Ribosomal RNA Depletion in silico

Question

[CL]

Hi, currently I have some Nanopore Direct RNA sequences. In the sequencing process rRNA should already be taken care of, although I did not perform the sequencing myself. Could anyone please help me see if the following code can remove rRNA?

# Index for Ribo Genome
minimap2 -d Tair10_Ribo_index.mmi TAIR10_ensembl_rRNA_tRNA.fa -Q -t 3 -k 14
# Map to Ribo, take unmapped reads out (samtools -f 4 does that)
minimap2 -t 10 -ax map-ont Tair10_Ribo_index.mmi nano.fastq | samtools fastq -n -f 4 - > nano_unmapped_ribo.fastq.gz
# Remap with unmapped reads 
minimap2 -t 10 -G2k -x splice -uf -a tair10chr.fasta.fa.mm2.idx nano_unmapped_ribo.fastq.gz > nano_riboDepleted.sam

I have used the code but not a single read has been removed, which is highly unlikely since there should be some rRNA remaining.

Thanks!

[naveennavinbeast]

Are you using publicly available datasets

[GenoMax]

I have some Nanopore Direct RNA sequences

Asking since I have not seen direct RNA nanopore sequences. Does the sequence contain U's in place of T's? If so that may be causing not alignment.

[CL]

Hi, no it is not public data. I used Dorado to do the basecalling, and the resulting fastq file contains "T"s.

[GenoMax]

minimap2 is unlikely to make an alignment error. If this run was from total RNA then it seems surprising that you have no rRNA in your data.

[CL]

Thanks so much! In your opinion, do you think my codes above look correct? (like the logic of mapping to ribosome, taking out the unmapped, and remap to the genome) I would like to make sure of this; if so, I think I probably need to look at my data to figure out where the issues arise.

[GenoMax]

Your operations are logical.