Correlational Tractography & Connectometry

Correlational tractography tracks the tract segments that have anisotropy correlated with a study variable in the group studies, and connectometry is the statical method that uses a permutation test to get statistical inference for correlational tractography.

This documentation introduces the steps to create a connectometry database for generating correlational tractography and testing its significance.

Step C1: Reconstruct SRC Files

Skip this step if you want to use MNI-space NIFTI files as the input

First, generate SRC files for your data and place all SRC files in a folder. Also make sure to run a [Diffusion MRI Analysis]Step T1a: Quality Control to exclude problematic data.

Click [Step C1: Reconstruct SRC Files for Connectometry] and select the folder to reconstruct all SRC files. DSI Studio will run QSDR for all SRC files using the HCP-1065 young adult template.

If you would like to use a study-specific anisotropy template instead of the built-in template, then you will need to reconstruct each SRC file by QSDR in [Step T2: Reconstruction] and assign a different template. Make sure to check [ODFs] in the output option.

Post-reconstruction quality check

Each FIB file generated from the previous step will have a file name such as *.R67.fib.gz. The R67 indicates that the R-squared value between the subject and the template’s QA map is 0.67. An R-squared value lower than 0.6 may require further inspection to confirm whether it is due to a registration error.

The most common cause for a low R2 is an inverse order of the axial slices, which can be corrected in the reconstruction step to flip the image volume at the Z direction.

The second common cause is an artifact in the background, which may be handled by introducing a brain mask and using [Edit][Trim Image] in the reconstruction step to clean up the background.

If still cannot solve the low R2 value problem, please feel free to send the data to me (uploaded provided in the webpage of the Discussion forum), and I will figure out a solution for you.

Step C2: Create a connectometry database

A connectometry database contains information about the subjects’ diffusion profiles that allows further connectometry analysis. It requires the ODF-containing fib files for all subjects and an atlas that serves as the sampling skeleton.

  1. Connectometry Dialog

Click on [Step C2: Create a Connectometry Database] to bring up the following database dialog.


  1. Open SRC files

Use [Search in Directory] or [Add] button to load FIB files constructed from the previous step. Alternatively, you can use MNI-space NIFTI files, and specify the name of the metrics.

You may need to make sure that the file orders match that of your demographic records. Use the “sort” function to sort the files.

If you are going to study the change in a longitudinal study, make sure that you place baseline and follow-up scans of the same subject together.

  1. Specify the template file

The default atlas is HCP1065 2-mm atlas provided in the DSI Studio package, which is available at

If your DWI were acquired at a high resolution (e.g. 1.4mm), QSDR will reconstruct data to 1-mm MNI space, and you will need to use HCP1065 1-mm template

If your data have both 2-mm MNI FIB file and 1-mm MNI FIB, DSI Studio will prompt an “inconsistency resolution” error. To solve it, downsample the high-resolution SRC files in STEP T2 Reconstruction using [Edit][Resample to isotropic] (assign 2 mm), and rerun QSDR reconstruction.

For animal or pediatric studies, you may need to create a group averaged template as the template file. The function is located at [Tools][P1:Create Template/Skeleton]. Select all fib files created using Step C1, and DSI Studio will generate a group-averaged template FIB file.

  1. Choose index of interest

The FIB file contains many diffusion metrics. The default choice is “QA” known as the quantitative anisotropy. You can also study other diffusion measures such as FA, AD, RD, MD, RDI, NRDI. Each metric will need its dedicated connectometry database file.

The QA here is the SDF values sampled at fiber orientations, and the fiber orientations here are defined by the template in the previous steps. The sampled values are similar to QA defined in the native space, but there are differences. QA in the native space is defined on the peaks of SDF, but here the QA values are extracted at the template fiber orientations. The physical meaning is the same, but their evaluation approach is different.

  1. Specify the output file name

Please assign the file extension as *.db.fib.gz

  1. Create connecometry database

Click the “Create Database” button to create the connectometry database as a db.fib.gz file. You can add or remove subjects from the database using [Diffusion MRI Connectometry][Edit Connectometry Database]

At the End of this step, you will have a connectometry database, which is a file name with the extension db.fib.gz

Optional: Create a population average template

The FIB files created from Step C1 can be averaged into a population average template using the following steps:

  1. Click [Tools][O8: Create template/skeleton]

  2. Add in all FIB files and specify the output file name. This dialog outputs two fib files, one without and one with the averaged ODFs. The one without the ODFs can be used as the template for creating a connectometry database.

Step C2a: Modify a Connecometry Database (Optional)

After creating the database file, you can still check the alignment of the database or add/remove subjects to/from the database using [Step C2a:Modify a Connecometry Database]. A quick visual checking on the database is highly recommended.

If you are going to study the change in a longitudinal study, use [Step C2a:Modify a Connecometry Database] to calculate the difference between baseline scan and follow-up scan for each subject (e.g. base of s#1, follow-up of #1, base of s#2, followup of s#2…etc.) and save a new database.

Additional processing for longitudinal studies

Skipping this if your study is a cross-sectional study (has no repeat scans of the same subjects).

If your study is a longitudinal study (e.g. comparing pre- and post-treatment of the same subject), then the scans of the same individuals need to be matched to calculate their difference. The following are the steps:

  1. In the main window, click [Step C2a:Modify a Connecometry Database] and select the database:

  1. In the top menu, select [Tools][Longitudinal scans…] to bring up the subject match window:

Click on the “Match consecutive scans” button to match scan 0 (base) and scan 1 (study), 2 and 3, 4 and 5, throughout the entire database.

Alternatively, you can use a text file to match scans. The text file should be the consecutive numbers of the matching pairs. The first number will be used as the baseline, and the second number will be the study scans. For example, a text file with “0 1 2 3 4 5” will match scan 0 (base) and scan 1(study), scan 2 and scan 3, scan 4 and scan 5. Please note that the first scan in the database is labeled by 0, not 1.

The “metric” group box allows you to define how the difference is calculated. “a-b” calculates the absolute difference (recommended) by “the study scan (a) - the baseline (b)”, whereas “(a-b)/(a+b)” calculates the difference as a ratio. “a-b” is recommended here. If your data were acquired from different scanners or sites, you may need to check “Normalize QA”.

Click “Ok” to calculate the difference between scans and go back to the database window.

Use [File][Save DB as..] to save the differences as a “modified connectometry db”.

The new connectometry db will contain only the difference between the paired scans for further analysis.

Step C3: Group Connectometry

Open the db.fib.gz file in [Step C3: Group Connectometry Analysis] in the main window.

If a dialog prompts a warning about poor image quality (low R2), you may need to remove problematic data. This can be done by (1) opening the connectometry db.fib.gz file using [Step C2a:Modify a Connecometry Database] provided in the main window and manually identifying and deleting subjects with a registration problem or motion artifact.

Step C3a: Load demographics

Prepare a comma-separated values (CSV) file that records any variables you would like to include in the regression model. The first row should be the name of the scalar values. The second row is the value for the first subject, and the rest follows. If there is any missing data, leave the field empty. DSI Studio will ignore subjects with missing data.

Click on the button labeled “open subjects demographics”. Load the text file that records all the scalar values that will be included in the regression model.

*If your data are from a longitudinal study and only want to look at longitudinal differences without considering any other variable, go directly to [Step C3d: Parameters].

Step C3b: Select variables

Please choose the variables to be considered. This can include all covariates to be considered such as sex and age and also your target study variable.

DSI Studio will use linear regression to eliminate the effect of covariates from the diffusion measures.

There are several tips in choosing the variables in the model:

*TIPS: *If you have multiple study variables, including too many of them in the model may lead to over-fitting unless you have enough sample size. Otherwise, include basic variables like the sex and age with the one study variable at a time.

*TIPS: *If your study variables are highly correlated to each other, consider using a PCA to get the principal components. (see You will need to interpret the meaning of each principal component by checking its coefficient.

Step C3c: Study variable

Choose the target variable to study, and DSI Studio will find any tracks correlated with this variable. The effect of other covariates selected in C3b will be regressed out.

[Nonparametric] will use Spearman rank-based correlation to consider the nonlinear effect between the study variable and the diffusion measure. The other covariates will still be regressed using a linear regression model.

If your data are from a longitudinal study and want to look at longitudinal differences after considering variables selected in the previous step, choose “Intercept”.

For example:

If you have a longitudinal connectometry database with “age”, and you don’t select anything for Step C3b and select “Intercept” Step C3c. This will show tracks with significant changes in the longitudinal study.

If you choose “age” for Step C3b and choose “Intercept” for Step C3c?. This will show tracks with significant changes in the longitudinal study, and the effect of age on the change will be eliminated.

If you choose “age” for Step C3b and choose “age” for Step C3c? This will show tracks with changes that are significantly correlated with age. A positive correlation means when age increases, the tracks show increased anisotropy.

Step C3d: Parameters

Parameters Descriptions
FDR Control If FDR control is checked, DSI Studio will output ONLY tracks with a significant correlation (i.e., FDR lower than the threshold).
If unchecked, DSI Studio will report the FDR value as the significance level to examine the hypothesis
My experience is that an FDR of 0.05 is highly confirmatory while 0.05~0.2 suggests a high possibility of positive findings. FDR is very different from the p-value. It is unreliable if the subject number is low (e.g., less than 10). Any findings with FDR lower than 0.2 may be worth reporting since the results have much more “true positive” findings than “false positive” findings. FDR is not sensitive to sample size. This is its strength (unlike p-value constantly improves with large sample size) and weakness (The result from small sample size may not be reliable even if the FDR is small).
Length Threshold Different length thresholds correspond to different null hypotheses, and a longer length is usually more specific (less sensitive) and tends to achieve a lower FDR (with fewer findings).
The length threshold can be increased to achieve a better FDR (not always).
T threshold Higher values will map tracks with a more substantial correlation effect, whereas lower values for weak correlation.
I would recommend running separate analyses with different T-threshold (e.g., t=2, 2.5, and 3) to map different levels of correlation. Each threshold is viewed as a different hypothesis and will receive an FDR.

study region (optional)

Adding regions as constraints will also need to increase permutation count (e.g., 8000) to avoid the discrete error.

The default study region for connectometry is “whole brain,” which means that connectometry will look at the whole-brain region. You can choose to use an ROI in the MNI space by loading a NIFTI file of the mask. You can also choose a region from the atlas. The region type can be ROI (select tracts passing the region), ROA (discard any track passing the region), End (select track ending in the region), Seed (Only start the seeds from the region). “Whole-brain” is most suitable for the exploratory purpose. If you have a specific region of interest, limiting the computation to an ROI can give a more specific result (e.g., assign cerebral peduncle as the ROI can investigate whether CST is affected). Using only one ROI is suitable for most cases unless for a specific need in the tracking routine.

After assigning the regions, you may need to increase seed count because the regions setting will remove part or most of the findings and leave only sparse results. The best setting can be case-dependent. If the count is too low, it can risk no findings or get very sparse meaningless findings. On the other hand, a high seed count can flood findings with less significant results. There is a sweet range, and in most cases, the range is quite extensive, and as long as the value is not too low, the value does not make much of a difference.

You can exclude cerebellum regions by adding cerebellum white matter and cortex as terminative regions:

select cohort (optional)

You can include/exclude a particular group of subjects in/from the analysis. Click on the [Select Cohort] button and select criteria to [Apply]. The criteria will be listed on the [Select If] line edit. You can use the [Check] button to visualize the selection results.

advanced options

Parameters Descriptions
Permutation count determines the total number of permutations applied to the FDR analysis. Higher values give a smoother and more accurate estimation of FDR curves, but it requires more computation time
Pruning helps remove noisy findings. Higher values may improve FDR with the expense of sensitivity.
Normalize QA Check this to homogenize data for potential scanner or site differences.
If your data contains scans acquired from different protocols (e.g. b-value, resolution …etc.), you will need to check “Normalize QA” to homogenize the data

Step C3e: Run connectometry and report the results

Click on the [Step C3d: Run connectometry] button and wait until the computation is finished.

After the analysis, DSI Studio will output several files to report the results. You may also click on [Step C3e: View Results in 3D] button to view the tracks with a substantial difference.

file outputs

Results for T-statistics is stored in a FIB file:

Output Files Descriptions
bmi.t_statistics.fib.gz A FIB file storing the t-statistics and can be opened in [Step T3:fiber tracking] to visualize T-statistics with tracks. The T statistics for positive correction is stored as “inc_t”, whereas negative correlation is stored as “dec_t”
bmi.t2.fdr.jpg the FDR with respect to tracking length.
bmi.t2.fdr_dist.values.txt the FDR values with respect to length. HTML file reporting the connectometry results

Results for positive correlation: ( negative correlation will have neg_corr in the file name)

Output Files Descriptions
bmi.t2.pos_corr.dist.jpg shows the histogram of track length. tractography file stores tracks that are positively correlated with the study variable. It can be open with the t-statistics FIB file
bmi.t2.pos_corr.jpg figure showing the tracks in four different views.


  1. To change how DSI Studio visualizes tracks, open any FIB file in [Step T3 Fiber Tracking] and change [Step T3(c) Option]. After closing the fiber tracking window, DSI Studio will memorize the setting and apply it.**

  2. There are several ways to improve the FDR, with the expense of losing sensitivity. You may increase the length threshold (e.g. 30 mm), increase the T threshold, increase pruning iterations in the advanced options.


  1. If the direction of the correlation is wrong, try checking/unchecking “normalize QA” to fix the problem.

  2. If you have very different FDR results at different permutation counts, increase the permutation count until the FDR converges.

  3. If the finding only shows very short tracks, try lowing the T threshold

  4. If the finding only contains very few tracks, please increase the permutation count in the advance option (may need more computation time).

  5. If the finding has many short fragments, consider increasing the length threshold or pruning count.

Report Connectometry Results

Please note that correlation does not necessarily imply causality. The results can be the “cause” or the “response” of the study variable.

The following are the strategies to report results.

  1. report FDR at different T-scores gives an overview of finding from high sensitivity (low T) to high specificity (high T)

You can visualize the results at different T thresholds (see the next section about visualization). The FDR value will be different for different track lengths. An FDR < 0.05 indicates a highly confirmative finding. An FDR > 0.2 suggests that there are substantial amounts of false findings. The result is thus inconclusive and not reliable. An FDR between 0.05 and 0.2 covers a wide spectrum of reliability.

  1. report FDR at different subject subgroups (e.g., male group versus female group)

  2. Convert the finding to ROI using [Tracts][Tract to ROI] to track the entire pathways. This allows you to confirm the anatomical structure of the findings.

Post Connectometry Analyses

There are additional analyses that could be done after connectometry to show more details about the findings. The following are some recommendations.

1. Segment findings into bundles

The connectometry results often include multiple pathways, and they can be segmented into different bundles manually (video) or through [Tract Misc][Recognize and Cluster].

I would recommend checking [Tract Misc][Recognize and Cluster] and fine-tuning it using manual segmentation.

2. Report FDR for each bundle

The FDR in report.html is for the overall results, but findings with a longer distance will have a lower FDR than the others. You can report the FDR of each bundle based on its length.

To do this, first, open the 3D interface at Step C4f or open *.t_statistics.fib.gz in Step T3 and load * files.

Segment findings into bundles as mentioned above. Get the length using [Tracts][Statistics]. If the length is 60mm, then the voxel distance is 30.

Last, check the FDR values in *.fdr_dist.values.txt and look up the row with voxel distance=30 to get the FDR values. There is one FDR for positive correlation and one for negative. Make sure you get the right one.

3. Scatter plots of bundle statistics and study variable

We can further plot a scatter plot between the study variable and the diffusion variable (e.g. QA or nQA) averaged from the bundle.

To do this, open the connectometry database (*.db.fib.gz) in [Step T3: Fiber Tracking] and load the tracks from * **(track positively correlated with the study variable)or **.neg_corr*.tt.gz (tracks negatively correlated) using [Tracts][Open Tracts]. Then use [Tracts][Statistics] to get along tracks metrics for each subject in the connectometry database. The metric (here, use QA as an example) has raw QA or normalized QA (used if you check [normalize QA] in the advance option). This step will take a while.

The QA value can be copied to the clipboard and pasted into an Excel sheet. We can place them with the demographics values in and plot a scatter plot.

4. Visualize bundle in mosaic T1w

The finding can be visualized in slices using the following steps:

  1. Switch slice image to “T1w”.

  2. In the main menu, add tracks to ROI by [Tracks][Tracks to ROI]

  3. In the Options window (right upper corner), under the “Region Window” node. Change the slice layout to Mosaic 2

  4. Change the contrast of the slices using the slider on the top of the region window to enhance the tracks regions.

5. Network Property Analysis for Connectometry Findings

The connectometry findings usually present a group of fiber bundles, and how these bundles affect the network topology can be further analyzed to better present the results.
The following are steps to carry out this analysis:

  1. After group connectometry analysis finishes, click on the “View Results in 3D” button or open the trk file in STEP3 Fiber Tracking. Alternatively, you can open the FIB file generated within the demographic folder in Step T3 Fiber Tracking, and load the trk files generated by group connectometry.

  2. Remove the all tracks using [Tracts][Delete Tracts][Delect all].

  3. Run whole-brain fiber tracking (No ROI or seed) to get a total of 100,000 tracks using default tracking settings ([Option][Restore Tracking Settings]).

  4. Click [Tracts][Connectivity matrix] to bring up the connectivity dialog.

  5. Select “FreesurferDKT_Cortical” as the parcellation atlas.

  6. Switch to the “Network measures” tab and copy the content (e.g. density, …etc.) to the Excel sheet.

  7. Close the connectivity dialog.

  8. Load the group connectometry tracks (trk files). There should be one file for positive correlation and one for the negative correlation after group connectometry finishes (unless there is no finding). Load the one you want to study using [Tracts][Open Tracts]

  9. Select the loaded tracks, and transform it into a region using [Tracts][Tract to ROI]. The region will appear on the left. Change region’s type from “ROI” to “ROA”. Remove the connectometry tracks after the conversion.

  10. Filter whole-brain tracks (generated in 4.) using the [Tracts][Filter Tracts by ROI/ROA/END. You should find some tracks being deleted using the connectometry regions.

  11. Now repeat 5. 6. 7. to get network measures, and you can then compare the difference between these two sets of measures

  12. Calculate the value differences as a percentage change in

  1. If the change of clustering_coeff_average(binary) is +5% and the finding is from a positive/negative correlation of a study factor X, then you may report: “The network topology analysis on connectometry results show that X increase/decrease 5% of clustering coefficient in the network topology.” Repeat the same reporting format for other network measures.

  2. Provide a table summarizing the results using the following:

Table: Effect of X, Y, Z on network topology measures

Study Variable Clustering Coefficient (%) Network Characteristic Path Length (%) Global Efficiency (%) Local Efficiency (%) Small Worldness (%)
X -0.30 -5.38 3.35 4.60 5.08
Y 62.27 -7.95 6.88 57.90 66.91
Z -18.65 -10.57 8.11 -7.47 -8.24

Get SDF values along the track

Once you get tracks that show significant difference or correlation. You can open the connectometry db at [STEP 3 Fiber tracking] and load the resulting tracks. Click on track statistics and you will get SDF values along the tracks for “each subject”. This allows you to plot the difference between the groups.