Question
Asked 23rd Oct, 2014
  • 30M Genomics Private Limited

Can anyone explain how to calculate methylation level from bisulfite sequencing results?

I have followed all the standard protocols for bisulfite sequencing like cloning, etc. I got the sequence and I can identify whether the CpG is methylated or unmethylated but I don't know how to calculate the percentage of methylation from the sequence electrophoregram. How do I do this?

Most recent answer

26th Oct, 2016
David Langenberger
ecSeq Bioinformatics GmbH
DNA Methylation Data Analysis Workshop 
How to use bisulfite-treated sequencing to study DNA methylation
When?
22. - 25. November 2016
Where?
Leipzig, Germany
Link?
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Popular Answers (1)

23rd Oct, 2014
Nicholas Wagner
Heinrich-Heine-Universität Düsseldorf
Yes, you are right! You have to sequence many of the clones to calculate the level of methylation (At the very least 10 clones, better 20 or more).
Then you look at the same CpG position in all seqeunces. Methylated cytosines will stay "C". Unmethylated cytosines will have been converted to "T" by the bisufilte treatment. Count the number of "C"s and "T"s at one position, and you can calculate the level of methylation.
examples:.
If 10/10 nucleotides at this position are "C", then the DNA is fully (100%) methylated at this position.
If 3/10 nucleotides at this position are "C", then the DNA was 30% methylated at this position. This is also called "partial methylation".
If 0/10 nucleotides at this position are "C", then the DNA is unmethylated (0%) at this position. 
-----
To explain "partial methylation" (also called "differential methylation") a bit more:
One CpG on one chromosome can only be methylated or unmethylated, but not a mix. However, you have two copies of each chromosome in one cell, so one cell can be 0%, 50% or 100% methylated at one CpG.
When you isolate DNA, you have a DNA mix of many thousand cells. Depending on how many of the cells are unmethylated (0%), hemimethylated (50%) or fully methylated (100%) , you can get a level of methylation from 0 - 100%.
Because bisulfite sequencing has a relatively high error rate, samples between 0% and 10% are usually classified as "unmethylated", and samples between 90% and 100% are classified as "fully methylated". [But these thresholds are not fixed. Some groups use 0-5% and 95-100%, others use 0-20% and 80-100%.]
"partial methylation" refers to a state where the DNA is not fully methylated and not unmethylated, so in most cases partial methylation is between 10% and 90%. [or for the examples from other groups 5%- 95%  and 20%-80%]
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All Answers (10)

23rd Oct, 2014
Antonella Sangalli
University of Verona
I use QUMA (QUantification tool for Methylation Analysis) (http://quma.cdb.riken.jp/).You can easily align, visualize and quantify
bisulfite sequence data for CpG methylation analysis. It also performes statistical analyses!
6 Recommendations
23rd Oct, 2014
Benet Dhas
30M Genomics Private Limited
Thanks Sangalli. QUMA shows methylation levels compared to the nearby CpGs. Is it possible to calculate the methylation level for a single CpG from bisulfite sequence? If not, how much this method QUMA is reliable?
23rd Oct, 2014
Nicholas Wagner
Heinrich-Heine-Universität Düsseldorf
Could you be more specific on what you did, please? What did you clone and when? Did you use NGS or Sanger?
For now, it sounds to me like you did Sanger sequencing of bisulfite-treated DNA from many clones. The Sanger sequence for each clone should be a definite result: either methylated or unmethylated. There should not be a mixture of methylated and unmethylated signal in the electropherogram of one clone. If you can see a mixed signal, something most likely went wrong in the cloning procedure. In short: you should not calculate the methylation from the electropherogram.
Once you have counted the number of methylated and unmethylated clones, you then calculate the percentage of methylation. (e.g. 15 methylated clones and 5 unmethylated clones = 75 % methylation)
6 Recommendations
23rd Oct, 2014
Benet Dhas
30M Genomics Private Limited
Thanks Wagner. I had done bisulfite specific PCR, gel eluted the specific product and cloned it. I got more than one clone but I sanger sequenced only one clone. Do I have to sequence all the clones to find out level of methylation?
I am bit confused. A CpG can be a methylated one or unmethylated one. How can it be a partially methylated. If I am looking into a single CpG, is it possible that I get partial methylation? Kindly clarify the term PARTIAL METHYLATION
23rd Oct, 2014
Nicholas Wagner
Heinrich-Heine-Universität Düsseldorf
Yes, you are right! You have to sequence many of the clones to calculate the level of methylation (At the very least 10 clones, better 20 or more).
Then you look at the same CpG position in all seqeunces. Methylated cytosines will stay "C". Unmethylated cytosines will have been converted to "T" by the bisufilte treatment. Count the number of "C"s and "T"s at one position, and you can calculate the level of methylation.
examples:.
If 10/10 nucleotides at this position are "C", then the DNA is fully (100%) methylated at this position.
If 3/10 nucleotides at this position are "C", then the DNA was 30% methylated at this position. This is also called "partial methylation".
If 0/10 nucleotides at this position are "C", then the DNA is unmethylated (0%) at this position. 
-----
To explain "partial methylation" (also called "differential methylation") a bit more:
One CpG on one chromosome can only be methylated or unmethylated, but not a mix. However, you have two copies of each chromosome in one cell, so one cell can be 0%, 50% or 100% methylated at one CpG.
When you isolate DNA, you have a DNA mix of many thousand cells. Depending on how many of the cells are unmethylated (0%), hemimethylated (50%) or fully methylated (100%) , you can get a level of methylation from 0 - 100%.
Because bisulfite sequencing has a relatively high error rate, samples between 0% and 10% are usually classified as "unmethylated", and samples between 90% and 100% are classified as "fully methylated". [But these thresholds are not fixed. Some groups use 0-5% and 95-100%, others use 0-20% and 80-100%.]
"partial methylation" refers to a state where the DNA is not fully methylated and not unmethylated, so in most cases partial methylation is between 10% and 90%. [or for the examples from other groups 5%- 95%  and 20%-80%]
27 Recommendations
28th Oct, 2014
Benet Dhas
30M Genomics Private Limited
Thank you so much Wanger for your detailed explanation.
28th Oct, 2014
Nicholas Wagner
Heinrich-Heine-Universität Düsseldorf
You are welcome :) Please upvote if the answer was helpful ;-)
4 Recommendations
4th Dec, 2015
Andrew Nkurunungi
Alabama A & M University
You can use getMethylationStats() function from https://code.google.com/p/methylkit/
Very easy to use.
8th Apr, 2016
Mary Chuah
Hi, has anyone tried Direct Bisulfite sequencing with Sanger without cloning and measuring peak heights of C's and T's at CpG to obtain percentage methylation? Any comments on the realibility of this method?
Thanks
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