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Hi Erik, These commands look fine to me. You might want to check the intermediate files ($USER/346137/r_seg_output/seeds_to_*) and see whether they are aligned with the original seed mask or the output segmentation file. This would at least tell us, whether the shifting happens in the probtrackx2 or the find_the_biggest command. It might also help to run the fslhd command on the seed file, intermediate files, and output segmentation files and check for differences in the output (particularly check for inconsistencies in the sform or qform matrices). Cheers, Michiel > On 1 Feb 2018, at 01:09, Erik <[log in to unmask]> wrote: > > Dear FSL Community > I am having issues with using probtrackx2 and find_the_biggest. I have defined the subcortical seed ROI and the cortical target ROIs in diffusion space. I have double-checked the registration and all ROIs appear to be accurate when overlaid with the nodif image from the diffusion dataset. However, after running probtrackx2 and find_the_biggest, the seed mask has clearly moved from what was input into probtrackx2 so the output segmented ROI is no longer in the same space as the original input seed ROI. I have included the calls below: > > > probtrackx2 -x $USER/346137/seg/r_seed.nii.gz -l --onewaycondition --pd -c 0.2 -S 2000 --steplength=0.5 -P 5000 --fibthresh=0.01 --distthresh=0.0 --sampvox=0.0 --forcedir --opd -s $USER/346137/diffusion.bedpostX/merged -m $USER/346137/diffusion.bedpostX/nodif_brain_mask --dir=$USER/346137/r_seg_output --targetmasks=$USER/346137/seg/r_targets.txt --os2t > > find_the_biggest $USER/346137/r_seg_output/seeds_to_* $USER/346137/r_seg_output/r_segment > > > > Thank you in advance!