I recently started analysing nanopore data and I'm using dorado for the basecalling and I then need to align the ont reads to the reference genome. I understand that dorado outputs an unaligned bam file by default, can I align this bam file directly to the reference genome? Should I run the dorado basecaller with the reference option? I guess the second option should be the most appropriate, but if I want to just basecall first, could I do the first option?
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written
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njornet •
1 answer
can I align this bam file directly to the reference genome?
No you will need to convert to fastq first by using samtools fastq. You can also ask dorado to emit fastq by using --emit-fastq option. You will get a warning about loss of some metadata if you generate fastq but that should not affect the sequence.
Should I run the dorado basecaller with the reference option?
You could do this as a one step process since dorado uses minimap2 to do the alignments.
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If your goal is simple alignment to a reference genome, you can use dorado. I think dorado can also still output fastq format directly.
If your goal is to assess methylation, you'll need to use the MM and ML tags explained somewhat by this software: https://github.com/nanoporetech/modkit