STHD 1.0.1

STHD: probabilistic cell typing of single Spots in whole Transcriptome spatial data with High Definition

STHD: probabilistic cell typing of Single spots in whole Transcriptome spatial data with High Definition

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• Quick start: notebooks/tutorial.ipynb
• Generates single-spot (2um) cell type labels and probabilities for VisiumHD data using a machine learning model.
• Input: VisiumHD data and reference scRNA-seq dataset with cell type annotation.
• Output: cell type labels and probabilities at 2um spot level.
• Visualization - STHDviewer: interactive, scalable, and fast spatial plot of spot cell type labels, in a HTML.

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• Author: Yi Zhang, PhD, [e-mail]
• Website: Yi Zhang Lab at Duke
• STHDviewer of VisiumHD colon cancer sample with near 9 million spots: STHDviewer_colon_cancer_HD:https://yi-zhang-compbio-lab.github.io/STHDviewer_colon_cancer_hd
• We provided test data. Download this folder and put as ./testdata/

Install

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• python version requirement: >=3.8.0
• How to use
  • create new python venv python3.8 -m venv sthd_env
  • activate the venv source sthd_env/bin/activate
  • Install STHD from pip: pip install STHD
  • Or:
    • download repo: git clone [e-mail]:yi-zhang/STHD.git
    • install dependencies: pip install -r STHD/requirements.txt
    • making sure ./STHD is in python path, e.g adding via sys.path.append('./STHD')
    • then in script: from STHD import {the module you need}

STHD Quickstart using a colon cancer VisiumHD patch:

• See notebooks/tutorial.ipynb
• The test data includes a patch crop from the VisiumHD file in testdata/crop10

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STHD pipeline on a larger VisiumHD region, or the full VisiumHD sample:

Step 1: prepare normalized gene expression profile (lambda) by cell type from reference scRNA-seq data.

• This step will generate the reference file. Details are in notebooks/s01_build_ref_scrna.ipynb
• We provided the processed file ./testdata/crc_average_expr_genenorm_lambda_98ct_4618gs.txt

Step 2: pre-processing of VisiumHD data files

• The test data includes a larger region from the VisiumHD file in testdata/crop10large/

Preparing the VisiumHD sample.

• 10X Genomics colon cancer sample can be downloaded from: https://www.10xgenomics.com/datasets/visium-hd-cytassist-gene-expression-libraries-of-human-crc
• Required input includes 2um level spatial expression: square_002um , which usually contains filtered_feature_bc_matrix.h5 and spatial/tissue_positions.csv . It is often from the downloaded folder "Binned outputs (all bin levels)". tissue positions in .parquet format can be converted using STHD/hdpp.py.
• Required input also includes full-resolution H&E image: Visium_HD_Human_Colon_Cancer_tissue_image.btf. It is often from the "Microscope image".
• The scale factor number will also be useful, which is usually in square_002um/spatial/scalefactors_json.json
• Our processed data files are available as in: testdata/VisiumHD/

Step 3: Patchify the large region

• This step will take a large region and split into patches. Details are in notebooks/s11_patchify.ipynb
• Or, use example command line:

# Spliting patches from a test large cropped data:
python3 -m STHD.patchify \
--spatial_path ./testdata/crop10large/all_region/adata.h5ad.gzip \
--full_res_image_path ./testdata/crop10large/all_region/fullresimg_path.json \
--load_type crop \
--dx 1500 \
--dy 1500 \
--scale_factor 0.07973422 \
--refile ./testdata/crc_average_expr_genenorm_lambda_98ct_4618gs.txt \
--save_path ./testdata/crop10large_patchify \
--mode split

• For full sample, example command line below (will take some space and time)

# Spliting patches from the full-size VisiumHD sample:
python3 -m STHD.patchify \
--spatial_path ./testdata/VisiumHD/square_002um/ \
--counts_data filtered_feature_bc_matrix.h5 \
--full_res_image_path ./testdata/VisiumHD/Visium_HD_Human_Colon_Cancer_tissue_image.btf \
--load_type original \
--dx 6000 \
--dy 6000 \
--scale_factor 0.07973422 \
--refile ./testdata/crc_average_expr_genenorm_lambda_98ct_4618gs.txt \
--save_path ./analysis/full_patchify \
--mode split

Step 4: Obtain training command line for the patch list

• This step trains STHD on each patch. The command can be flexibly modified to submit to different slurm jobs on a HPC. Details are in notebooks/s12_per_patch_train.ipynb ,Or,
• Example command is:

python3 -m STHD.train --refile ./testdata/crc_average_expr_genenorm_lambda_98ct_4618gs.txt \
--patch_list ./testdata/crop10large/patches/52979_9480 ./testdata/crop10large/patches/57479_9480 ./testdata/crop10large/patches/52979_7980 ./testdata/crop10large/patches/55979_7980 ./testdata/crop10large/patches/57479_7980 ./testdata/crop10large/patches/54479_9480 ./testdata/crop10large/patches/55979_9480 ./testdata/crop10large/patches/54479_7980

Step 5: Combine the patch results

• This step combines STHD patch-wise results together. Details are in notebooks/s13_combine_patch.ipynb, Or

#Combine predictions
python3 -m STHD.patchify \
--refile ./testdata/crc_average_expr_genenorm_lambda_98ct_4618gs.txt \
--save_path ./testdata/crop10large_patchify \
--mode combine

Step 6: Visualize!

• This step takes STHD results on a large region and generate STHDviewer for interactive exploration. Details are in notebooks/s21_visualize.ipynb

Step 7: Downstream analyses

• One example is STHD-guided binning using a size of choice for —nspot
• Details are in notebooks/s04_STHD_cell_type_guided_binning.ipynb; Or.

python -m STHD.binning_fast --patch_path ./testdata/crop10/ --nspot 4 --outfile ./testdata/crop10_STHDbin_nspot4.h5ad

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Dependencies

requirements.txt

Reference

Sun C*, Yi Zhang*#. "STHD: probabilistic cell typing of single Spots in whole Transcriptome spatial data with High Definition". (2024) bioRxiv 2024.06.20.599803. Preprint link

Issues

Please contact Dr. Yi Zhang (yi.zhang[at]duke.edu) for desired addon features. You are welcome to follow our work by checking the Zhang lab at Duke!