I am trying to take trimmed and qc filtered paired RNA seq data and run it through HiSat2 > Stringtie > Cuffdiff for DE analysis. I am getting some interesting behavior with the HiSat2 alignment percentages. When the Report alignments tailored specifically for Cufflinks option is checked in galaxy or the --dta-cufflinks for command line users, the mapped percentage fall substantially on the same dataset. Does anyone have any idea what could be causing this?
For example with cufflinks option: 36198885 reads; of these: 36198885 (100.00%) were paired; of these: 17307719 (47.81%) aligned concordantly 0 times 15647243 (43.23%) aligned concordantly exactly 1 time 3243923 (8.96%) aligned concordantly >1 times
Same Dataset without: 36198885 reads; of these: 36198885 (100.00%) were paired; of these: 3167919 (8.75%) aligned concordantly 0 times 27036679 (74.69%) aligned concordantly exactly 1 time