The following output files from Xenium Onboard Analysis (XOA) are required as inputs for Xenium Ranger:

1. transcripts.zarr.zip or transcripts.zarr
2. cells.zarr.zip or cells.zarr
3. morphology.ome.tif
4. morphology_mip.ome.tif
5. morphology_focus.ome.tif
6. experiment.xenium
7. metrics_summary.csv
8. analysis_summary.html
9. gene_panel.json (only required for XOA v1.0 - 1.4)

These files should be located in one directory, which will be specified by the --xenium-bundle argument. For descriptions of these files, see the Xenium Onboard Analysis documentation.

The following file formats are compatible for use with the import-segmentation pipeline:

• Labeled mask in TIFF (32-bit) or NumPy NPY format
• Polygons in GeoJSON format (FeatureCollection type)
• Baysor output CSV and GeoJSON file formats

This pipeline has been tested using segmentation files generated by Cellpose v2.2.2, QuPath v0.4, and Baysor v0.6.

The polygon GeoJSON should be exported as a FeatureCollection. Features should be continuous with no holes and have geometry of type Polygon (i.e., no type MultiPolygon). The cells and nuclei can be saved in the same or separate GeoJSON files. The format should look similar to this example:

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{
   "type": "FeatureCollection",
   "features": [
     {
       "type": "Feature",
       "id": "12059192-3b27-4438-96dc-97b41ca84717",
       "geometry": {
         "type": "Polygon",
         "coordinates": [
           [
             [3418.94, 2],
             [3414.62, 3.85],
             [3414.12, 22.65],
             [3415.06, 27.26],
             [3420.1, 35.15],
             [3428.03, 40.22],
             [3437.01, 42.94],
             [3440.69, 45.87],
             [3445.32, 46.71],
             [3468.85, 46.71],
             [3477.88, 44.35],
             [3534.35, 44.31],
             [3546.79, 37.83],
             [3552.28, 30.11],
             [3552.94, 25.45],
             [3552.93, 6.63],
             [3550.68, 2.49],
             [3546, 2],
             [3418.94, 2]
           ]
         ]
       }
     }
   ]
}

Segmentation results from QuPath are compatible with Xenium Ranger and have these specifications:

• The feature objectType should be cell, as features with a non-cell objectType will be ignored (i.e., annotations).
• The --nuclei argument will use the nucleusGeometry polygon if it exists in the GeoJSON, otherwise it will use the geometry polygon. The --cells argument will use the geometry polygon.
• QuPath exports nucleus and cell segmentation results in one file, so the same GeoJSON file should be specified for both --cells and --nuclei. The format should look similar to this example:

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{
  "type":"FeatureCollection",
  "features": [
    {
      "type":"Feature",
      "id":"fd0c3d4e-6146-427d-9696-97fbe7adb63d",
      "geometry":{
        "type":"Polygon",
        "coordinates":[
          [
           [4348.52, 0],
           [4344.02, 1.37],
           [4341.18, 10.23],
           [4341.8, 24.31],
           [4346.73, 32.28],
           [4353.27, 38.99],
           [4361.18, 44],
           [4379.94, 44.48],
           [4388.64, 40.97],
           [4396.11, 35.26],
           [4404.71, 21.36],
           [4404.27, 2.55],
           [4400.29, 0.06],
           [4348.52, 0]
           ]
          ]
      },
      "nucleusGeometry":{
        "type":"Polygon",
        "coordinates":[
          [
           [4373.91, 4.81],
           [4366.61, 9.07],
           [4364.28, 12.61],
           [4364.3, 16.85],
           [4370.37, 22.75],
           [4378.35, 20.24],
           [4382.75, 13.06],
           [4380.87, 9.26],
           [4373.91, 4.81]
           ]
          ]
      },
      "properties":{
        "objectType":"cell"
      }
    }
  ]
}

Segmentation results from Baysor are compatible with Xenium Ranger and have these specifications:

• Baysor segmentation GeoJSON should be of type GeometryCollection, where each geometry is of type Polygon.
• The Polygon should contain an extra field called cell to identify which cell the polygon corresponds to.

Here is an example of the format:

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{
  "geometries": [
    {
      "coordinates": [
        [
          [15.891722, 21.441122],
          [13.95383, 21.565407],
          [14.233208, 25.427168],
          [15.927658, 26.113512],
          [17.464699, 25.211208],
          [16.642977, 22.83285],
          [15.891722, 21.441122]
        ]
      ],
      "type": "Polygon",
      "cell": 5
    }
  ],
  "type": "GeometryCollection"
}