Question: Bismark Methyl-Seq Analysis
0
gravatar for mosharrofgeb
3 months ago by
mosharrofgeb30
mosharrofgeb30 wrote:

We have sequenced a genome using Illumina's True-seq bisulfite sequencing kit. After getting back the seq, we are analyzing methylation rate using Bismark. I Need help with the interpretation of the result and proper way of normalization.

Before sequencing: Sample DNA was divided into 2 groups: 1. Bisulfite treatment was carried out and DNA was subsequently sequenced (group 1, methylated group) 2. DNA was sequenced without bisulfite treatment (group 2, control group)

Both group was sequenced in paired-end fashion.

I am using Bismark to analyze the seq and trying to get the methylation rate in this particular genome. After running Bismark on Methylated files I got this finale percentages:

C methylated in CpG context: 0.6%

C methylated in CHG context: 0.5%

C methylated in CHH context: 0.7%

Whereas after running Bismark on my Control files I got these percentages:

C methylated in CpG context: 99.6%

C methylated in CHG context: 99.3%

C methylated in CHH context: 99.9%

So, how would I interpret my data?

a. Is 0.6 % (CpG) the actual methylation percentage in my genome?

b. I have found in some literatures that if CpG, CHG, and CHH percentages are very close, that means that genome actually does not do methylation. Is it true?

c. What was the purpose of using the control group (group 2)? Do I still need any spike-in control to normalize the data? If so, what that could be?

Thank you very much for reading this long post!!

Bests!!!

ADD COMMENTlink modified 3 months ago by mark.ziemann900 • written 3 months ago by mosharrofgeb30

what are your samples?

ADD REPLYlink written 3 months ago by Santosh Anand3.0k

Genomic DNA sequenced in miseq!

ADD REPLYlink written 3 months ago by mosharrofgeb30
1
gravatar for mark.ziemann
3 months ago by
mark.ziemann900
Australia/Mebourne/Monash University
mark.ziemann900 wrote:

Those bismark results would suggest that

  • Informatics pipeline is working well (result from group 2)
  • Bisulfite conversion is working well (result from group 1 CHG/CHH)
    • There is negligible CpG methylation (result from group 1 CpG)

The 0.6% methylation percent is too close to the Illumina error rate (1/200) to be very meaningful. There is probably no cytosine methylation in your sample. But perhaps there are a few specific sites with higher levels. An orthogonal method could be used to be certain.

ADD COMMENTlink written 3 months ago by mark.ziemann900

Ya, that actually makes very sense. Thanks for pointing this out...

ADD REPLYlink written 3 months ago by mosharrofgeb30
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