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seed-analysis

Abstracted system for doing seed-based correlation analysis with any given set of groups and covariates. Uses AFNI 3dttest++.

I. How to setup this pipeline for any project.

Below is a general guide to setting up this pipeline for any project. We assume that you will clone this repository and, in that directory, follow these instructions.

This script creates a number of recipes based on the number of groups and seeds. The number of recipes it has to create is:

(seeds * groups + seeds * contrasts) * 2

where contrasts is:

seeds^2 - seeds

So, the time it takes make to prepare rises with the square of groups and linearly with the number of seeds. As these increase, make will take more time to get ready to be invoked, and might look like it's hanging. Try make test-contrasts to see how long it takes to compile.

The number of recipes is multiplied by two to account for the cluster correction recipes, which are separate from the base processing.

Important: Do not use a hyphen (-) in group, seed, or covariate names. Or anywhere, really. The system relies in many places on hyphens being used exclusively to identify contrasts (e.g. patient-control). I have built in a few first-order checks against hyphens in bad places, but the system will fail spectacularly if there are hyphens in unexpected places.

1. Create list of seeds

The makefile requires that the list of seeds and their locations be read in from a file. That file must be named allseeds.txt and be located in the top-level analysis directory and have the project seed directory, and all the seeds used in the project listed in a single column like so:

/mnt/praxic/pdnetworksr01/lib/SVC_seeds/
DANRaIPS
DANLpIPS
DANRFEF
DANLFEF
...

The makefile reads from the directory listed in the first line and looks for the seeds on the n>1 lines. These files can have any notes following the seed name in the file name, but must have an .nii.gz extension. For example, DANRaIPS_sphereroi.nii.gz is acceptable, but ROI_DANRaIPS.nii wouldn't be.

In other words, the DANRaIPS seed must match the file glob DANRaIPS*.nii.gz.

Important: All seeds must be generated for all subjects (see next section). If they aren't, the t-test for any seed where one or more subjects is missing the file will fail (with an error from 3dttest++).

2. Create group-*.txt group files

The makefile identifies groups by looking for files that match the regex group-[[:alpha:]]*.txt. For example, these files would create the groups "control" and "patient."

group-control.txt
group-patient.txt
Makefile

Please ensure that there are no other files matching this pattern in the working directory. Reminder: Do not use "-" in group names.

Group files must contain lines the following format:

140593 /mnt/praxic/pdnetworksr01/subjects/140593/session1/mcvsa/SVC_MEICA/
140605 /mnt/praxic/pdnetworksr01/subjects/140605/session1/mcvsa/SVC_MEICA/

Each line consists of a subject identifier, followed by a space, then followed by the absolute path to the seed-based connectivity maps for that subject. It is important to label each subject because the syntax of 3dttest++ requires each map to be labeled with a subject identifier.

For info on the [[:alpha:]] syntax, see this page. It is equivalent to [a-zA-Z].

Important: All of these directories must contain all of the required files. 3dttest++ will choke if it is given a non-existent file, and will not continue without it. I suggest you create a script to manage the creation of these files based on the existence of the map directories.

3. Set variables

The first fifty or so lines of the makefile set a number of variables for make execution. However, all the project-specific variables are set in the auxiliary file analysis/settings.conf.

In general, the convention is ALL CAPS variables should be modified on a project basis and left alone. PascalCase variables can be given a default, but can be overridden on the command line (any variable can be so overridden, but these are likely to be.) All lowercase variables should not be modified.

To override the default value of any variable, simply set a new value for them on the command line, like so:

make SINGLEGROUP_PD ANALYSIS=-ETAC

settings.conf

  1. PROJECT_DIR: The top-level directory for your project (in IBIC standard convention, this contains bin/, subjects/, etc.)
  2. STANDARD_MASK: The MNI-space mask used in 3dttest++. Using a mask reduces the number of comparisons and speeds up processing. Make sure the resolution (Xmm) matches the space your files are registered to. Default: 2mm
  3. SVCSUFFIX: Depending on how meica was set up, the maps aren't necessarily given consistent names. Set this to whatever your project chose as the output. We're looking for the Z-transformed correlation map.
  4. COVFILE: The covariates file (see section XXX) to read from. Leave blank if there are no covariates.
  5. ANALYSIS: Which type of 3dttest++ cluster correction to use. Available options are -ETAC or -Clustsim. Note that the number of cores can be specified as an argument to these flags, or left off to use all available cores. See section 7 for a discussion of cluster correction. Important: The leading dash must be included.

Makefile

  1. Paired: By default, an unpaired t-test is executed. To do a paired t-test, changed the value of Paired to anything other than the empty string (for example, "yes"). 3dttest++ will fail if your subject lists aren't properly paired.
  2. seedsdir: This variable is read in from the first lne of allseeds.txt and contains all the ROIs used in later analyses.
  3. allseeds: By default, all the seeds are read in from the file allseeds (see section 1). This can be overriden here if you have another method to identify the names of the seeds.
  4. groups: This pulls the group names out of the group-*.txt files (see section 2). Don't override this setting, as later recipes assume the existence of the same group-*.txt files.
  5. contrasts: This variable automatically creates all the contrasts based on the given group, and filters out any 0 contrasts (A - A). Don't change this declaration! There should be N(N-1) total contrasts
  6. covariate: This variable is set automatically based on whether there is a COVFILE. It is later read by 3dttest++, and adds the option -covariates for the proper syntax. Don't change this logic statement.
  7. pairflag: Adds the -paired flag to the GROUPDIFF t-test if the Paired variable is set to true.

4. Test your setup

Check to make sure your setup is done correctly. You can run make test-<variable> to get the contents of a variable and how many "words" it has; useful in checking that you have exported the correct number of contrasts, etc.

Run bin/check-config.sh, which will check that the appropriate files exist in your analysis/ directory and that they are formatted correctly. It isn't perfect, so do not count a successful result here as proof positive your project is ready to go.

Additionally, check the number of degrees of freedom for your analysis:

make SINGLEGROUP_<group>_DoF

make GROUPDIFF_<contrast>_DoF

Note though, that due to the constraints of Make, the calculation has to be written out twice. So it is possible that there is a discrepancy between the test calculation and the value actually passed to cluster-correct.sh. Please let me know if you discover any such issues.

5. Set up covariates

A covariate file takes the following format, including up to 32 whitespace-separated columns (including the subject identifier column).

idnum   age gender  ...
RRF01   18  0       ...
RRF02   20  1       ...
RRF04   21  1       ...
RRF05   22  0       ...
...     ... ...		...

From the 3dttest++ manual:

* A maximum of 31 covariates are allowed. If you have more, then seriously consider the likelihood that you are completely deranged.

The column headers will be part of the output filenames, so ensure that they are descriptive, but not too cumbersome. For example, you might end up with files named DMNLPCC_PDa-PDr_mean_age.nii.gz.

You can only add one covariate file, so the covariates for all subjects in all groups must be present in this one file.

There is no required name for the covariate file (COVFILE is empty by default), but covariates.txt works just fine.

Additionally from the 3dttest++ manual:

* There is no provision for missing values -- the entire table must be filled!

6. Run analyses

There are two primary ways of running this makefile: single-group and between-group analyses.

  • Run SINGLEGROUP_<g>, where g is one of the groups set in step 2 to run for a single group. Note that this requires a minimum of 14 subjects (an AFNI 3dttest++ requirement).

For example, you could type:

make SINGLEGROUP_patient

to create a directory called patient with the single group analyses results for the subjects in the file group_patient.txt.

  • Run GROUPDIFF_<g1>-<g2>, where g1 and g2 are two different groups from step 2. This also requires 14 subjects, but between the two groups.

Analogously, this would be called using

make GROUPDIFF_control-patient

to create the contrast between patients and controls.

7. Run cluster correction

The basic function of cluster correction is to determine what areas of activation are interesting by grouping regions with enough contiguous voxels above a certain threshold, and discarding those areas that are too small.

While the math behind the methodologies is beyond the scope of this README, the basic effect is to remove noise.

Important: It is possible to have no voxels survive cluster correction. In this case, the cluster corrected maps will be empty, this isn't necessarily a symptom of program failure.

Cluster correction is a separate step because it takes a while, even when parallelized (see section 8), and this setup allows you to examine intermediate results before dedicating entire machines to cluster correction.

To run cluster correction, call either single or difference cluster correction:

make SINGLEGROUP_patient_clustcorr

make GROUPDIFF_control-patient_clustcorr

Either of these will use the cluster correction method identified in Section 3.

Note that if run asynchronously, the QA image creation program might try to take pictures of temporary files that are deleted as the makefile progresses. You might see such "unable to read file" message. These are harmless; only the appropriate files are created, but you can avoid this messages by running sequentially (e.g. make SINGLEGROUP_patient_clustcorr -k.)

7.1 Cluster correction results

This will create a new directory at the same level as nifti and headbrik: clustcorr, which will contain six files for each seed:

  • Two NIfTI images: one for positive-signed clusters and one for negative-signed clusters
  • Two GIFs: one QA picture for each neuroimage
  • Two text files: These contain the cluster size thresholds used

For each sign, there is the actual image, a QA picture for quick checking, and a text file with all the clusters in said image, along with their size in voxels and coordinates.

7.2 Clustering methods

7.2.1 ClustSim

The ClustSim algorithm has its own AFNI program: 3dClustSim but is also built into 3dttest++ as -Clustsim.

From that page: The ClustSim method works by "simulates the noise volumes by randomizing and permuting input datasets, and sending those volumes into 3dClustSim directly. There is no built-in math model for the spatial [auto-correlation function]."

Importantly, the cluster size is the same for all brain regions with ClustSim. This is at the core of Eklund's 2016 [1] critique of fMRI false-positive rates.

7.2.2 ETAC

Equitable Tresholding and Clustering (ETAC) is a response to the abovementioned concerns about false-positive rates. Its 3dttest++ option is -ETAC.

See Cox et al.'s response [2] for details about how ETAC works, but at its core, it uses different cluster thresholds for different regions of the brain based what we know about how large functional clusters are in one area of the brain compared to another.

8. Notes on parallelizing

You might want to (a) parallelize processing of a single ROI to speed it up (see 7.1); or (b) parallelize processing of multiple ROIs to decrease total competion time (see 7.2).

8.1 Parallelizing locally

The easiest way to parallelize locally is to override Analysis to be -Clustsim, without an integer argument. Then, 3dttest++ will use all available cores.

Use this to test one ROI, as testing multiple will rapidly overwhelm your machine, but this is useful for rapid testing over waiting an hour+ for one-core execution to complete.

8.2 Parellelizing on the grid engine

Check that the grid engine is set up properly for your user/machine. See here for instructions.

You can submit the jobs on the queue with qmake:

qmake -cwd -V -- -j <n> -k <TARGET>

Where "n" is the number of jobs (which you can get with make test-allseeds to see how many you can parallelize), and TARGET is the make target you want to build (like SINGLEGROUP_PD). The -cwd flag to qmake tells it to work in the current directory, and -V passes along all environment variables.

Or, you can submit the jobs using rmake:

rmake -T <TARGET>

II Script documentation

This project contains four scripts in bin/ that you may need to run. Three of these scripts are automatically called by the Makefile, and check-config.sh is for your information.

1. check-config.sh

Run check-config.sh from the root directory (i.e. where the Makefile is) to check your configuration.

2. cluster-correct.sh

cluster-correct.sh takes the output of 3dttest++ and cluster corrects given certain p and alpha values. Here are the mandatory and optional arguments:

Mandatory:

  • -i * Input prefix, path to HEAD/BRIK files without +{orig,tlrc}.{HEAD,BRIK} suffixes.
  • -d * Set the degrees of freedom. (Previously not a mandatory option.)
  • -o * Output directory.
  • -D Group mode. Use this when comparing two groups, not just one.

Optional:

  • -1/2 Use a 1- or 2-sided t-test (default 2)
  • -a * Set custom alpha value (default 0.5)
  • -h Display help menu
  • -k Keep mode, don't delete intermediate files for debugging
  • -L Creates a log file in the output directory with parameters from the run.
  • -n [123] Which neighbor mode to use (default 3)
    • 1: Faces only
    • 2: Faces and edges
    • 3: Faces, edges, and corners
  • -p * Set custom p threshold (default 0.05), set value between 0.0-1.0. Not all p-values can be used, and cluster-correct.sh will warn you if you try to use one.

3. extract-all-briks.sh

This script extracts all the AFNI bricks to individual NIFTI files and names them appropriately. It takes 2+ arguments. The first is an output directory, and the second and beyond are BRIK files. All bricks from all BRIK files will be extracted to the given directory.

Each BRIK file must have a corresponding HEAD file, or else it will fail.

4. slice-all-that-match.sh

This script creates images using slicer for every NIFTI file you give it and outputs them to the same place as the input. It also annotates "EMPTY" onto empty files so you can be sure they are actually empty and not just missing from the given view.

III Citations

[1] Eklund, A., Nichols, T. E., & Knutsson, H. (2016). "Cluster failure: Why fMRI inferences for spatial extent have inflated false-positive rates. Proceedings of the National Academy of Sciences, 113(28), 7900–7905. https://doi.org/10.1073/pnas.1602413113

[2] Cox, R. W., Chen, G., Glen, D. R., Reynolds, R. C., & Taylor, P. A. (2017). FMRI Clustering in AFNI: False-Positive Rates Redux. Brain Connectivity, 7(3), 152–171. https://doi.org/10.1089/brain.2016.0475