meaning of "total otherC considered"?

[raj...@gmail.com]

Hi, I ran methylKit in R to read in a couple of sorted SAM files, and I am getting output of this sort in the console:

calling for metylation percentage per base for sample: test  

3773531 3770164

total otherC considered (>95% C+T): 7543695

average conversion rate = 97.8812569207141

total otherC considered (Forward) (>95% C+T): 3773531

average conversion rate (Forward) = 97.8811194326097

total otherC considered (Reverse) (>95% C+T): 3770164

average conversion rate (Reverse) = 97.8813945316043

Reading methylation percentage per base for sample: test 

I don't understand the meaning of "total otherC considered", and how the average conversion rate is calculated.

Any help in correctly interpreting these numbers would be much appreciated,

Best,

Rajat

Postdoc, Ontario Cancer Institute

[Altuna Akalin]

OtherC refers to the non-CpG Cs. Converstion rate is calculated as number of Ts(non-methylated Cytosines) divided by coverage for each non-CpG cytosine. And the average conversion rate is the average of those values. For a cytosine to be included in this calculation it has to have at least 10X  coverage and the bases generating that coverage should have at least 20 phred score (if you haven't changed the default arguments).  If you believe non-CG methylation is prevalent in your organism and/or tissue you need to do spike-in controls to reliably measure the conversion rate. 

Best,

Altuna

[raj...@gmail.com]

Hi Altuna,

I wanted to follow up on the question of calculating the average conversion rate a while ago.  So is it the case that methylKit is not using CpG cytosines to get the rate?  If so, why not, i.e., why pick only non-CpG cytosines?

Thank you,

Rajat

[Kalyan K Pasumarthy]

Here are few points to consider why non-CpGs are important in cacluating conversion:

1. In a human genome, CpGs are the ones that get methylated (non CpG methylation is an extremely rare event).
   
2. Methylation analysis involves Bisulfite reaction, that converts unmethylated Cs into U and methylated Cs remain Cs

In a bisulfite reaction, we intend to convert all the unmethylated Cs into Us. So, if we take CpGs for conversion ratio, we will always get a low bisulfite conversion efficiency (because a large number of them are already methylated and they remain as C). If we consider nonCpGs, we will get a better conversion efficiency.

Regards,
Kalyan

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[raj...@gmail.com]

Thank you Kalyan, that makes sense now!  Rajat

Regards,
Kalyan

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