Scoring CpG methylation by peak height - (Dec/04/2006 )
I am getting nice sequences on BSP products and I am wondering what is the best way to score the peak height (I am doing it by eye so far). I have seen previously on this forum someone scoring with a score ranging from 0 to 4 (0=no methylation, 1=<25%.......and 4=75%, the way I understand is that when the C peak is about 75% as high as the T peak, then it is 4, am I right ?);
But I am wondering how to score when both peaks are the same height, or when the C peak is higher than the T peak. Is is relevant to have scores ranging from 0 to 8 ? And is it relevant to do it buy eye (I don't have any software to do that).
Thanks in advance for your help.
to calculate the amount of methylation, I do the following:
(1) Measure the peaks of C and T - I do it with the Sequencing Analysis Software
(2) Calculate %methylated=C/C+T
(3) Score your calculated value according to the scheme.
75% methylation would mean the C peak being three times higher than the T peak!
The scoring is needed to account for all the variabilities from the PCR reactions etc.
Thanks Krümel for your fast reply,
I understand now the relation between the score and the % methylation.
Is it possible to download Sequencing Analysis for free ? I have Sequence Scanner from Applied Biosystems, but I cannot find how the get peak height values, unless by pointing the peak with the mouse which gives a x and y value. I am not sure it is precise enough.
Well, I also use ABI Software (DNA sequencing analysis 5.1) - I don't know, if you can get more precise applications, but as you score your raw values afterwards, I think that the y-value serves well for our purpose.
In the paper by Han et al (PubMed-ID: 16797472) they compare direct sequencing with cloning and sequencing results (also incorporating standards - mixture of unmethylated and methylated DNA) and found identical results, using the raw-values! They also measured peak heigths and generated a percent value - so, I think this should be okay.
sequence scanner from ABI will also suit your needs and it's free.
Thanks for the reference Krumelmonster!