I’m doing a fixel-based analysis on healthy controls and patients with tumours that are on either the right side or left side of the brainstem and vary in size and compression of the brainstem. I want to negate the risk of poor subject-template registration if, for example, a subject has a large left-sided tumour compressing the brainstem, but the template better reflects the brain with a small right-sided tumour that doesn’t touch the brainstem. My aim is to compare the left and right sides of the brain between the group with left-sided tumours and healthy controls, right-sided tumours and healthy controls, and left-sided tumours vs right-sided tumours. Right now, I am using a symmetric template with a subset (randomly selected) of the left-sided tumour patients’ original FOD and flipped FODs, the right-sided tumour patients’ original and flipped FODs, and the healthy controls’ original and flipped FODs. I then use a hemisphere mask derived from the symmetric template to compare the fixel-based metrics in each subjects’ flipped image to their original image. I was wondering if this is the best approach in template creation to account for the wide variability in tumour size, side, and degree of brainstem compression in my patient cohort?