bbsplit : How to configure ambiguous/ambiguous2 parameters in order to consider as unmapped reads, reads mapped on both human/mouse references.

Question

[ZheFrench]

I reopen an old topic for me that I need to clarify.

From the code of bbsplit : https://github.com/BioInfoTools/BBMap/blob/master/sh/bbsplit.sh

In Rnaseq Nextflow pipeline, the parameter was set as followed :

ambiguous2=all

My understanding is that it keeps both reads mapped in both genomes.

https://github.com/nf-core/rnaseq/issues/1408

So I set up ambiguous2=toss but I get more reads that previously with "ambiguous2=all".
That's not what we should expected.
I should have less reads because these reads are considered unmapped if you set ambiguous2=toss.
Am I understanding something wrong ?
What would be the good set up to count only reads map unambigously on one reference only ?

[GenoMax]

From the code of bbsplit : https://github.com/BioInfoTools/BBMap/blob/master/sh/bbsplit.sh

This is NOT the official repository of BBTools. SF is the official repo : https://sourceforge.net/projects/bbmap/

So I set up ambiguous2=toss but I get more reads that previously with "ambiguous2=all".

What does this mean? Can you post output of the results stats for both runs :

refstats=<file>     Write statistics on how many reads were assigned to which reference to this file.