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Nicole Repina edited this page Mar 24, 2023 · 27 revisions

Welcome to the scMultipleX Wiki!

Installation

Create new conda environment with python 3.8+:

conda create -n scmpx python=3.8

Activate the conda environment:

conda activate scmpx

Install scMultipleX:

pip install git+https://github.com/fmi-basel/gliberal-scMultipleX.git

Demo Run of scMultipleX

  1. SSH into [email protected]:

ssh [email protected]

Note to Windows users: use power shell, or Putty.

  1. Create an output directory (ex. username/scMultiplex-demo-test)

mkdir /tungstenfs/scratch/gliberal/Users/MY_SAVE_DIR

  1. Copy demo config file (demo.ini) to own user folder

cp -t /tungstenfs/scratch/gliberal/Users/MY_SAVE_DIR /tungstenfs/scratch/gliberal/Code/Common/Repositories/gliberal-scMultipleX/resources/scMultipleX_testdata/demo.ini

  1. Check that demo.ini it is copied over:

ls /tungstenfs/scratch/gliberal/Users/MY_SAVE_DIR

You should see the demo.ini file listed in directory

  1. Edit this config file:
  • Remote into favorite virtual machine
  • Navigate to demo.ini and open it in favorite text editor (e.g. Notepad++)
  • Change base_dir_save to MY_SAVE_DIR path, save.
  1. This step needs to be performed only once per machine and user Make symlink in user home bin directory that points to run_scmultiplex executable:
  • cd $HOME
  • mkdir -p bin
  • ln -s -t bin /tungstenfs/scratch/gliberal/Code/Common/Repositories/gliberal-scMultipleX/run_scmultiplex
  • ls -l bin
  1. Run scMultipleX on test dataset

run_scmultiplex --help

run_scmultiplex --cpus 12 --config /tungstenfs/scratch/gliberal/Users/MY_SAVE_DIR/demo.ini --tasks 0 1 2 3 4 5 6 7

  1. Check output folder!

Configuring scMultipleX

[00BuildExperiment]

General parameters for initializing FAIM-HCS experiment structure

well_pattern = Regex pattern for recognizing well ID

raw_ch_pattern = Regex pattern for recognizing channel ID in raw image files

mask_ending = Suffix of organoid segmentation image

base_dir_raw = Path to raw data directory (folder contains rounds)

base_dir_save = Path to save directory

spacing = Z,Y,X pixel spacing of region-extracted data in um/pix, comma-separated

overview_spacing = Y,X pixel spacing of well overview images, comma-separated

round_names = Names of multiplexing rounds, comma-separated

[00BuildExperiment.round_R0]

Round-specific parameters for initializing FAIM-HCS experiment structure. Include this subsection for each round and update name, e.g. round_R1

name = Round name

nuc_ending = Suffix of nuclear segmentation image

mem_ending = Suffix of membrane segmentation image

root_dir = Path to raw data directory for this round

fname_barcode_index = Number of underscores in Yokogawa barcode, integer

organoid_seg_channel = Image channel used for organoid segmentation, e.g. C01

nuclear_seg_channel = Image channel used for nuclear segmentation, e.g. C01

membrane_seg_channel = Image channel used for membrane segmentation, e.g. C04

[01FeatureExtraction]

Parameters used during feature extraction

excluded_plates = Folder name of plate (e.g. day2,day3) to exclude from analysis, comma-separated

excluded_wells = Well ID to exclude from analysis (e.g. A01,C06), comma-separated

ovr_channel = Image channel used for organoid segmentation, e.g. C01

name_ovr = Naming of regionprops file; always keep as regionprops_ovr_

iop_cutoff = Float value 0 to 1 for cutoff threshold for calling a nucleus inside a membrane. Recommended value is 0.6
            iop = number of pixels in intersection of membrane and nuclear label / number of pixels in nuclear label
            Closer to 1 means better match

[02OrganoidLinking]

Parameters used during organoid linking

iou_cutoff = _ Float value 0 to 1 for cutoff threshold for matching RX to R0 object. Recommended value is 0.2_
            iou = number of pixels in intersection of R0 and RX object label / number of pixels in union of R0 and RX object label
            Closer to 1 means better match

Running scMultipleX

On virtual machines

scMultipleX is installed on tungsten at: scratch/gliberal/Code/Common/Repositories/gliberal-scMultipleX

and can be run on any Linux machine.

run_scmultiplex --cpus [NUM CORES, INT] --config [PATH TO .INI CONFIG] --tasks [TASKS TO RUN]

Use run_scmultiplex --help for details on arguments.

Note:

  • --cpus default is number of cores available for the process on the machine
  • --tasks available are integers 0 - 7

On CPU cluster

To run on CPU cluster, configure the clusterme.sh file From vcl1043, navigate to folder containing clusterme.sh and run:

sbatch clusterme.sh

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