User’s guide#

The following user’s guide provides a detailed steps to be able to analyze white matter tissue features from dMRI data using the White Matter Analysis Pipeline (WMAP) tool.


The assumptions in this pipeline are as follows:

  • All raw data is available in DICOM format.

  • All participants are assumed to have AP/PA dMRI acquisitions.

  • All participants are assumed to have field map acquisitions.

  • All participants are assumed to have T1w acquisitions.

  • All participants are assumed to have $b = 3000$ s/mm^2 dMRI data.

  • The data layout is kept consistent across runs: the only change may be in the participant identifier in the folders and filenames.

  • Generally, it is assumed that scripts are launched from a path where they are known.

  • The only steps that can process all participant data at once are those in the Data organization/standardization and Preprocessing sections; the rest of the steps need to be run individually for each participant.

Note that:

  • Data file paths may need to be adjusted.

  • The path to the 3D Slicer executable and its extensions may need to be adjusted across scripts depending on the OS and installation path.

    As an example, for 3D Slicer version 5.2.2, on a Linux machine, the 3D Slicer executable path, if installed under /opt, it might be:


    On a macOS machine, the path may be:


    Similarly, for the UKFTractography extension module, the path to the executable file would be:


    The same extension on a macOS machine could be located at:


The help for all scripts provided can be retrieved by typing in a terminal:

$ [my_script] -h

where [my_script] corresponds to the name of the script.

Pipeline description#

Data organization/standardization#

The purpose of this step is to produce a consistent layout and NIfTI-centered data format from the DICOM data files obtained from the scanner. The BIDS standard is followed to accomplish this task.

The QSIprep preprocessing tool expects the input data to be BIDS-compliant.


Note that organizing and standardizing the data layout according to BIDS is increasingly important for efficient work and collaboration, beyond the requirement by QSIprep.


Its purpose, among others, is denoise the T1w and dRMI data to correct for different artifacts and prepare the data for downstream analysis.


QSIprep uses a number of other software tools under the hood, transparently to the user, such as DIPY, FreeSurfer, FSL or MRtrix, among others. The user is not required to install them, except for FreeSurfer, if the installation using containers is followed.

Data format accommodation#

This pipeline uses the UKF tractography reconstruction method. The input dMRI data required corresponds to the $b = 3000$ s/mm^2 shell data, in addition to the reference $b = 0$ s/mm^2 shell data.

All MRI data written by QSIprep is written using the NIfTI format. The dMRI data analysis tools (tractography and white matter bundle identification) require files to be in NRRD format, so the NIfTI files need to be converted to NRRD files.

Finally, the brainmask data used to constrain the tractography method to the brain tissue requires also a particular pixel type.



Its purpose is to reconstruct the white matter fibers from the preprocessed dRMI data.



The pipeline was developed on an Ubuntu 22.04.3 LTS machine using Python 3.10.

Although not shown below, advanced users are encouraged to install the Python packages within a virtual environment. This minimizes the risk of incompatibilities across different Python package versions required by the tools used.

Data organization/standardization#

  1. Install heudiconv following the heudiconv installation instructions

    Although a local installation was used, Singularity would work in the same way; only the way the tool is called would need to be changed.

  2. Install the BIDS validator tool following the BIDS validator quickstart guide

    Note that the JavaScript version (“Command line version”) has been used here; other versions should work equally well and should give the exact same result.

    It assumes Node.js is installed.


Versions used are:

  • heudiconv: 0.13.1

  • bids-validator: 1.12.0 (Node.js v20.5.1)


  1. Install FreeSurfer (only required to point to a valid license file, as the software itself is used from the Apptainer container) following the FreeSurfer Download and Install instructions.

  2. Install Apptainer (formerly known as Singularity) using their Quick Installation guide.

    For macOS and Windows systems, Apptainer needs to be installed in a Virtual Machine (VM) or a Windows Subsystem for Linux (WSL).

    Apptainer is an open-source equivalent to Docker; these both container systems allow to ship all necessary dependencies.

ℹ️ Note

Apptainer containers have access only to a limited set of paths in the host machine (called “bind points”), so users may need to specify other paths so that the container obtains access to the data hosted on such paths. The relevant information can be found at the Bind Paths and Mounts section.

  1. Build the QSIprep Apptainer (Singularity) container (output is a file with the extension sif).

    Pick an appropriate path (the folder must exist) to host the *.sif file, e.g.:

    $ singularity build /mnt/data/containers/qsiprep/qsiprep-0.19.0.sif docker://pennbbl/qsiprep:0.19.0

    If the following error appears when building the container:

    INFO:    Creating SIF file...
    FATAL:   While performing build: while creating squashfs: create command failed: exit status 1: Write failed because No space left on device
    FATAL ERROR: Failed to write to output filesystem

    it means that when building the container the filesystem has run out of disk space to store temporary data required in the build process. In order to solve the issue, we need to specify a temporary folder where we have sufficient disk space using the --tmpdir option. Pick an appropriate path for this purpose (the folder must exist), e.g.:

    $ singularity build --tmpdir /mnt/data/containers/.singularity /mnt/data/containers/qsiprep/qsiprep-0.19.0.sif docker://pennbbl/qsiprep:0.19.0

    The tested version corresponds to 0.19.0.


Versions used are:

  • Apptainer: 3.11.3 (Singularity)

  • FreeSufer: freesurfer-linux-ubuntu22_x86_64-7.3.2-20220804-6354275

  • QSIprep: 0.19.0

ℹ️ Note

The scripts provided were developed Singularity instead of Apptainer. Users may need to change the singularity command to apptainer in the future. Scripts should work transparently.

Data format accommodation#

  1. Install the Scilus Apptainer container following the Singularity for TractoFlow instructions.

    The SCILPY tools will be shipped inside this container, which requires Apptainer to be available on the system (see Preprocessing).

ℹ️ Note

The Scilus containers were primarily developed to support Tractoflow. Tractoflow is a dMRI data processing pipeline in itself; Tractoflow is not used here, but some relevant documentation about the Scilus containers may be found on its website.

  1. Install SlicerDMRI (see Analysis-installation): the diffusion data conversion tool (DWIConvert) is installed together with SlicerDMRI.

  2. Install SimpleITK using the Python wheels


Versions used are:

  • SCILPY (Scilus container): scilus 1.5.0

  • DWIConvert: (see SlicerDMRI version in Analysis-versions)

  • SimpleITK: 2.2.1


  1. Download and install 3D Slicer following the instructions in the Installing 3D Slicer section.

    Older releases can be downloaded by using the offset parameter in the download page. For example, the download page from 7 days ago is

  2. Install SlicerDMRI following the instructions in the Download section

  3. Install UKFTractography from the 3D Slicer Extensions Manager following the How to section

  4. Install White Matter Analysis (WMA) following the instructions in the WMA Installation and Usage section

  5. Download the version of the ORG Atlas that is intended to be used from the 8082481 Zenodo record


Versions used are:

  • 3D Slicer: 5.2.2 r31382 / fb46bd1

  • SlicerDMRI: commit 6207e52

  • UKFTractography: commit fcf83e2

  • White Matter Analysis (WMA): commit 173f241

  • ORG Atlas: v1.2


Data organization/standardization#

  1. Run the following command from a terminal:

    $ heudiconv -f [heuristic_name_or_path_to_file] --bids -o [path_to] --files [in_data_path]

    The [heuristic_name_or_path_to_file] is the name of a known heuristic or a file that contains some heuristics to be able to identify the relevant information in the study. The file used here is hosted in this repository. Note that if the study contains acquisitions whose naming contains different conventions, the file will need to be modified. One of the most crucial parts for a correct identification of the data is the protocols2fix dictionary in that file. Some documentation is provided here. Additionally, the documentation in the file header should be read carefully.

    The input data path [in_data_path] must be set to the place where the raw DICOM data corresponding to all available participants exists, and pick an appropriate location for the output [path_to] directory, e.g.

$ heudiconv \
    -f /mnt/data/study_name/bids_config/ \
    --bids \
    -o /mnt/data/study_name_bids_data \
    --files /mnt/data/study_name_raw_data

Note that all raw imaging data needs to be in DICOM format; heudiconv does not deal with raw imaging data in NIfTI format, and it will error if such data is found in the input data path.

Checking BIDS compliance#

As QSIprep expects the data to be BIDS-compliant, it is recommended that we ensure that heudiconv has accomplished this by running a BIDS validator tool.

  1. Check that the data that has been written conforms to BIDS running:

    $ bids-validator [path_to]

    Set the input data path [path_to] to the place where the above step has written the data to, e.g.

    $ bids-validator /mnt/data/study_name_bids_data

    No warnings or errors should be present, e.g.:

    $ bids-validator /mnt/data/study_name_bids_data
    This dataset appears to be BIDS compatible.
            Summary:                  Available Tasks:        Available Modalities:
            21 Files, 512.32MB                                MRI
            1 - Subject
            1 - Session
      If you have any questions, please post on


  1. Run the script from a terminal:

    $ [in_bids_path] [out_path] [fs_license_fname] [qsiprep_singularity_fname] [work_dir]


    $ \
        /mnt/data/study_name_bids_data/heudiconv \
        /mnt/data/study_name_bids_data/qsiprep \
        /usr/local/freesurfer/license.txt \
        /mnt/data/containers/qsiprep/qsiprep-0.19.0.sif \

    The above command assumes that we have changed the directory to the path where the script lies, and that it has the appropriate permissions (most notably, execution permissions, which may be granted running chmod +x [my_file] from a terminal and providing the appropriate filename).

    The [in_bids_path] argument needs to point to the dirname where the BIDS dataset_description.json file lies.

    Some steps of the script may take a long time to complete even for a single participant.

ℹ️ Note

Apptainer uses an intermediate work directory (--work_dir) where intermediate files are written as the processing take place, and before the final data gets written to the destination path. This work directory may grow considerably, even for a single participant. Similarly, if some jobs (e.g. processing some participant data) were terminated unexpectedly, and for some reason, dirnames (e.g. the root dirname) are manually changed between runs, since qsiprep will try to pick and re-run unfinished jobs (which are traced through the data stored in the work directory), this may give rise to exceptions derived from FileNotFoundError: No such file or no access errors, as previous filenames and dirnames will be present in the work directory.

ℹ️ Note

The brainmask may need to be adjusted manually (e.g. using 3D Slicer) if the result is not accurate enough.

ℹ️ Note

The denoising step in the preprocessing operation is chosen to be applied to all combined dMRI data. Further information related to this choice can be found in the Merging multiple scans from a session and Denoising and Merging Images sections of the QSIprep documentation.

Data format accommodation#

b-value shell data extraction#

  1. Extract the $b = {0, 3000}$ s/mm^2 shell data from the preprocessed diffusion data. For the $b = 0$ s/mm^2 (b0) data, an average across all volumes that have no diffusion-weighting is computed. The computed mean b0 data is a 3D volume, as the b0 data has no diffusion-encoding direction; the fourth dimension is added back on the mean b0 data, and a pair of bval and bvec files are generated to accompany the mean b0 volume data. Finally, the extracted data (mean b0 and b3000 shell) are concatenated and merged into a single file: tractography will be performed on the $b = 3000$ s/mm^2 shell data, the mean b0 serving as the reference.

    $ [in_nifti_fname] [in_bval_fname] [in_bvec_fname] [out_dirname] [scilus_singularity_fname] [data_dirname]


    $ \
        /mnt/data/study_name_bids_data/qsiprep/sub-001/dwi/sub-001_acq-dir99_space-T1w_desc-preproc_dwi.nii.gz \
        /mnt/data/study_name_bids_data/qsiprep/sub-001/dwi/sub-001_acq-dir99_space-T1w_desc-preproc_dwi.bval \
        /mnt/data/study_name_bids_data/qsiprep/sub-001/dwi/sub-001_acq-dir99_space-T1w_desc-preproc_dwi.bvec \
        /mnt/data/study_name_bids_data/prepare_shell_data \
        /mnt/data/containers/scilus/containers_scilus_1.5.0.sif \

    Note: When executing the above script the shell data concatenation step may raise the following warning:

    UserWarning: Detected only 1 direction on your bvec file.
    For diffusion dataset, it is recommended to have at least 3 directions.
    You may have problems during the reconstruction step.

    The above warning is expected since the b0 data contains only one 0-direction in the corresponding bvec file. No action is required following this warning in this case.

Data format conversion#

  1. QSIprep uses a data format called NIfTI; UKF, however requires the data to be in NRRD (or NHDR) format. 3D Slicer’s tool DWIConvert tool is used to perform the NIfTI to NRRD conversion:

    $ [in_nifti_fname] [in_bval_fname] [in_bvec_fname] [out_nrrd_fname]


    $ \
        /mnt/data/study_name_bids_data/prepare_shell_data/sub-001_acq-dir99_space-T1w_desc-preproc_dwi_b0_mean-b3000.nii.gz \
        /mnt/data/study_name_bids_data/prepare_shell_data/sub-001_acq-dir99_space-T1w_desc-preproc_dwi_b0_mean-b3000.bval \
        /mnt/data/study_name_bids_data/prepare_shell_data/sub-001_acq-dir99_space-T1w_desc-preproc_dwi_b0_mean-b3000.bvec \
  2. Change the brainmask pixel type: the brainmask computed by QSIprep has to have its pixel type changed (to either signed char, unsigned char, short, and unsigned short) so that UKF can process it. SimpleITK is used for such purpose:

    $ [in_fname] [out_fname]


    $ \
        /mnt/data/study_name_bids_data/qsiprep/sub-001/anat/sub-001_acq-dir99_space-T1w_desc-brain_mask.nii.nii.gz \

    The script converts the source brainmask NIfTI data format file to an NRRD format file.



  1. Compute the tractography from the preprocessed diffusion data:

    $ [in_dmri_fname] [in_mask_fname] [out_tractography_fname]


    $ \
        /mnt/data/study_name_bids_data/nifti2nrrd/sub-001_acq-dir99_space-T1w_desc-preproc_dwi_b0_mean-b3000.nrrd \
        /mnt/data/study_name_bids_data/nifti2nrrd/sub-001_acq-dir99_space-T1w_desc-brain_mask.nrrd \

Data accommodation#

  1. Limit the size of the computed tractogram by randomly subsampling the streamlines so that WMA can perform the bundle identification with a reasonably sized tractogram. The general recommendation is to set an upper bound of $500000$ streamlines:

    $ [in_dirname] [out_dirname]


    $ \
        /mnt/data/study_name_bids_data/ukftractography \

White matter bundle identification#

  1. Launch the bundle identification step using WMA:

    $ [in_tractography_fname] [atlas_dirname] [out_dirname]


    $ \
        /mnt/data/study_name_bids_data/tractography_subsample/sub-001_acq-dir99_space-T1w_desc-preproc_dwi_b0_mean-b3000_pp.vtp \
        /mnt/data/atlas/org_atlas/ORG-Atlases-1.2 \

    ℹ️ Note

    If the bundle statistics computation step fails with the following error:

    ERROR: Reporting diffusion measurements of fiber clusters. failed. No diffusion measurement (.csv) files generated.

    the statistics can be computed from the 3D Slicer Graphical User Interface: by selecting the Diffusion/Quantify/Tractography Measurements menu, the folder that contains the identified bundles (typically names AnatomicalTracts after WMA has run), can be selected, and an output filename be given to obtain the statistics of interest. Selecting the Column_Hierarchy option allows to lay out the statistics across columns.

Relevant notes#


By default, QSIprep output data are in subject space aligned to AC-PC: AC-PC alignment changes the coordinates from the native scanner coordinates to a new system where $0, 0, 0$ is where the midline intersects the anterior commissure. This is the same $0, 0, 0$ as in MNI space, so the brains will look somewhat aligned if you open the MNI template and the AC-PC image, as long as it is an adult brain, and despite no (MNI) template warping taking place by default.

Further documentation can be found at the Specifying outputs section of the QSIPrep documentation.


The data is currently set to be resampled to a resolution of $1 x 1 x 1$ mm at the QSIPrep preprocessing step, in line with other notable dMRI processing pipelines, such as Tractoflow.

The resampling step, added to the combination of the AP/PA data into a single file, together with any multi-shell data, explain the dMRI data large file size that it is seen at the output of the QSIPrep preprocessing workflow.


Not all parameters of the underlying tools are exposed by the scripts; some of their parameters have fixed values in the scripts (e.g. the output data resolution in the script, or the tractography parameters in the script), and other parameters are kept to their default values. The proposed values were found to be appropriate for the data used during the development. Users are encouraged to adjust the parameter values if results are suboptimal after visual inspection.

Python packages#

Scilus 1.5.0 requires NumPy < 1.25.0: if NumPy is installed system-wide, and it is picked up when running (despite using the container), if NumPy’s version is >= 1.25.0, the following error will be raised:

Traceback (most recent call last):
  File "/usr/local/bin/", line 33, in <module>
    sys.exit(load_entry_point('scilpy', 'console_scripts', '')())
  File "/usr/local/bin/", line 25, in importlib_load_entry_point
    return next(matches).load()
  File "/usr/lib/python3.10/importlib/metadata/", line 171, in load
    module = import_module('module'))
  File "/usr/lib/python3.10/importlib/", line 126, in import_module
    return _bootstrap._gcd_import(name[level:], package, level)
  File "<frozen importlib._bootstrap>", line 1050, in _gcd_import
  File "<frozen importlib._bootstrap>", line 1027, in _find_and_load
  File "<frozen importlib._bootstrap>", line 1006, in _find_and_load_unlocked
  File "<frozen importlib._bootstrap>", line 688, in _load_unlocked
  File "<frozen importlib._bootstrap_external>", line 883, in exec_module
  File "<frozen importlib._bootstrap>", line 241, in _call_with_frames_removed
  File "/scilpy/scripts/", line 14, in <module>
    from dipy.core.gradients import gradient_table
  File "/usr/local/lib/python3.10/dist-packages/dipy/", line 41, in <module>
    from numpy.testing import Tester
ImportError: cannot import name 'Tester' from 'numpy.testing' (/home/<username>/.local/lib/python3.10/site-packages/numpy/testing/

In order to solve this, NumPy needs to be downgraded, i.e. pip install --upgrade numpy==1.24.


Please cite appropriately each of the packages used in this pipeline.