[BioC] lumi for Illumina methylation data - understanding the colour adjustment

Lavinia Gordon lavinia.gordon at mcri.edu.au
Tue Dec 21 05:20:41 CET 2010

Hi Pan

Thank you for your reply, very prompt at this busy time of year!
I think I am getting confused with how the HumanMethylation27 array 
works, or perhaps I don't understand the colour adjustment properly.
My query is really, if the methylated and unmethylated are the same 
colour, and that colour is adjusted (so I assume Cy3 is adjusted 
differently to Cy5, as Cy5 is incorporated differently), why are the 
intensities of the methylated adjusted differently to the intensities of 
the unmethylated if they are the same colour?

Many thanks,


On 21/12/2010 2:56 PM, Pan Du wrote:
> Hi Lavinia
> It is hard to answer your question without knowing the quality of your data.
> My suggestion is performing visually color bias check first before applying
> color bias adjustment. If the color bias is not severe, then only perform
> conservative adjustment (scaling and shift adjust) or not perform any color
> adjustment at all. Need to know the smooth quantile color adjustment has
> strong assumption of the data (same distribution of two color channels).
> Quantile normalization may bring bias after adjustment, this is the same as
> the expression microarray normalization. If you would like to, please send
> me the plot produced by plotColorBias2D of this sample. Also, I will add
> more detailed description of this in the vignette.
> Thanks for reporting this!
> Pan
> On 12/20/10 7:06 PM, "Lavinia Gordon"<lavinia.gordon at mcri.edu.au>  wrote:
>> Dear Dr Du
>> I am a big fan of /lumi/ and was delighted to see that you have made it
>> compatible with methylation arrays.  I have used these new functions on
>> several of my datasets and am very happy with the alternative method of
>> working with M values.  I just have one query regarding the colour
>> adjustment.
>> So, for example, probe A is a red probe, and has a (GenomeStudio)
>> unmethylated intensity of 2205 and a methylated intensity of 2822.
>> 2822/(2822+2205)
>> beta = 0.5613686
>> After colour adjustment, it has an unmethylated intensity of 1718.882
>> and a methylated intensity of 2576.6539:
>> 2576.6539/(2576.6539+1718.882)
>> beta = 0.5998446
>> Why, if the methylated is the same colour as the unmethylated, has the
>> unmethylated intensity decreased by 23% but the methylated by only 9%?
>> with thanks for your time,
>> Lavinia Gordon.

Senior Bioinformatics Officer
Murdoch Childrens Research Institute
Royal Children's Hospital
Flemington Road
Victoria 3052

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