Methlydacel methylation bias weird output - methylation almost 100% with strange spikes

Hi all,

I’m working on an RRBS methylation analysis pipeline and have run into an issue.

I am using methyldackel to assess methylation bias. In most of the literature showing these methylation bias plots, the average methylation is anywhere from 20-80%, with the distribution across each strand being relatively constant, except for the beginning/end of the strands.

My plots look different (see example attached) - I’m getting >95% methylation for all my samples, and almost all of them have a spike somewhere in the middle. For most of the OT strands, the spike is at position 41, and for most of the OB strands, the spike is at position 84.

I expect my methylation to be high as I’m using RRBS, but 95% seems excessive, plus the strange spikes give me pause (plus further in my analysis, metilene is not identifying any differential methylation). Does anyone know what might be causing this?

Thank you so much!

Hi @Shanna
Maybe consider posting this question to other forum(s) dedicated to methylation analysis or methyldackel. Your question is about data analysis and interpretation, and you need an advise from a person with experience in the field.
Kind regards,

Also, consider using this tutorial as a guide