[BioC] Analysis of many Flagged spots

[Davide Valentini]

Sorry for the delay in my reply, But I haven't noticed you message 
before ! Thanks a lot for your suggestions.
I submitted your indications to the bio lab, Me too, I was wondering why 
so many flags, we are discussing the situation with the group.
Thanks a lot

Davide

J.delasHeras at ed.ac.uk wrote:
> 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
>
>   


-- 
Davide Valentini
PhD - Biostatistician
Department of Medical Epidemiology and Biostatistic
Karolinska Institute
Box 281
SE-171 77 Stockholm
SWEDEN
Tel: +46-8-524 82294
Fax: +46-8-31  4975