10x Genomics Support/Space Ranger 2.1/Analysis/

Understanding Space Ranger Outputs

This page provides an overview of Space Ranger outputs for various supported workflows.

All pipelines produce all of their output in a single pipeline output directory, whose name depends on the pipeline:

  • For spaceranger mkfastq, the --id argument is used or if unspecified the flow cell ID is used (e.g., HAWT7ADXX)
  • For spaceranger count and spaceranger aggr, the --id argument is used

Output files appear in the outs/ subdirectory within this pipeline output directory. For example, a typical spaceranger count analysis may display the following after completion:

cd /home/jdoe/runs spaceranger count --id=sample345 \ --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \ --transcriptome=/opt/refdata-gex-GRCh38-2020-A \ --image=/home/jdoe/runs/image.tif \ --slide=V19J01-123 \ --area=B1 Running preflight checks (please wait)... Outputs: - Run summary HTML: /home/jdoe/runs/sample345/outs/web_summary.html - Outputs of spatial pipeline: aligned_fiducials: /home/jdoe/runs/sample345/outs/spatial/aligned_fiducials.jpg detected_tissue_image: /home/jdoe/runs/sample345/outs/spatial/detected_tissue_image.jpg scalefactors_json: /home/jdoe/runs/sample345/outs/spatial/scalefactors_json.json tissue_hires_image: /home/jdoe/runs/sample345/outs/spatial/tissue_hires_image.png tissue_lowres_image: /home/jdoe/runs/sample345/outs/spatial/tissue_lowres_image.png cytassist_image: null aligned_tissue_image: null tissue_positions: /home/jdoe/runs/sample345/outs/spatial/tissue_positions.csv spatial_enrichment: /home/jdoe/runs/sample345/outs/spatial/spatial_enrichment.csv barcode_fluorescence_intensity: null - Run summary CSV: /home/jdoe/runs/sample345/outs/metrics_summary.csv - Correlation values between isotypes and Antibody features: null - BAM: /home/jdoe/runs/sample345/outs/possorted_genome_bam.bam - BAM BAI index: /home/jdoe/runs/sample345/outs/possorted_genome_bam.bam.bai - BAM CSI index: null - Filtered feature-barcode matrices MEX: /home/jdoe/runs/sample345/outs/filtered_feature_bc_matrix - Filtered feature-barcode matrices HDF5: /home/jdoe/runs/sample345/outs/filtered_feature_bc_matrix.h5 - Unfiltered feature-barcode matrices MEX: /home/jdoe/runs/sample345/outs/raw_feature_bc_matrix - Unfiltered feature-barcode matrices HDF5: /home/jdoe/runs/sample345/outs/raw_feature_bc_matrix.h5 - Secondary analysis output CSV: /home/jdoe/runs/sample345/outs/analysis - Per-molecule read information: /home/jdoe/runs/sample345/outs/molecule_info.h5 - Loupe Browser file: /home/jdoe/runs/sample345/outs/cloupe.cloupe - Feature Reference: null - Target Panel file: null - Probe Set file: null Pipestance completed successfully!

In this case:

  • /home/jdoe/runs/ is where the pipeline was run
  • /home/jdoe/runs/sample345/ is the top-level output directory containing pipeline metadata
  • /home/jdoe/runs/sample345/outs/ contains the final pipeline output files

More information about the contents of the pipeline output directory can be found in the Pipestance Structure page.

Space Ranger maintains compatibility with common analysis tools by using standard output file formats whenever possible. For example, the barcoded BAM files can be viewed in standard genome browsers such as IGV to verify alignment quality and other features. Visium-specific data, including spot and molecular barcodes, can be accessed using any third-party tools or scripts that can parse the additional elements utilized by Space Ranger.

The outputs generated from the primary analysis pipeline spaceranger count for Gene Expression (GEX) contained in the outs/ directory are summarized below:

web_summary.htmlData summary with images, metrics, and plots that can be used for quality assessment as well as errors and warnings related to data quality
metrics_summary.csvMetrics for quality assessment
cloupe.cloupeLoupe Browser file for data visualization and analysis
Secondary analysis results (clustering, DGE, Moran's I)
spatial/Folder containing Visium-specific outs: QC images to check image processing pipeline, downsampled input images, and files that describe spot barcode locations in the images
molecule_info.h5Molecule level information used in additional pipelines (spaceranger aggr, spaceranger targeted-compare)
Barcoded BAM (optional)
Read alignment files
Filtered GEX Matrix
MEX: filtered_feature_bc_matrix
HD5F: filtered_feature_bc_matrix.h5
Feature barcode matrices that contains both tissue-associated as well as background barcodes and are used in secondary analysis in R/Python
Unfiltered GEX Matrix
MEX: raw_feature_bc_matrix
HD5F: raw_feature_bc_matrix.h5
Feature barcode matrices that contains both tissue-associated as well as background barcodes and are used in secondary analysis in R/Python
deconvolution/Folder containing outputs of LDA-based spot deconvolution for the range of K topics

The spaceranger count pipeline for Gene Expression and Protein Expression analysis will output each of the types of files listed above. There are additional output files for Visium CytAssist-enabled Gene Expression and Protein Expression libraries. For specific analysis output descriptions, see this page.

The spaceranger aggr pipeline outputs metrics, web summary, filtered feature-barcode matrices, and a .cloupe file within the outs/ directory. Refer to the aggr outputs page for more details.

Space Ranger v2.1 supports protein expression analysis with built-in isotype normalization to correct for technical noise that is inherent in antibody-based assays. The filtered feature barcode matrices contain the normalized counts while the raw feature barcode matrices contain raw UMI counts. For convenience, the normalization factor is provided in the outputs.

The isotype_normalization_factors.csv has the following columns:

Column NameDescription
barcodeAll spot barcodes in the Visium capture area.
in_tissueBoolean that specifies whether the spot was detected under tissue.
normalization_factorThe normalization factor used to calculate isotype normalized protein expression from the raw antibody UMI counts. The value is equivalent 10,000 / (1+sum(isotype control UMIs)).