API usage

In [1]:

import spatialdata
import sopa.segmentation
import sopa.io

import spatialdata import sopa.segmentation import sopa.io

Create a SpatialData object¶

For this tutorial, we use a generated dataset. You can expect a total runtime of a few minutes.

To load your own data, see the commented lines below, or read the sopa.io API.

In [2]:

# The line below creates a toy dataset for this tutorial
# To load your own data, such as MERSCOPE data, you can do `sdata = sopa.io.merscope("/path/to/region_0")`
# For more details, see https://gustaveroussy.github.io/sopa/api/io/
sdata = sopa.io.uniform()

sdata

# The line below creates a toy dataset for this tutorial # To load your own data, such as MERSCOPE data, you can do `sdata = sopa.io.merscope("/path/to/region_0")` # For more details, see https://gustaveroussy.github.io/sopa/api/io/ sdata = sopa.io.uniform() sdata

[INFO] (sopa.utils.data) Image of size ((4, 2048, 2048)) with 400 cells and 100 transcripts per cell

Out[2]:

SpatialData object with:
├── Images
│     └── 'image': SpatialImage[cyx] (4, 2048, 2048)
├── Points
│     └── 'transcripts': DataFrame with shape: (<Delayed>, 4) (3D points)
└── Shapes
      └── 'cells': GeoDataFrame shape: (400, 1) (2D shapes)
with coordinate systems:
▸ 'global', with elements:
        image (Images), transcripts (Points), cells (Shapes)
▸ 'microns', with elements:
        transcripts (Points)

Before starting, we create the variables below that denotes the names of the image and transcripts that we want to use, as displayed in the SpatialData object above:

In [3]:

image_key = "image"
points_key = "transcripts" # (ignore this for multiplex imaging)
gene_column = "genes" # (optional) column of sdata[points_key] containing the gene names

image_key = "image" points_key = "transcripts" # (ignore this for multiplex imaging) gene_column = "genes" # (optional) column of sdata[points_key] containing the gene names

Segmentation¶

Option 1: Cellpose¶

First, we generate the bounding boxes of the patches on which Cellpose will be run. Here, the patches have a width and height of 1500 pixels and an overlap of 50 pixels. We advise bigger sizes for real datasets (see our default parameters in one of our config files). On the toy dataset, this will generate 4 patches.

In [16]:

patches = sopa.segmentation.Patches2D(sdata, image_key, patch_width=1200, patch_overlap=50)
patches.write();

patches = sopa.segmentation.Patches2D(sdata, image_key, patch_width=1200, patch_overlap=50) patches.write();

[INFO] (sopa.segmentation.patching) 4 patches were saved in sdata['sopa_patches']

The following channels are available for segmentation. Choose one or two channels used by Cellpose.

In [17]:

from sopa._sdata import get_spatial_image

print(get_spatial_image(sdata, image_key).c.values)

from sopa._sdata import get_spatial_image print(get_spatial_image(sdata, image_key).c.values)

['DAPI' 'CK' 'CD3' 'CD20']

Then, we initialize the Cellpose model. Here, we run segmentation using DAPI only, and we set the cell diameter to be about 35 pixels:

In [18]:

channels = ["DAPI"]

method = sopa.segmentation.methods.cellpose_patch(diameter=35, channels=channels, flow_threshold=2, cellprob_threshold=-6)
segmentation = sopa.segmentation.StainingSegmentation(sdata, method, channels, min_area=2500)

# The cellpose boundaries will be temporary saved here. You can choose a different path
cellpose_temp_dir = "tuto.zarr/.sopa_cache/cellpose"

channels = ["DAPI"] method = sopa.segmentation.methods.cellpose_patch(diameter=35, channels=channels, flow_threshold=2, cellprob_threshold=-6) segmentation = sopa.segmentation.StainingSegmentation(sdata, method, channels, min_area=2500) # The cellpose boundaries will be temporary saved here. You can choose a different path cellpose_temp_dir = "tuto.zarr/.sopa_cache/cellpose"

Sequential running¶

If desired, you can run Cellpose sequentially, as in the lines below, but this is slower than the "Parallel running" section below.

In [ ]:

segmentation.write_patches_cells(cellpose_temp_dir)

segmentation.write_patches_cells(cellpose_temp_dir)

Parallel running¶

Here, we show how to run Cellpose in parallel for all the patches. It's up to you to choose the way to parallelize it: for instance, if you have a Slurm cluster, you can run one job per patch.

Below, we run segmentation on each patch, and save the results in a temporary directory (here, tuto.zarr/.sopa_cache). On this example, we performed a "for-loop", so you should paralellize this yourself using multiple jobs or multi-threading (see this discussion for more details). Note that you can also use our Snakemake pipeline that will handle the parallelization for you.

In [7]:

# parallelize this for loop yourself (or use the Snakemake pipeline)
for patch_index in range(len(sdata['sopa_patches'])):
    segmentation.write_patch_cells(cellpose_temp_dir, patch_index)

# parallelize this for loop yourself (or use the Snakemake pipeline) for patch_index in range(len(sdata['sopa_patches'])): segmentation.write_patch_cells(cellpose_temp_dir, patch_index)

[INFO] (sopa.segmentation.shapes) Percentage of non-geometrized cells: 0.71% (usually due to segmentation artefacts)
[INFO] (sopa.segmentation.shapes) Percentage of non-geometrized cells: 0.00% (usually due to segmentation artefacts)
[INFO] (sopa.segmentation.shapes) Percentage of non-geometrized cells: 1.00% (usually due to segmentation artefacts)
[INFO] (sopa.segmentation.shapes) Percentage of non-geometrized cells: 1.37% (usually due to segmentation artefacts)

Resolving conflicts¶

At this stage, you executed 4 times Cellpose (once per patch). Now, we need to resolve the conflict, i.e. where boundaries are overlapping due to segmentation on multiple patches:

In [20]:

cells = sopa.segmentation.StainingSegmentation.read_patches_cells(cellpose_temp_dir)
cells = sopa.segmentation.shapes.solve_conflicts(cells)

shapes_key = "cellpose_boundaries" # name of the key given to the cells in sdata.shapes

sopa.segmentation.StainingSegmentation.add_shapes(sdata, cells, image_key, shapes_key)

cells = sopa.segmentation.StainingSegmentation.read_patches_cells(cellpose_temp_dir) cells = sopa.segmentation.shapes.solve_conflicts(cells) shapes_key = "cellpose_boundaries" # name of the key given to the cells in sdata.shapes sopa.segmentation.StainingSegmentation.add_shapes(sdata, cells, image_key, shapes_key)

Reading patches: 100%|██████████| 4/4 [00:00<00:00, 434.64it/s]
[INFO] (sopa.segmentation.stainings) Found 388 total cells
Resolving conflicts: 100%|██████████| 72/72 [00:00<00:00, 7784.85it/s]
[INFO] (sopa.segmentation.stainings) Added 367 cell boundaries in sdata['cellpose_boundaries']

Option 2: Baysor¶

In [4]:

shapes_key = "baysor_boundaries" # the name that we will give to the baysor "shapes"

shapes_key = "baysor_boundaries" # the name that we will give to the baysor "shapes"

Baysor needs a config to be executed. You can find official config examples here.

You can also reuse the Baysor parameter we have defined for each machine, as in our Snakemake config files. Note that, our Snakemake config is a .yaml file, but the Baysor config should still be a .toml file.

For this tutorial, we will use the config below. Instead of a dictionnary, you can also have a .toml file and provide config_path to the patchify_transcripts function.

In [23]:

config = {
    "data": {
        "force_2d": True,
        "min_molecules_per_cell": 10,
        "x": "x",
        "y": "y",
        "z": "z",
        "gene": "genes",
        "min_molecules_per_gene": 0,
        "min_molecules_per_segment": 3,
        "confidence_nn_id": 6
    },
    "segmentation": {
        "scale": 3,  # Important parameter: typical cell diameter, in microns (see our configs)
        "scale_std": "25%",
        "prior_segmentation_confidence": 0,
        "estimate_scale_from_centers": False,
        "n_clusters": 4,
        "iters": 500,
        "n_cells_init": 0,
        "nuclei_genes": "",
        "cyto_genes": "",
        "new_component_weight": 0.2,
        "new_component_fraction": 0.3
    }
}

config = { "data": { "force_2d": True, "min_molecules_per_cell": 10, "x": "x", "y": "y", "z": "z", "gene": "genes", "min_molecules_per_gene": 0, "min_molecules_per_segment": 3, "confidence_nn_id": 6 }, "segmentation": { "scale": 3, # Important parameter: typical cell diameter, in microns (see our configs) "scale_std": "25%", "prior_segmentation_confidence": 0, "estimate_scale_from_centers": False, "n_clusters": 4, "iters": 500, "n_cells_init": 0, "nuclei_genes": "", "cyto_genes": "", "new_component_weight": 0.2, "new_component_fraction": 0.3 } }

Then, we generate the bounding boxes of the patches on which Baysor will be run. Here, the patches have a width and height of 3000 microns and an overlap of 50 microns. We advise bigger sizes for real datasets (see our default parameters in one of our config files). On the toy dataset, this will generate 1 patch.

In [24]:

# The cellpose boundaries will be temporary saved here. You can choose a different path
baysor_temp_dir = "tuto.zarr/.sopa_cache/baysor"

patches = sopa.segmentation.Patches2D(sdata, points_key, patch_width=3000, patch_overlap=50)
valid_indices = patches.patchify_transcripts(baysor_temp_dir, config=config)

# The cellpose boundaries will be temporary saved here. You can choose a different path baysor_temp_dir = "tuto.zarr/.sopa_cache/baysor" patches = sopa.segmentation.Patches2D(sdata, points_key, patch_width=3000, patch_overlap=50) valid_indices = patches.patchify_transcripts(baysor_temp_dir, config=config)

[INFO] (sopa.patches.patches) Writing sub-CSV for transcript segmentation

[########################################] | 100% Completed | 211.89 ms

[INFO] (sopa.patches.patches) Patches saved in directory tuto.zarr/.sopa_cache/baysor

Now, we can run Baysor on each individual patch. It's up to you to choose the way to parallelize it: for instance, if you have a Slurm cluster, you can run one job per patch.

Below, we run segmentation on each patch, and save the results in a temporary directory (here, tuto.zarr/.sopa_cache/baysor). On this example, we performed a "for-loop", so you should paralellize this yourself using multiple jobs or multi-threading (see this discussion for more details). Note that you can also use our Snakemake pipeline that will handle the parallelization for you.

NB: depending on you Baysor installation, you may need to update the baysor_executable_path variable to locate the Baysor binary executable

In [25]:

import subprocess

baysor_executable_path = "~/.julia/bin/baysor"

import subprocess baysor_executable_path = "~/.julia/bin/baysor"

In [ ]:

for patch_index in valid_indices:
    command = f"""
    cd {baysor_temp_dir}/{patch_index}
    {baysor_executable_path} run --save-polygons GeoJSON -c config.toml transcripts.csv
    """
    subprocess.run(command, shell=True)

for patch_index in valid_indices: command = f""" cd {baysor_temp_dir}/{patch_index} {baysor_executable_path} run --save-polygons GeoJSON -c config.toml transcripts.csv """ subprocess.run(command, shell=True)

At this stage, you executed Baysor on each patch. Now, we need to resolve the conflict, i.e. where boundaries are overlapping due to segmentation on multiple patches (although, for this tutorial, there is one patch so there is no conflict):

In [27]:

from sopa.segmentation.transcripts import resolve

resolve(sdata, baysor_temp_dir, gene_column, min_area=10)

from sopa.segmentation.transcripts import resolve resolve(sdata, baysor_temp_dir, gene_column, min_area=10)

[INFO] (sopa.segmentation.transcripts) Cells whose area is less than 10 microns^2 will be removed
Reading transcript-segmentation outputs: 100%|██████████| 1/1 [00:00<00:00, 21.16it/s]
Resolving conflicts: 0it [00:00, ?it/s]
/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/spatialdata/_core/_elements.py:92: UserWarning: Key `baysor_boundaries` already exists. Overwriting it.
  self._check_key(key, self.keys(), self._shared_keys)
/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/spatialdata/_core/_elements.py:112: UserWarning: Key `table` already exists. Overwriting it.
  self._check_key(key, self.keys(), self._shared_keys)
[INFO] (sopa.segmentation.transcripts) Added sdata.tables['table'], and 373 cell boundaries to sdata['baysor_boundaries']

Aggregate¶

This mandatory step turns the data into an AnnData object. We can count the transcript inside each cell, and/or average each channel intensity inside each cell boundary.

NB: Baysor already counts the transcripts inside each cell to create a cell-by-gene table, so you don't need to provide gene_column

In [28]:

aggregator = sopa.segmentation.Aggregator(sdata, image_key=image_key, shapes_key=shapes_key)

aggregator.compute_table(gene_column=gene_column, average_intensities=True)

aggregator = sopa.segmentation.Aggregator(sdata, image_key=image_key, shapes_key=shapes_key) aggregator.compute_table(gene_column=gene_column, average_intensities=True)

[INFO] (sopa.segmentation.aggregate) Using existing table for aggregation
[WARNING] (sopa.segmentation.aggregate) sdata.table is already existing. Transcripts are not count again.
[INFO] (sopa.segmentation.aggregate) Averaging channels intensity over 373 cells with expansion 0.0

[########################################] | 100% Completed | 234.10 ms

/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/spatialdata/_core/_elements.py:92: UserWarning: Key `baysor_boundaries` already exists. Overwriting it.
  self._check_key(key, self.keys(), self._shared_keys)
/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/spatialdata/_core/_elements.py:112: UserWarning: Key `table` already exists. Overwriting it.
  self._check_key(key, self.keys(), self._shared_keys)

In [29]:

sdata

sdata

Out[29]:

SpatialData object with:
├── Images
│     └── 'image': SpatialImage[cyx] (4, 2048, 2048)
├── Points
│     └── 'transcripts': DataFrame with shape: (<Delayed>, 4) (3D points)
├── Shapes
│     ├── 'baysor_boundaries': GeoDataFrame shape: (373, 1) (2D shapes)
│     └── 'cells': GeoDataFrame shape: (400, 1) (2D shapes)
└── Tables
      └── 'table': AnnData (373, 5)
with coordinate systems:
▸ 'global', with elements:
        image (Images), transcripts (Points), baysor_boundaries (Shapes), cells (Shapes)
▸ 'microns', with elements:
        transcripts (Points), baysor_boundaries (Shapes)

Now, sdata.tables["table"] is an AnnData object.

• If you count the transcripts, then adata.X are the raw counts
• If you average the channel intensities, then adata.X are the channels intensities
• If you both count the transcript and average the intensities, then adata.X are the raw counts, and adata.obsm["intensities"] are the channels intensities

Annotation¶

Option 1: Transcript-based (Tangram)¶

Tangram is a transcript-based annotation that uses an annotated single-cell reference. Let's suppose your reference AnnData object is stored in a file called adata_reference.h5ad (preferably, keep raw counts), and the cell type is in adata.obs["cell_type"]. Then, you can annotate your spatial data as follows:

In [6]:

from sopa.annotation.tangram import tangram_annotate
import anndata

from sopa.annotation.tangram import tangram_annotate import anndata

In [ ]:

adata_reference = anndata.read_h5ad("adata_reference.h5ad")

tangram_annotate(sdata, adata_reference, "cell_type")

adata_reference = anndata.read_h5ad("adata_reference.h5ad") tangram_annotate(sdata, adata_reference, "cell_type")

Option 2: Staining-based¶

For now, our fluorescence-based annotation is very simple. We provide a dictionary where a channel is associated with a population. Then, each cell is associated with the cell type whose corresponding channel is the brightest (according to a certain Z-score). In this tutorial example, we can annotate Tumoral cells, T cells, and B cells:

In [23]:

from sopa.annotation import higher_z_score

marker_cell_dict = {
    "CK": "Tumoral cell",
    "CD20": "B cell",
    "CD3": "T cell"
}

higher_z_score(sdata.tables["table"], marker_cell_dict)

from sopa.annotation import higher_z_score marker_cell_dict = { "CK": "Tumoral cell", "CD20": "B cell", "CD3": "T cell" } higher_z_score(sdata.tables["table"], marker_cell_dict)

[INFO] (sopa.annotation.fluorescence) Annotation counts: cell_type
Tumoral cell    128
T cell          121
B cell          118
Name: count, dtype: int64

Pipeline report¶

You can optionally create an HTML report of the pipeline run (in the example below, we save it under report.html). It contains some quality controls for your data.

In [24]:

sopa.io.write_report("report.html", sdata)

sopa.io.write_report("report.html", sdata)

[INFO] (sopa.io.report.generate) Writing general_section
[INFO] (sopa.io.report.generate) Writing cell_section
[INFO] (sopa.io.report.generate) Writing channel_section
[INFO] (sopa.io.report.generate) Writing transcripts_section
[INFO] (sopa.io.report.generate) Writing representation_section
[INFO] (sopa.io.report.generate) Computing UMAP on 367 cells
/Users/quentinblampey/mambaforge/envs/sopa/lib/python3.10/site-packages/scanpy/plotting/_tools/scatterplots.py:394: UserWarning: No data for colormapping provided via 'c'. Parameters 'cmap' will be ignored
  cax = scatter(
[INFO] (sopa.io.report.generate) Writing report to report.html

Visualization¶

With the Xenium Explorer¶

The Xenium Explorer is a software developed by 10X Genomics for visualizing spatial data, and it can be downloaded freely here. Sopa allows the conversion to the Xenium Explorer, whatever the type of spatial data you worked on. It will create some files under a new tuto.explorer directory:

In [25]:

sopa.io.write("tuto.explorer", sdata, image_key, points_key=points_key, gene_column=gene_column)

sopa.io.write("tuto.explorer", sdata, image_key, points_key=points_key, gene_column=gene_column)

[INFO] (sopa.io.explorer.table) Writing table with 5 columns
[INFO] (sopa.io.explorer.table) Writing 3 cell categories: region, slide, cell_type
[INFO] (sopa.io.explorer.shapes) Writing 367 cell polygons
[INFO] (sopa.io.explorer.points) Writing 40000 transcripts
[INFO] (sopa.io.explorer.points)    > Level 0: 40000 transcripts
[INFO] (sopa.io.explorer.points)    > Level 1: 10000 transcripts
[INFO] (sopa.io.explorer.images) Writing multiscale image with procedure=semi-lazy (load in memory when possible)
[INFO] (sopa.io.explorer.images)    (Loading image of shape (4, 2048, 2048)) in memory
[INFO] (sopa.io.explorer.images)    > Image of shape (4, 2048, 2048)
[INFO] (sopa.io.explorer.images)    > Image of shape (4, 1024, 1024)
[INFO] (sopa.io.explorer.images)    > Image of shape (4, 512, 512)
[INFO] (sopa.io.explorer.images)    > Image of shape (4, 256, 256)
[INFO] (sopa.io.explorer.images)    > Image of shape (4, 128, 128)
[INFO] (sopa.io.explorer.images)    > Image of shape (4, 64, 64)
[INFO] (sopa.io.explorer.converter) Saved files in the following directory: tuto.explorer
[INFO] (sopa.io.explorer.converter) You can open the experiment with 'open tuto.explorer/experiment.xenium'

If you have downloaded the Xenium Explorer, you can now open the results in the explorer: open tuto.explorer/experiment.xenium (if using a Unix operating system), or double-click on the latter file.

With spatialdata-plot¶

spatialdata-plot library is a static plotting library for SpatialData objects

In [20]:

import spatialdata_plot

import spatialdata_plot

In [31]:

sdata\
    .pl.render_points(size=0.01, color="r", alpha=0.5)\
    .pl.render_images()\
    .pl.render_shapes(shapes_key, outline=True, fill_alpha=0, outline_color="w")\
    .pl.show("global")

sdata\ .pl.render_points(size=0.01, color="r", alpha=0.5)\ .pl.render_images()\ .pl.render_shapes(shapes_key, outline=True, fill_alpha=0, outline_color="w")\ .pl.show("global")

INFO     Value for parameter 'color' appears to be a color, using it as such.                                      

/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/anndata/_core/aligned_df.py:67: ImplicitModificationWarning: Transforming to str index.
  warnings.warn("Transforming to str index.", ImplicitModificationWarning)
/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/spatialdata/_core/_elements.py:102: UserWarning: Key `transcripts` already exists. Overwriting it.
  self._check_key(key, self.keys(), self._shared_keys)
/Users/quentinblampey/mambaforge/envs/spatial/lib/python3.10/site-packages/spatialdata_plot/pl/render.py:320: UserWarning: No data for colormapping provided via 'c'. Parameters 'cmap', 'norm' will be ignored
  _cax = ax.scatter(
Clipping input data to the valid range for imshow with RGB data ([0..1] for floats or [0..255] for integers).

Save your SpatialData object¶

You can save your SpatialData object for later use. This will create a .zarr directory.

In [ ]:

sdata.write("tuto.zarr")

sdata.write("tuto.zarr")

You can then open the data with spatialdata:

In [ ]:

import spatialdata

spatialdata.read_zarr("tuto.zarr")

import spatialdata spatialdata.read_zarr("tuto.zarr")

Further analyses¶

• You can read this tutorial on spatial statistic and geometric analysis.
• You can use Squidpy which operates on both the SpatialData object or the AnnData object, or use other tools of the scverse ecosystem such as Scanpy.
• You can also try the CLI or the Snakemake pipeline of Sopa.