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OK, I just verified it is a left-right flip. I created an ROI in fslview, saved it and then did: fslmaths test -x y z testflip When I loaded in testflip.nii.gz, it was positioned where test.nii.gz was. But when I remove test.nii and reload it, it is now on the other hemisphere (and mirrors the original file). There is no y or z shift. What implications does using the testflip.nii.gz ROI have for the resulting tractography?