⬅️ read the published article here
⬅️ read the preprint here
⬅️ Preprocessed Multiplexed Image Data and UTAG results

This package implements segmentation of multiplexed imaging data into microanatomical domains. Multiplexed imaging data types are typically imaging mass cytometry (IMC), co-detection by indexing (CODEX), multiplexed ion beam imaging by time of flight (MIBI-TOF), cyclic immunofluorescence (CyCIF), and others. The package also provides functions for the downstream analysis of the detected micro-anatomical structure.

pip install git+https://github.com/ElementoLab/utag.git@main

Installation should take less than 10 seconds.

There are no specific hardware requirements.

Software requirements:

• UTAG has been tested on Mac OS, Linux, and Windows WSL (Windows Subsystem for Linux).
• Python 3.7+ (tested on 3.8.2)
• Python packages (automatically installed by pip):
  • numpy
  • pandas
  • anndata
  • scanpy
  • parc
  • squidpy
  • scipy
  • matplotlib
  • tqdm
  • networkx
  • parmap
  • scikit-learn
  • setuptools_scm (may require manual installation for MacOS through pip)

Specific versions of Python packages have been pinned to the setup.cfg file.

Check out new tutorial for how to run and visualize results with UTAG on your own data with minimal required input (numeric cell feature matrix and xy coordinates).

The UTAG process can be run with a single function call utag.utag. The input is a AnnData object which should have the position of cells (typically centroids) in the spatial slot of adata.obsm. The function will output domain classes for each cell stored in the obs slot of the returned AnnData object.

Please refer to the notebook directory, and to the notebook on running UTAG on healthy lung data for a reproducible example. All data and respective results used for analysis can be downloaded from .

All data could alternatively be downloaded through command line:

pip install zenodo_get
zenodo_get -d 10.5281/zenodo.6376767 -o data

To run the method on multiple images/slides in batch mode:

from utag import utag

# Use Scanpy to get a h5ad file with provided data
import scanpy as sc
adata = sc.read(
    'data/healthy_lung_adata.h5ad',
    backup_url='https://zenodo.org/record/6376767/files/healthy_lung_adata.h5ad?download=1')

# Run UTAG on provided data
utag_results = utag(
    adata,
    slide_key="roi",
    max_dist=20,
    normalization_mode='l1_norm',
    apply_clustering=True,
    clustering_method = 'leiden', 
    resolutions = [0.05, 0.1, 0.3]
)

UTAG should take around ~2 min on a local machine for the batch mode on the data.

To run the method on a single image, pass None to the slide_key argument:

from utag import utag
utag_results = utag(
    adata,
    slide_key=None,
    max_dist=20,
    normalization_mode='l1_norm',
    apply_clustering=True,
    clustering_method = 'leiden', 
    resolutions = [0.05, 0.1, 0.3]
)

To visually inspect the results of the method:

import scanpy as sc
for roi in utag_results.obs['roi'].unique():
    result = utag_results[utag_results.obs['roi'] == roi].copy()
    sc.pl.spatial(result, color = 'UTAG Label_leiden_0.1', spot_size = 10)

Although UTAG greatly reduces manual labor involved in segmentation of microanatomical domains across, successful application of UTAG depends on three key user inputs. First is the max_dist parameter which defines the threshold distance. Second is the clustering resolution (under list of resolutions) to determine the coarsity of the clustering. Last is user interpretation of the resulting clusters to identify the structure.

We intentionally leave the optimization of max_dist open to users to maximize the applicability of UTAG to unseen datasets. This is because this parameter is tightly related with the resolution or magnification of the data under use. In our manuscript, we apply the method on IMC data and optical imaging-based CyCIF, which have different per unit area pixel densities. In the case of IMC, we suggest that a well working max_dist is between 10 and 20 as 1 pixel exactly maps to 1 micrometer. With an imaging-based technique like CyCIF, the optimal distance can vary with magnification, focal lengths, distance to tissue, and other factors, which make it hard to suggest a one-fits-all rule. Also there might be nuanced differences for the exact tissue of interest that may vary across specimens under examination.

We believe that the optimal clustering resolution is a hyperparameter that should be explored to suit their biological question of interest. For such reasons, we provide a list of resolutions as default to be explored by the users. A general rule here is that increasing the resolution parameter will return more refined substructures, while decreasing it will return coarser, more broad structures. We also recommend users to use a higher resolution parameter when screening for a rare structure, as a higher resolution will capture more structures, and vice versa. In our benchmarking, we saw that with the exception of extreme hyperparameter values, UTAG’s performance was fairly robust across various clustering resolutions (Extended Data Figure S3).

For more detailed usage of the package and downstream analysis, please refer to IMC Healthy Lung.ipynb in the documentation folder.

To make UTAG available to R users, we port the python code to using the reticulate package. The code was tested under after installing UTAG natively for python 3.8.10 and under conda environment with python 3.10.4, on Ubuntu 20.04.3 LTS and R 4.2.0.

Nonetheless, we highly recommend that users use our package in python for more involved analysis as the package has been developed and tested more thoroughly in python.

install.packages('reticulate')
library(reticulate)

# grab python interpreter
use_python('/usr/bin/python3') # in case UTAG is installed on native python
# use_condaenv('utag') # in case UTAG is installed using conda
# use_virtualevn('utag') # in case UTAG is installed under virtualenv


# import necessary python packages
utag <- import('utag')
scanpy <- import('scanpy')

# read anndata with cell expressions and and locations 
adata <- scanpy$read(
    'data/healthy_lung_adata.h5ad',
    backup_url='https://zenodo.org/record/6376767/files/healthy_lung_adata.h5ad?download=1'
)

# show general content of the data
print(adata)

# run UTAG
utag_results <- utag$utag(
    adata,
    slide_key = "roi",
    max_dist = 20,
    normalization_mode = 'l1_norm',
    apply_clustering = TRUE,
    clustering_method = 'leiden', 
    resolutions = c(0.05, 0.1, 0.3)
)

# show content of the data that now includes UTAG results for various resolutions
print(utag_results)

Also available as a R markdown file in.

We are happy to receive community contributions to UTAG through pull requests on Github.

Please run tests after re-installing the package:

pytest --pyargs utag

or before by:

pytest utag/tests/utag_test.py