BANKSY: Spatial Clustering Algorithm that Unifies Cell-Typing and Tissue Domain Segmentation (version 1.1.1)

Vipul Singhal*, Nigel Chou*, Joseph Lee, Yifei Yue, Jinyue Liu, Wan Kee Chock, Li Lin, YunChing Chang, Erica Teo, Hwee Kuan Lee, Kok Hao Chen^# and Shyam Prabhakar^#

Overview

This repository contains the code base and examples for Building Aggregates with a Neighborhood Kernel and Spatial Yardstick developed for: BANKSY: A Spatial Clustering Algorithm that Unifes Cell Typing and Tissue Domain Segmentation. BANKSY is a method for clustering spatial transcriptomic data by augmenting the transcriptomic profile of each cell with an average of the transcriptomes of its spatial neighbors.

By incorporating neighborhood information for clustering, BANKSY is able to:

1. Improve cell-type assignment in noisy data

2. Distinguish subtly different cell-types stratified by microenvironment

3. Identify spatial zones sharing the same microenvironment

BANKSY is applicable to a wide variety of spatial technologies (e.g. 10x Visium, Slide-seq, MERFISH) and scales well to large datasets. For more details on use-cases and methods, see the preprint.

This Python version of BANKSY (compatible with Scanpy), we show how BANKSY can be used for task 1 (improving cell-type assignment) using Slide-seq and Slide-seq V2 mouse cerebellum datasets. The R version of BANKSY is available here (https://github.com/prabhakarlab/Banksy).

Prerequisites

System requirements:

Machine with at least 16 GB of RAM. BANKSY is extremely scalable and fast even on CPU for large datasets.

Software requirements:

This software requires the following packages and has been tested on the following versions:

1. Python >= 3.8
2. Scanpy >= 1.8.1
3. Anndata >= 0.7.1
4. numpy >= 1.21
5. scipy >= 1.6
6. umap >= 0.5.1
7. scikit-learn >= 0.24.2
8. python-igraph >= 0.9
9. leidenalg

(Optional) As alternatives to leiden clustering, we also support mclust and louvain. To use these clustering algorithms, you need these additional packages:

For mclust

• R == 4.2.3
• r-mclust == 6.0.0
• rpy2 >= 3.4.0

For louvain (via sc.tl.louvain)

• louvain

Getting Started

Installation via Anaconda (recommended)

To use Banksy_py, we recommend setting up a conda environment and installing the prequisite packages, then cloning this repository.

  (base) $ conda create --name banksy
  (base) $ conda activate banksy
  (banksy) $ conda install -c conda-forge scanpy python-igraph leidenalg
  (banksy) $ git clone https://github.com/prabhakarlab/Banksy_py.git
  (banksy) $ cd Banksy_py

To run the examples presented in juypter notebooks, install the extensions for juypter.

  (banksy) $ conda install -c conda-forge jupyter

Try out BANKSY by running the examples in the provide ipython notebooks: slideseqv1_analysis.ipynb and/or slideseqv2_analysis.ipynb. More details on running BANKSY are provided within the notebooks.

To run the slideseq_v2 dataset, please to download the data from the original source and save it in the data/slide_seq/v2 folder.

Installation from environment.yml file

Users can directly install the prequisite packages (which replicates our Anaconda environment) from environment.yml here after cloning in this repository:

  (base) $ git clone https://github.com/prabhakarlab/Banksy_py.git
  (base) $ cd Banksy_py
  (base) $ conda env create --name banksy --file=environment.yml
  (base) $ conda activate banksy

Installation using pip from requirements.txt file

Users who have python=3.8-3.11 and pip can also install our environment from requirements.txt here after cloning in this repository:

  $ git clone https://github.com/prabhakarlab/Banksy_py.git
  $ cd Banksy_py
  $ pip install -r requirements.txt

Installation via PyPI (Coming Soon)

We are working on depositing this package in the PyPI repository for future users via pip install banksy.

General Steps of the BANKSY algorithm

To run BANKSY on a spatial single-cell expression dataset in anndata format:

1. Preprocess the gene-cell matrix using Scanpy. This includes filtering out cells and genes by various criteria, and (for sequencing-based technologies e.g. 10X Visium or Slide-seq) selecting the most highly variable genes.
2. intitalize_banksy to generate the spatial graph (stored in banksy_dict object).
3. run_banksy_multiparam to perform dimensionality reduction and clustering.

Note that individual BANKSY matrices (for given hyperparameter settings) can be accesed from the banksy_dict object. For example, to access the BANKSY matrix generated using scaled_gaussian decay and lambda = 0.2, use banksy_dict['scaled gaussian'][0.2]["adata"].

(optional) For advanced users who want to understand the entire BANKSY pipeline, you also can run individual steps below:

1. Preprocess gene-cell matrix (as above). z-score by gene using banksy.main.zscore or scanpy.pp.scale. Functions provided in the Scanpy package handle most of these steps. Parameters and filtering criterion may vary by spatial technology and dataset source.

2. Constructing the spatial graph which defines spatial neighbour relationships using banksy.main.generate_spatial_weights_fixed_nbrs. This outputs a sparse adjacency matrix defining the graph. Visualize these with banksy_utils.plotting.plot_graph_weights.
   Some parameters that affect this step are:

   • The spatial graph can be generated via the $k_{geom}$ parameter, which connects a cell to its $k_{geom}$ nearest neighbours. This spatial graph is the basis in which the neighbourhood matrix $M$ and the azimuthal gabor filter (AGF) matrix $G$ is constructed.

   • decay types: By default, we recommend scaled_gaussian, which weights a cell's neighbour expression as a gaussian envelope. Alternative methods include uniform which weights all neighbours equally, reciprocal weights neighbours by $1/r$ where $r$ is the distance from neighbouring cell to the index cell. ranked ranks neighbouring cells by distance with farther cells having higher rank, then sets Gaussian decay by rank. Sum of neighbour weights are always normalized to 1 for each cell.

   • generate_spatial_weights_fixed_radius (not used in paper) generates a spatial graph where each cell is connected to all cells within a given radius. This leads to variable numbers of neighbours per cell.

3. Generate neighbour expression matrix $N$ (n_cells by n_genes) using spatial graph to average over spatial neighbours. The neighbourhood matrix can be computed by sparse matrix multiplication of the spatial graph's adjacency matrix with the gene-cell matrix. Similarly, the AGF matrix $G$ (n_cells by n_genes) which represents the magnitude of expression gradient is also generated from the azimuthal transform.

4. Scale original expression matrix by $√(1 - λ)$ and neighbour expression matrix by √(λ) and concatenate matrices to obtain neighbour-augmented expression matrix (n_cells by 2n_genes) using banksy.main.weighted_concatenate with neighbourhood_contribution = $λ$. These operations are performed on the numerical data adata.X; use banksy.main.bansky_matrix_to_adata to recover Anndata object with the appropriate annotations.

The following steps are identical to single cell RNA seq analysis:

5. Dimensionality reduction, particularly PCA to reduce expression matrix (either neighbour-augmented or original for comparison) to (n_cells by n_{PCA_dims}). As a default, we set $PCA_{dims}$ = 20.

6. Clustering cells by finding neighbours in expression space and cluster using graph-based clustering. Here we find expression-neighbours and perform Leiden clustering following the implemenation in Giotto.

7. Refinement (Optional) In the prescene of noisy clusters, we offer an optional refinement step via banksy_utils.refine_clusters to smooth labels in the clusters exclusively for domain segmentation tasks. However, we do not recommend the use of excessive refinement as it wears out fine-grained domains.

Other useful tools in package

• banksy.main.LeidenPartition
  Finds neighbours in expression space and performs Leiden clustering. Aims to replicate implementation from the Giotto package as of 2020 to align with R version of the code. Note that scanpy also has a Leiden clustering implemenation with a different procedure for defining expression neighbours that can be used as an alternative. BANKSY is compatible with any clustering algorithm that takes a feature-cell matrix as input.

• labels.Label
  Object for convenient computation with class labels. Converts labels to sparse one-hot vector for fast computation of connectivity across clusters with spatial graph. To obtain an array of integer labels in the usual format (e.g. [1, 1, 5, 2, ...]), use Label.dense.

Examples to get started

We recommend the following examples to get started with the BANKSY package

1. Analyzing Slideseqv1 dataset with BANKSY
2. Analyzing Slideseqv2 dataset with BANKSY
3. Analyzing Starmap dataset

Reproducing results from our manuscript

To reproduce the results from our manuscript, please use the branch BANKSY-manuscript.

Contributing

Bug reports, questions, request for enhancements or other contributions can be raised at the issue page. Our team will attempt to resolve them best the we could.

Authors

• Nigel Chou (https://github.com/chousn)

• Yifei Yue (https://github.com/yifei-1021)

Acknowledgments

• Vipul Singhal - developed R version of BANKSY, compatible with seurat - (https://github.com/vipulsinghal02)

• Joseph Lee - developed R version of BANKSY, compatible with seurat - (https://github.com/jleechung)

Refer to requirements.txt for the supported versions of different packages

Citations

If you want to use or cite BANKSY, please refer to the following paper:

BANKSY unifies cell typing and tissue domain segmentation for scalable spatial omics data analysis 

which can be accessed here: [https://www.nature.com/articles/s41588-024-01664-3]

License

This project is licensed under The GPLV3 license. See the LICENSE.md file for details.