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Hi Mark, I did some pos tprocessing and I think I have problem again in this step. So I came back to this question. I checked the scripts of run_first_all and first_flirt. It seems that the matrix is the concatened matrix of registration in stage 1 and stage2. So, you mean that I should use only mat_stage1.mat or only mat_stage2.mat or -concat <mat_stage1.mat>, <mat_stag2.mat>? I have another question that may be related to above again. FSL does the segmentation on images that their dimention and orientation is not necessary the same as MNI152_T1_1mm. The dimention and orientation of the first and second steps of registration change to MNI reference image as I checked in first_flirt (by debugging), but the final segmented image has the same orientation and dimention of the original image. I tracked the scripts run_first_all, first_flirt and run_first. I didn't find any step that this resampling to original grid and orientation occurs. Could you please guide me in this regard? Siamak