Dear all,

to test the alignment procedure, I inserted a genome in reverse sense into an artificial set of fastq files and aligned against the reference genome in normal orientation. The resulting alignment does not show any mapping.

Is it normal that the aligner could not map the reads because they were in the reverse orientation? I thought that orientation could not affect the alignment procedure...

Thank you
Reference genome orientation of course matters. What is interchangable would be the reference genome in its standard representation and its reverse complement but not its reverse. That would completely ignore that DNA has a 5'->3' polarity that cannot simply be changed at will.