Running pyHiM#

Prepare run#

  • Copy the TIFF images you want to process into a single folder.

    Note

    This folder will be called the input_directory

  • Copy or create a file named parameters.json into your input_directory.

    Note

    This file contains all the input parameters required to run pyHiM. You can download and unzip an example here.

  • Update the parameters.json file to indicate the reference cycle used for drift correction.

    Note

    This can be done by manually editing the parameters.json file or by running the graphical user interface provided in the script: pyhim_parameters (tutorial here).

Basic run#

  • Open a terminal (Linux) or a Anaconda Prompt (windows)

  • Move to the input_directory :

      cd input_directory

  • Activate your conda environment:

      conda activate pyHiM

  • To run the basic pyHiM pipeline :

    pyhim

    Note

    The basic pyHiM pipeline will:

    1. Project 3D images

    2. Calculate drift based on fiducial images

    3. Correct drift for nuclei and barcode images (i.e. registration)

    4. Segment and localize barcode spots

    5. Segment nuclei and masks

    6. Match spots to masks to build chromatin traces

    7. Build single-trace and ensemble pairwise distance maps

  • Each module will create and store its results in a single dedicated folder inside the input_directory. A summary of all outputs will also be provided as a markdown file HiM_Analysis<DDMMYYYY_HHMMSS>.md saved in the input_directory.

Optional arguments#

If you require help, you can call pyHiM with the help option :

pyhim -h

The output should look as follows:

usage: pyhim [-h] [-C CMD] [-F ROOTFOLDER] [-P PARAMETERS] [-T THREADS]

optional arguments:
  -h, --help            show this help message and exit
  -C CMD, --cmd CMD     Comma-separated list of routines to run. DEFAULT: 
                        project,register_global,register_local,mask_2d,
                        localize_2d,mask_3d,localize_3d,filter_localizations,
                        register_localizations,build_traces,build_matrix
  -F ROOTFOLDER, --rootFolder ROOTFOLDER
                        Folder path with input images. DEFAULT: Current
                        directory
  -P PARAMETERS, --parameters PARAMETERS
                        Path of the parameters.json file. DEFAULT: pyHiM
                        expect this file inside your current directory
  -T THREADS, --threads THREADS
                        Thread number to run with parallel mode. DEFAULT: 1
                        (sequential mode)

  • -F or --rootFolder indicates the rootFolder where pyHiM expects to find the dataset.

  • -C or --cmd is an optional argument that can be used to run a specific set of functions detailed as a comma separated list without space.

    Note

    The mode of action will be determined from the parameters.json configuration file.

  • --threads will ask pyHiM to run in parallel using multiple threads in your computer or computer cluster.

    Note

    To visualize the progress of your run, open your browser in http://localhost:8787 and make sure you connect by

    ssh -L 8787:localhost:8787 username@servername

Running in multiple folders#

To run pyHiM in multiple folders we recommend that you create a BASH script and provide the location of each input_directory that needs to be processed. For this, run pyHiM with this way:

pyhim -F <input_directory>

Where <input_directory> is the relative or absolute path to the folder containing your input data.