Searching for RNA modifications (m6A, Y, m5C, etc).

[assetdaniyarov]

Good day, colleagues,

I have a question regarding the search for several RNA modifications, including:

• N6-methyladenosine (m6A)
• pseudouridine (Y)
• 5-methylcytosine (m5C)
• 5-hydroxymethylcytosine (hm5C)
• 1-methyladenosine (m1A)
• N3-methylcytosine (m3C)
• N4-acetylcytosine (ac4C)
• 7-methylguanosine (m7G)

Can I search for these modifications using the Master of Pores 2? Does nanocompore allow you to search when running mop_mod.nf? If yes, what reference value should be used?

What references can be used at all? The references from Piano DB (http://180.208.58.19/%ce%a8-WHISTLE/index.html) and RMVar (https://rmvar.renlab.org/download.html) do not fit the format.

So far, I am only using the full human transcriptome - GRCh38_latest_rna.fna and the annotation GRCh38_latest_genomic.gff.gz.

Could you help to search for the above modifications?

[enovoa]

Hi @aset8 , sorry, it is unclear what you mean with "search for these modifications" in MasterOfPores. The mop_mod module, will identify what is distinct between two given samples that you are feeding it. So if you feed it with WT and METTL3 KO, to put an example, it will identify m6A sites that are placed by METTL3. MasterOfPores does not predict what RNA modification type it is identifying, it just tells you that there is a difference in that position between the two samples. It can detect in principle any RNA modification type that is changing between your two conditions, but it doesn't perform "de novo" prediction on each individual sample, just "differential prediction" between two conditions.

With regards to your second question - the reference should be a FASTA genome or a FASTA transcriptome. I am not sure what is the format of the PianoDB and RMVar that you are mentioning, but I assume it is not about FASTA transcriptomes/genomes, but rather RNA modification annotation text files. You may wish to intersect your results from mop_mod with these files that you refer to, but you don't feed them to MasterOfPores.

Hope that helped!

[adalisan]

@aset8 indeed some of those modifications may be detected in mop_mod. and there's a specific nanoconsensus workflow (mop_consensus) which combines the output of different RNA mod. methods. As far as I could see there were no ways to compare detections with ground truth, if GT exists.

[rugilemat]

Hi everyone, I just wanted to follow up on this.

Am I correct to understand that in order to run MOP_MOD I need to have KD/KO samples? As some of the tools now have options to run pre-trained datasets rather than KD/KO comparison, do you have any plans to include that functionality?