This page describes raw output (decoded transcript counts and morphology images) and other standard output files derived from them, which are included in the Atera Onboard Analysis output bundle.

All run data will be stored in the output/ directory on the Atera Instrument computer and will be accessible on the Desktop. Refer to the Atera Instrument User Guide (CG001673) for instructions to export run data from the instrument.

Within the output/ directory, the data from individual runs are stored as subfolders and include the user-defined run name in the folder name. Within the top-level run folder, there are subfolders for each user-defined region of interest (ROI) on the Atera slides. The overall organization of subfolders is shown below:

Copy
output/customer/<USER_ID>/
└── <yyyymmdd>__<hhmmss>__<RUN_NAME>
    └── <INST_SN>__<SLIDE_NAME>__<ROI_NAME>__<yyyymmdd>__<hhmmss>

The RUN_NAME and ROI_NAME strings are user-defined; the other components of the directory names are auto-generated. <yyyymmdd> is the start date and <hhmmss> is the start time (in UTC). The separators between the strings in the directory name are two underscores. Spaces in RUN_NAME and ROI_NAME will be replaced by an underscore (_) in the output directory name.

Output bundle example directory structure:

With default output 2D images

Copy
└── <INST_SN>__<SLIDE_NAME>__<ROI_NAME>__<yyyymmdd>__<hhmmss>
    ├── analysis_summary_<INST_SN>_<SLIDE_NAME>_<ROI_NAME>.html
    ├── binned_transcripts.zarr.zip
    ├── cell_feature_matrix.zarr.zip
    ├── cells.zarr.zip
    ├── csc_cell_feature_matrix.zarr.zip
    ├── diffexp_genes
    │   ├── gene_expression_graphclust.csv
    │   └── gene_expression_azimuth.csv # for Human WTA assays
    ├── experiment.spatial
    ├── metrics_summary_<INST_SN>_<SLIDE_NAME>_<ROI_NAME>.csv
    ├── morphology_2d
    │   ├── ch0000_dapi.ome.tif
    │   └── ch0001_18s.ome.tif
    ├── morphology_3d
    │   └── ch0000_dapi_3d.ome.tif
    ├── morphology_fov_locations.json
    ├── overview_scan.png
    ├── overview_scan_fov_locations.json
    ├── panel_config.json
    ├── qc_images.html
    └── transcripts.zarr.zip

With 2D images for all cell segmentation stain images

Copy
└── <INST_SN>__<SLIDE_NAME>__<ROI_NAME>__<yyyymmdd>__<hhmmss>
    ├── analysis_summary_<INST_SN>_<SLIDE_NAME>_<ROI_NAME>.html
    ├── binned_transcripts.zarr.zip
    ├── cell_feature_matrix.zarr.zip
    ├── cells.zarr.zip
    ├── csc_cell_feature_matrix.zarr.zip
    ├── diffexp_genes
    │   ├── gene_expression_graphclust.csv
    │   └── gene_expression_azimuth.csv # for Human WTA assays
    ├── experiment.spatial
    ├── metrics_summary_<INST_SN>_<SLIDE_NAME>_<ROI_NAME>.csv
    ├── morphology_2d
    │   ├── ch0000_dapi.ome.tif
    │   ├── ch0001_atp1a1_cd45_e-cadherin.ome.tif
    │   ├── ch0002_18s.ome.tif
    │   └── ch0003_alphasma_vimentin.ome.tif
    ├── morphology_3d
    │   └── ch0000_dapi_3d.ome.tif
    ├── morphology_fov_locations.json
    ├── overview_scan.png
    ├── overview_scan_fov_locations.json
    ├── panel_config.json
    ├── qc_images.html
    └── transcripts.zarr.zip

The remaining sections describe the Atera output bundle files for each analysis run.

The experiment.spatial is an experiment manifest file in JSON format that includes experiment metadata and relative file paths to other data files in the output folder needed by 10x Explorer to visualize results. Click the link below for metadata descriptions.

The pipeline generates interactive HTML files to help with data QC, analysis interpretation, and troubleshooting. Click the link below to learn when to use these summaries and how to interpret the results.

A series of tissue morphology images are output by the pipeline, including nuclei-stained (DAPI) and multi-tissue stained (default: interior cell; optional: cell boundary, interior protein) images in OME-TIFF format. These files include a pyramid of resolutions and tiled chunks of image data, which allows for efficient interactive image visualization (JPEG-2000 compression, 16-bit grayscale).

All morphology images are oriented with the origin (0,0) in the top left corner (image coordinate system). For reference, the image axes are labeled in the Summary and Image QC tabs of the analysis summary file. The transcript X, Y, and Z locations use the same image coordinate system orientation.

• The morphology_3d/ch0000_dapi_3d.ome.tif is a 3D Z-stack of the DAPI image that can be useful to resegment cells, assess segmentation quality, and view data.
• The morphology_2d/ directory contains the 2D focus projection images for the nuclei DAPI stain image, as well as additional stain images if Atera Cell Segmentation Staining Reagents are used. These files are in multi-file OME-TIFF format. They each contain a pyramid of images including full resolution and downsampled images. The image order is specified in OME-XML metadata.

The OME-TIFF morphology image files can be viewed in 10x Explorer. The image orientation is the same as shown in the analysis summary, with the origin in the top left corner. This is relevant for image alignment (e.g., in 10x Explorer).

Multi-file OME-TIFF images can also be viewed in community-developed visualization software such as QuPath, Napari, or Fiji/ImageJ. To view all focus images in these programs, simply open one of the files (i.e., open program, drag and drop ch0000_dapi.ome.tif file). Since each focus image file's metadata specifies that all the focus files are stored in the morphology_2d/ directory, they do not need to be imported separately.

The overview_scan.png is the full-resolution (2670 x 6670 pixels) image of the entire sample on the slide.

FOV location files:

• The morphology_fov_locations.json file contains the field of view (FOV) name, height, width, and XY positions in the space of the region of interest's (ROI) morphology image. This is the same space used to compute transcript and cell locations and the units are in microns. The FOVs are 4819 x 4819 with an overlap of 113 pixels of overlap on each edge. The position information is useful for determining where FOV boundaries are to assess transcript deduplication and any FOV edge effects.
• The overview_scan_fov_locations.json file contains the FOV name, height, width, and approximate XY positions in the space of the overview scan image. This is the space that contains all the ROIs and the units are in pixels. The accuracy of the ROI coordinates have a 5 - 10 µm error. This position information is useful for approximating where multiple ROIs are located on an overview scan image.

The output bundle provides cell, transcript, cell-feature matrix, and secondary analysis outputs in Zarr format. Click the link below to learn what information is provided in each file and how to use them.

The pipeline outputs a diffexp_genes/ directory. These files contain the list of cluster-specific features that are differentially expressed in each cluster relative to all the other clusters for unsupervised graph-based clustering and annotated cell types for human WTA assays.

The pipeline outputs key metrics in text format as metrics_summary_<INST_SN>_<SLIDE_NAME>_<ROI_NAME>.csv. This file contains metrics that are useful for assessing decoding and cell segmentation quality programmatically.

The panel_config.json file is a copy of the panel configuration file, which describes the complete set of gene panel codebooks used in your experiment on the Atera Instrument.

The JSON schema contains metadata and payload objects. The payload object contains the following: