Any help on how to detect viral transcripts in ONT RNAseq data

Question

[j.thomas93]

Hiya,

Im quite new to bioinformatics and linux in general. I have generated some RNAseq data using ONT minion 1d cDNA kit (PCS-109) to compare the transcriptome of cells expressing viral proteins which are stably transfected into the cells. As a control i just need to confirm the expression of these proteins within the cells and im not sure on the best method. I seen one way is to map to a human reference and then take the unmapped sequences and map them to the viral genome. But im not sure exactly how to do this and what alignment tools to use. Any help on this would be hugely appreciated!

Thanks!

Answer 1

[WouterDeCoster]

I would suggest taking a look at kraken2 for species identification, which you could indeed do after identifying which reads do not map to the human genome. You can download pre-built databases from here.

If you already know which viral genome to expect you can make a fasta file of both the human genome and the viral genome, and use minimap2 to align to this reference. Including both simultaneously will give you the most accurate results.

[j.thomas93]

Thanks for the help! I do indeed know what virus to look for so I will try the simultaneous alignment as you suggested! Thanks again!

[j.thomas93]

I was wondering if you could help me set up the command? What parameters would you recommend for minimap2 for my data and why?

[WouterDeCoster]

Mapping cDNA is discussed in the manual.