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MicheleBortol edited this page Mar 21, 2021 · 20 revisions

Running SIMPLI

SIMPLI is run as a Nextflow pipeline:

nextflow run PATH_TO_SIMPLI/main.nf [COMMAND_LINE_ARGUMENTS] [NEXTFLOW_OPTIONS]

The most recent version of SIMPLI be downloaded and run from directly from this repository with:
nextflow run ciccalab/SIMPLI [COMMAND_LINE_ARGUMENTS] [NEXTFLOW_OPTIONS]

See the Nextflow documentation for more details.

Configuration file

In alternative all command line parameters can be specified through a configuration file: nextflow run main.nf -c CONFIGURATION_FILE

Parameters in the configuration file are specified in this format:

params.PARAMETER_NAME ="VALUE"

SIMPLI Parameters

SIMPLI accepts the following parameters (specified through the command line or a configuration file).

Input

These parameters specify the paths to the metadata files defining the samples and their associated images and channels:

  • sample_metadata_file = Metadata file defining all the samples used in the analysis.
  • tiff_input_metadata_file = Metadata file for TIFF input (single- or multi-channel).
  • raw_metadata_file = Metadata file for in input of Imaging Mass Cytometry data.
  • channel_metadata = Metadata file defining the channels for Imaging Mass Cytometry input data.

See the input page for their fields and formatting.

Selection of analysis steps

Choose which steps of the analysis to skip. Valid values are false and true.

  • skip_conversion = Do not perform the extraction of TIFF images from Imaging Mass Cytometry input data.
  • skip_normalization = Do not perform the 99th percentile normalization of the images by channnel and by sample.
  • skip_preprocessing = Do not perform image preprocessing with CellProfiler4.
  • skip_area = Do not perform the pixel-level analysis.
  • skip_segmentation = Do not perform cell segmentation with CellProfiler4.
  • skip_cell_type_identification = Do not perform cell masking.
  • skip_cell_clustering = Do not perform cell phenotyping by unsupervised clustering.
  • skip_cell_thresholding = Do not perform cell phenotyping by expression thresholding.
  • skip_homotypic_interactions = Do not perform homotypic interaction analysis.
  • skip_heterotypic_interactions = Do not perform heterotypic interaction analysis.
  • skip_visualization = Do not perform any of the following visualization steps.
  • skip_area_visualization = Do not plot the results of the pixel-level analysis.
  • skip_type_visualization = Do not plot the results of the cell masking.
  • skip_cluster_visualization = Do not plot the results of the cell phenotyping by unsupervised clustering.
  • skip_thresholding_visualization = Do not plot the results of the cell phenotyping by expression thresholding.
  • skip_homotypic_visualization = Do not plot the results of the homotypic interaction analysis.
  • skip_heterotypic_visualization = Do not plot the results of the heterotypic interaction analysis.

See the analysis page for details on each step of the analysis.

Step Specific input files and metadata:

  • normalized_metadata_file = Metadata file for normalized tiff images.
  • preprocessed_metadata_file = Metadata file for preprocessed tiff images.
  • single_cell_data_file = File with single cell data.
  • annotated_cell_data_file = File with single cell data annotated with cell identities.
  • clustered_cell_data_file = File with single cell data annotated with cell phenotypes from unsupervised clustering.
  • thresholded_cell_data_file = File with single cell data annotated with cell phenotypes from expression thresholding.
  • homotypic_interactions_file = File with single cell data annotated with spatial cell clusters from the homotypic interaction analysis.
  • heterotypic_interactions_file = File with cell-cell distances from the heterotypic interaction analysis.
  • single_cell_masks_metadata = Metadata file associating each sample to a UINT16 TIFF cell mask.
  • area_measurements_metadata = Metadata file with the parameters for the pixel-level analysis.
  • cell_clustering_metadata = Metadata file with the parameters for the cell phenotyping by unsupervised clustering.
  • cell_thresholding_metadata = Metadata file with the parameters for the cell phenotyping by expression thresholding.
  • cell_masking_metadata = Metadata file with the parameters for cell masking.
  • homotypic_interactions_metadata = Metadata file with the parameters for the homotypic interaction analysis.
  • heterotypic_interactions_metadata = Metadata file with the parameters for the heterotypic interaction analysis.

See the analysis page for the required inputs of each step of the analysis and their required fields and formats.

CellProfiler4 pipelines

These parameters specify the paths to the CellProfiler4 pipeline files:

  • cp4_preprocessing_cppipe =
  • cp4_segmentation_cppipe =

See the CellProfiler4 pipelines page for the requirements of a SIMPLI compatible CellProfiler4 pipeline.

Visualization

These parameters specify the colors used to generate the gradients representing different levels of gene expression in UMAPs and Heatmaps:

  • high_color = "'#FF0000'"
  • mid_color = "'#FFFFFF'"
  • low_color = "'#0000FF'"

Accepted values are color names or hexadecimal #RGB or #RGBA format ("#RRGGBB" or "#RRGGBBAA").

Output

  • output_folder = Specifies the path where all output will be collected.
  • tiff_type = single or ome. Output the preprocessed images as multiple single-channel tiff files, or as a single multi-channel .ome.tiff image.

See the output page for details on output paths and formatting.

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