Introduction

WEHI-SODA-Hub/spatialproteomics is a pipeline for running cell segmentation on COMET and MIBI data. For COMET, background subtraction can be performed followed by patched cellpose segmentation, or non-patched mesmer segmentation. For MIBI, mesmer segmentation can be run. Whole-cell and nuclear segmentations are run separately, and then consolidated into whole cells with nuclei with full shape and intensity measurements per compartment. The output GeoJSON files can be viewed in QuPath.

The pipeline uses the following tools:

• Background_subtraction -- background subtraction tool for COMET.
• MesmerSegmentation -- a CLI for running Mesmer segmentation of MIBI and OME-XML TIFFs.
• cellmeasurement -- a Groovy app that matches whole-cell segmentations with nuclei, and uses the QuPath API to calculate compartment measurements and intensities.
• sopa -- we use the sopa CLI tool to patchify images and perform cellpose segmentation.

Please see the docs for more detailed information on pipeline usage and output

Usage

[!NOTE] If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test (to test cellpose segmentation) or -profile test_mesmer to test mesmer segmentation before running the workflow on actual data.

If you are running this pipeline from WEHI, it has been set up to run on Seqera Platform.

[!NOTE] If you don't have a .gradle directory in your home, make sure you create it with mkdir $HOME/.gradle before runnning the pipeline. You con't need to do this if you are running via WEHI's Seqera Platform mentioned above.

Usage will depend on your desired steps. See usage docs for more detailed information.

Background subtraction

[!NOTE] This step will only work with COMET OME-TIF files.

Prepare a sample sheet as follows:

samplesheet.csv:

csv sample,run_backsub,run_mesmer,run_cellpose,tiff sample1,true,true,false,/path/to/sample1.tiff sample2,true,false,true,/path/to/sample2.tiff

You may also prefer to use YAML for your samplesheet, either is supported:

samplesheet.yml:

yaml - sample: sample1 run_backsub: true run_mesmer: true run_cellpose: false tiff: /path/to/sample1.tiff - sample: sample2 run_backsub: true run_mesmer: true run_cellpose: false tiff: /path/to/sample2.tiff

[!WARNING] Please ensure that your image name and all directories in your file path do not contain spaces.

If you don't specify any segmentation algorithm to run (mesmer or cellpose), the pipeline will run a background subtraction only.

Now, you can run the pipeline using:

bash nextflow run WEHI-SODA-Hub/spatialproteomics \ -profile <docker/singularity/.../institute> \ --input samplesheet.csv \ --outdir <OUTDIR>

[!WARNING] Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

Mesmer segmentation

Before running Mesmer, ensure that you have a deepcell access token and that you have set it in your Nextflow secrets:

bash nextflow secrets set DEEPCELL_ACCESS_TOKEN $YOUR_TOKEN

If you want to run Mesmer as your segmentation algorithm, you can specify a config file like so:

csv sample,run_backsub,run_mesmer,tiff,nuclear_channel,membrane_channels sample1,true,true,/path/to/sample1.tiff,DAPI,CD45:CD8 sample2,false,true,/path/to/sample2.tiff,DAPI,CD45

Nuclear channels only support one entry; membrane channels may have multiple values separated by : characters. If your channels have spaces in them, make sure that you surround your channel name with quotes. For example, CD45:"HLA I".

You can also set the segmentation parameters for mesmer either via CLI (e.g., --combine_method prod or in a config file pass to the workflow via -c. See usage for a full list.

[!WARNING] You cannot run both Mesmer and Cellpose segmentation on the same sample (with the same name). If you want to run both on a sample, put it on a different line and give it a different sample name.

Cellpose segmentation

If you want to run Cellpose as your segmentation algorithm, you can specify a config file like so:

csv sample,run_backsub,run_cellpose,tiff,nuclear_channel,membrane_channels sample1,true,true,/path/to/sample1.tiff,DAPI,CD45:CD8 sample2,false,true,/path/to/sample2.tiff,DAPI,CD45

As with Mesmer, nuclear channels only support one entry; membrane channels may have multiple values separated by : characters. You can also set the following parameters, either via CLI (e.g., --combine_method prod or in a config file pass to the workflow via -c. See usage for a full list.

Cellpose will run in a parallelised patched workflow using sopa. To control the patching process, you can use the patch_width_pixel and patch_overlap_pixel parameters.

If you want to skip measurements (this may take some time for large images), you can use set the parameter skip_measurements to true.

Dealing with large images

You can run the pipeline with different profiles for different size images:

• small: for images <150GB
• medium: for images <300GB
• large: for images <600GB

[!WARNING] If you are combining many membrane channels, using prod as the combine method may lead to large memory usage. In these cases, it is recommended to use max instead.

Credits

WEHI-SODA-Hub/spatialproteomics was originally written by the WEHI SODA-Hub.

We thank the following people for their extensive assistance in the development of this pipeline:

• Michael McKay (@mikemcka)
• Emma Watson

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

This pipeline was created using the nf-core template. You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.