A question about the result

[Tomcxf]

Hi! Thanks for your nanocompore to detect direct RNA modification. But I have some question about the output file. I got the normal output like:

pos	chr	genomicPos	ref_id	strand	ref_kmer	GMM_logit_pvalue	KS_dwell_pvalue	KS_intensity_pvalue	GMM_cov_type	GMM_n_clust	cluster_counts	Logit_LOR
873	NA	NA	TraesCS1A03G0224700.1	NA	GGATG	0.9636307191264709	0.9999969564924992	0.7237053561013628	full	2	WT_1:29/1__WT_2:40/0__mutant_1:41/0__mutant_2:31/0	-0.7351113796589768
894	NA	NA	TraesCS1A03G0224700.1	NA	TGCCA	nan	0.9999969564924992	0.6230859296888032	full	1	NC	NC
895	NA	NA	TraesCS1A03G0224700.1	NA	GCCAA	0.9724399160938189	0.9999969564924992	0.9531035022795072	full	2	WT_1:30/0__WT_2:42/0__mutant_1:45/1__mutant_2:33/0	0.6141587692413149
896	NA	NA	TraesCS1A03G0224700.1	NA	CCAAG	0.9250138233257315	0.9999969564924992	0.3286551634689277	full	2	WT_1:15/15__WT_2:30/13__mutant_1:29/17__mutant_2:27/6	-0.40365187098398375
897	NA	NA	TraesCS1A03G0224700.1	NA	CAAGG	nan	0.5916486177990066	0.5183734953514848	full	1	NC	NC
898	NA	NA	TraesCS1A03G0224700.1	NA	AAGGC	0.9079812410280157	0.9999969564924992	0.9964425319972754	full	2	WT_1:10/20__WT_2:15/27__mutant_1:9/38__mutant_2:11/22	0.4532469535634805
901	NA	NA	TraesCS1A03G0224700.1	NA	GCCTG	nan	0.9459273492344533	0.4516958290503259	full	1	NC	NC
908	NA	NA	TraesCS1A03G0224700.1	NA	TCTGA	0.9823659884224117	0.9999969564924992	0.5079364659616982	full	2	WT_1:3/27__WT_2:3/40__mutant_1:3/44__mutant_2:5/29	-0.1667570402528384

I just wonder what's the meaning of pos. I check the transcriptome file and find its the relative location. So is the pos start from 0, like python? I don't know whether it means the start of motif or the base's location of modification. And how can I make sure whether modification is more reasonable? Nanocompore aims to detect modification, not a exact kind of modification. But I want to classifiy them (like m6A, 5mC). Is there any solution? Thanks!

[lmulroney]

Hi @Tomcxf,

If you're using the default context settings, then pos is defined as the position of the first nucleotide in the kmer (ref_kmer column in the results tsv), using 0-based reference coordinates. If you change context to say, 2, then pos is defined as the pos of the middle nucleotide in the kmer, using 0-based reference coordinates.

As to your second question, it really depends on what experimental conditions you used for your reference condition. If the sample you compared against is a METTL3 KO/KD, like what was done in the manuscript, then you would find METTL3-dependent RNA modifications (m6A). And all other modifications will not be detected. If instead you KO/KD NUSUN2 or NSUN6, then you would find m5C and not any other modification.

I hope this helps, Logan