Interpreting direction of differential methylation

[NStrowbridge]

In previous discussions and on your documentation you mention you can use xpore postprocessing to "filter out those positions whose mod_assigment values are not in line with those of the majority in order to restrict ourselves with one modification type per kmer in the analysis."

From my understanding if I want to analyze m6a differential methylation I would use this and then filter for DRACH motifs. After that if I want to interpret direction of differential modification between treatments do I use "diff_mod_rate" column and if so, how do I interpret this?

Sorry if it is not clear what I am asking.

Kind regards,

Nic

[NStrowbridge]

Also, a further question. If I want to interpret just m6a modification differences between my two treatment groups can I just filter sites with DRACH motifs, via converting Us to Ts in DRACH motifs? Would I do this after postprocessing?

If so, would all combinations of DRACH motifs be appropriate or should I filter only ones with single A nucleotide in the 3rd position as other motifs with multiple As could also be modified?

To clarify, I have multiple different phenotypes from my samples, not treatment with KO.

Thanks again!

Nic

[yuukiiwa]

Hi Nic (tagging you here @NStrowbridge),

Sorry for the delayed reply! Just came back from vacation.

For your first comment, you can filter with xpore postprocessing and select for DRACH motifs. To interpret the direction of differential modification between treatments, we would suggest you to use the z-score, which also indicates the statistical significance of the difference.

For your second comment, you can filter for DRACH sites and convert U to T after xpore postprocessing. We find analysis restricting to DRACH motifs giving us the strongest m6A signal.

xpore is able to handle multiple different phenotypes, so it is okay to not include knockout as one of your samples.

Thanks!

Best wishes,
Yuk Kei