having only a relatively small  
number of spots you will have to be extra careful about your  
normalisation procedure... but that's another matter.

 From what you describe, I would want to know first of all why I am  
getting so many "bad" spots: is there a problem with the  
hybridisation/slides, or do my samples only light up a small % of spots?

Jose

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