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.

Hi Valerie,

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.

Krümel

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.
Valerie

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.

Krümel

Valerie,

sequence scanner from ABI will also suit your needs and it's free.

Thanks for the reference Krumelmonster!