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nanoporetech / tombo

Tombo is a suite of tools primarily for the identification of modified nucleotides from raw nanopore sequencing data.
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Direct nanopore RNA sequencing tombo resquiggle FASTA reference genome #386

Open ky66 opened 2 years ago

ky66 commented 2 years ago

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 commented 2 years ago

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 commented 2 years ago

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 commented 2 years ago

@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.