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Table of contents
  1. Input directory structure
    1. Note for MCMICRO users
  2. YAML configuration file
    1. General configurations
    2. Module configurations
  3. Markers.csv

Input directory structure

CyLinter can analyze any multiplex imaging data so long as they conform to the expected file formats and folder structure. In the below example, <sample#> corresponds to the name of a particular tissue sample.

<INPUT DIR>
├── cylinter_config.yml
├── csv/
│   ├── <sample1>.csv
│   └── <sample2>.csv
├── markers.csv
├── mask/
│   ├── <sample1>.ome.tif (or .tif)
│   └── <sample2>.ome.tif (or .tif)
├── seg/
│   ├── <sample1>.ome.tif (or .tif)
│   └── <sample2>.ome.tif (or .tif)
└── tif/
    ├── <sample1>.ome.tif (or .tif)
    └── <sample2>.ome.tif (or .tif)

Note for MCMICRO users

CyLinter can parse whole slide image (WSI) and tissue microarray (TMA) multiplex imaging data generated by the MCMICRO image-processing pipeline in their native file structure. In these cases, the MCMICRO output directory serves as the CyLinter input directory.

YAML configuration file

cylinter_config.yml is the YAML configuration file passed to the cylinter command on program execution. It specifies general program configurations and module-specific parameters for a given analysis and should be stored in the top level CyLinter input directory. The cylinter_config.yml file downloaded with the program is pre-configured for use with Example Data used to demonstrate CyLinter. On MacOS, this file is located here: /Users/<user>/miniconda3/envs/cylinter/lib/python3.10/site-packages/cylinter/cylinter_config.yml.

General configurations

ParameterDefaultDescription
inDir/Users/user/Desktop/cylinter_demoCyLinter input directory; contains multi-channel image files (TIFF/OME-TIFF), segmentation outline files (TIFF/OME-TIFF), cell ID masks (TIFF/OME-TIFF), single-cell spatial feature tables (CSV), cylinter_config.yml, and markers.csv organized according to the input directory structure or as native MCMICRO output structure.
outDir/Users/user/Desktop/cylinter_demo/outputCyLinter output directory path; created on program execution.
sampleMetadata“Filename”:
[“15”, “Glioblastoma”, “GBM”, “CANCER-TRUE”, 1]		Sample metadata dictionary: keys = Filenames (str); values = list of strings. First elements: sample names (str, may differ from Filename). Second elements: descriptive text of experimental condition (str). Third elements: abbreviation of experimental condition (str). Fourth elements: comma-delimited string of arbitrary binary declarations for computing t-statistics between two groups of samples (str). Fifth elements: replicate number specifying biological or technical replicates (int).
samplesToExclude[ ](list of strs) Sample names (i.e., first elements in sampleMetadata values) to exclude from analysis.
markersToExclude[ ](list of strs) Markers to exclude from analysis (not including nuclear dyes).

Module configurations

For module-specific configuration settings, see Modules

Markers.csv

markers.csv is a standard input file into the MCMICRO image-processing pipeline also used by CyLinter to index marker channels in a batch multiplex images labeled with the same markers. The file takes the following format and must be included in the top level CyLinter input directory:

channel_number,cycle_number,marker_name
1,1,<DNA1>
2,1,<abx1>
3,1,<abx2>
4,1,<abx3>
5,2,<DNA2>
6,2,<abx4>
7,2,<abx5>
8,2,<abx6>
.
.
.
  • Additional metadata columns may be present in the file, but are not currently read by CyLinter.