Direct nanopore RNA sequencing tombo resquiggle FASTA reference genome

[ky66]

So, I performed direct RNA sequencing on nanopore and got fast5 files for them. When I do tombo resquiggle, should me reference fasta file by DNA or RNA sequence in fasta?

For instance, if I sequenced "AUG" in nanopore, should my reference be "ATG" or "AUG"?

Thanks

[marcus1487]

Tombo can handle either U or T bases in the reference, but direct RNA data must be mapped to the transriptome reference and not the genome (spliced mapping is not enabled).

[wenluo711]

Hi @marcus1487 I'm also trying to use direct RNA seq to run resquiggle and methylation analysis. but where can I find human transcriptome reference file? Do we have to find a transcriptome reference with U but not T for direct RNA data?

Thanks! Wen

[Evelynn-H]

@wenluo711 When I resquiggle RNA sequencing data, I didn't transfer U to T in the sequencing result. The base included in the transcriptome reference is consist of ATCG not AUCG. You can find transcriptome reference here http://genome.ucsc.edu/ . Hope it will help you.