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10x Genomics
Visium Spatial Gene Expression

Single-Library FFPE Analysis with spaceranger count

Table of Contents

Space Ranger's pipelines analyze sequencing data produced from Visium Spatial Gene Expression for Formalin Fixed Paraffin Embedded (FFPE) and Fresh Frozen (FF) tissue samples. The workflow diagram shows all the required inputs and the corresponding spaceranger flags for FFPE tissue samples:

Input files

The spaceranger count is run on each individual Capture Area in the Visium slide. The required inputs are

Run spaceranger count

For a list of accepted arguments, see the Command Line Argument Reference below, or run spaceranger count --help.

Select the tab corresponding to the imaging workflow.


To generate spatial feature counts for a single library FFPE sample using automatic fiducial alignment and tissue detection on a brightfield image input, run spaceranger count with the following arguments. The code in red indicates user-specific inputs.

$ cd /home/jdoe/runs
$ spaceranger count --id=sample345 \ #Output directory
                   --transcriptome=/home/jdoe/refdata/GRCh38-2020-A \ #Path to Reference
                   --probe-set=/home/jdoe/spaceranger-2.0.0/probe_set/Visium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.csv\ #Path to Probe Set
                   --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \ #Path to FASTQs
                   --sample=mysample \ #Sample name from FASTQ filename
                   --image=/home/jdoe/runs/images/sample345TIFF \ #Path to brightfield image input
                   --slide=V19J01-123 \ #Slide ID
                   --area=A1 \ #Capture area
                   --localcores=8 \ #Allowed cores in localmode
                   --localmem=64 #Allowed memory (GB) in localmode

To generate spatial feature counts for a single library FFPE sample using a fiducial alignment and tissue assignment JSON file generated in Loupe Browser on a brightfield image input, run spaceranger count with the following arguments. The code in red indicates user specific inputs.

$ cd /home/jdoe/runs
$ spaceranger count --id=sample345 \ #Output directory
                   --transcriptome=/home/jdoe/refdata/GRCh38-2020-A \ #Path to Reference
                   --probe-set=/home/jdoe/spaceranger-2.0.0/probe_set/Visium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.csv\ #Path to Probe Set
                   --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \ #Path to FASTQs
                   --sample=mysample \ #Sample name from FASTQ filename
                   --image=/home/jdoe/runs/images/sample345TIFF \ #Path to brightfield image input
                   --slide=V19J01-123 \ #Slide ID
                   --area=A1 \ #Capture area
                   --loupe-alignment=sample345.json \  #Manual alignment file
                   --localcores=8 \ #Allowed cores in localmode
                   --localmem=64 #Allowed memory (GB) in localmode

The arguments to specify the fluorescence image can be either --darkimage or --colorizedimage depending on the image format. Details about the different supported fluorescence image formats are described in Image Recommendations.


Following a set of preflight checks to validate input arguments, spaceranger count pipeline stages will begin to run:
Running preflight checks (please wait)...
Checking sample info...
Checking FASTQ folder...
Checking reference...
Checking reference_path
Checking optional arguments...
...

Probe set reference CSV

Probe set reference CSV file for human can be found in the probe_sets directory in the Space Ranger package. Note that only the highlighted probe set reference CSV files are compatible with Visium Spatial Gene Expression for FFPE workflow.

spaceranger-2.0.0/probe_sets/
├── Visium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.csv
├── Visium_Human_Transcriptome_Probe_Set_v2.0_GRCh38-2020-A.csv
└── Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv

The CSV files for both human and mouse

Output files

A successful spaceranger count run concludes with a message similar to this:

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:                                            /home/jdoe/runs/sample345/outs/probe_set.csv
 
Pipestance completed successfully!

The output of the pipeline is contained in a folder named with the sample ID you specified (such as sample345). If this folder already exists, in a rerun with the original parameters, spaceranger will assume it is an existing pipestance and attempt to resume running it. The subfolder named outs contains the main pipeline output files.

Once spaceranger count has successfully completed, you can further explore the results by

Command-line argument reference

The flags unique to FFPE analysis are highlighted in bold.

ArgumentDescription
--idRequired. A unique run ID string (such as sample345).
--fastqsRequired. Either:
Path of the fastq_path folder generated by spaceranger mkfastq
(such as /home/jdoe/runs/HAWT7ADXX/outs/fastq_path). This contains a directory hierarchy that spaceranger count will automatically traverse.
- OR -
Any folder containing fastq files, for example if the fastq files were generated by a service provider and delivered outside the context of the mkfastq output directory structure.
Can take multiple comma-separated paths, which is helpful if the same library was sequenced on multiple flowcells.
Doing this will treat all reads from the library, across flowcells, as one sample.
--sampleOptional. Sample name as specified in the sample sheet supplied to spaceranger mkfastq.
Can take multiple comma-separated values, which is helpful if the same library was sequenced on multiple flowcells and the sample name used (and therefore fastq file prefix) is not identical between them.
Doing this will treat all reads from the library, across flowcells, as one sample.
Allowable characters in sample names are letters, numbers, hyphens, and underscores.
--probe-setRequired to enable FFPE analysis. Path to the probe set reference CSV file (such as /opt/spaceranger-2.0.0/probe_sets/Visium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.csv).
--transcriptomeRequired. Path to the Space Ranger compatible transcriptome reference (such as /opt/GRCh38-2020-A).
--imageRequired for brightfield image input. Brightfield tissue H&E image in JPEG or TIFF format. For more information on image file constraints, encoding and formats refer the Image Recommendations.
--darkimageRequired for dark background fluorescence image input. Multi-channel, dark-background fluorescence image as either a single, multi-layer TIFF file or as multiple TIFF or JPEG files (which can provided as a comma separated list or by invoking the --darkimage parameter multiple times). Details on image file constraints, encoding and formats are described in the Image Recommendations.
--colorizedimageRequired for color composite fluorescence image input. A color composite of one or more fluorescence image channels saved as a single-page, single-file color TIFF or JPEG. Please see the Image Recommendations for information on input image file formats.
--slide Visium slide serial number. Refer to the Slide Parameters for information on supported slide versions.Required unless --unknown-slide is passed.
--area Visium capture area identifier. Required unless --unknown-slide is passed. Options for Visium are A1, B1, C1, D1.
--slidefile Slide layout file indicating capture spot and fiducial spot positions. Required when no internet access unless --unknown-slide is passed.
--unknown-slideOptional. Set this if the slide serial number and area identifier are unknown. The value passed to this depends of the slide version. Possible value for Visium Spatial for FFPE is visium-1. Setting this will cause Space Ranger to use default spot positions. Not compatible with --slide, --area, or --slidefile. Refer to the Slide Parameters for more information.
--reorient-imagesDefault:true. Whether the pipeline should rotate and mirror the image to align the upright hourglass fiducial pattern in the top left corner. Set --reorient-images=false if you are certain your image is already oriented with the upright hourglass in the top left corner.
--loupe-alignmentRequired for manual alignment. Alignment file produced by the manual Loupe alignment step. A --image, --darkimage or --colorizedimage, the same image(s) that were used for manual alignment, must be supplied in this case.
--filter-probesDefault:true. Include all non-deprecated probes listed in the probe set reference CSV file. Probes that are predicted to have off-target activity to homologous genes are excluded from analysis by default. Setting --filter-probes=false will result in UMI counts from all non-deprecated probes, including those with predicted off-target activity, to be used in the analysis. An alternative and preferable method to including all probes is described in this article. Probes whose ID is prefixed with DEPRECATED are always excluded from the analysis.
--no-bamOptional. Add this flag to skip generating BAM file. This will reduce the total computation time for the pipestance and the size of the output directory. If unsure, we recommend not using this option, as BAM files can be useful for troubleshooting and downstream analysis.
--nosecondaryOptional. Disable secondary analysis, such as dimensionality reduction, clustering, spatial enrichment calculation (Moran's I) and visualization.
--r1-lengthOptional. Limit the length of the input R1 sequence to the first N bases where N is the value supplied by the user. Note that the length includes the Barcode and UMI sequences so do not set this below 28. This and --r2-length are useful for determining the optimal read length for sequencing.
--r2-lengthOptional. Limit the length of the input R2 sequence to the first N bases where N is the value supplied by the user. From Space Ranger v2.0 onwards the length of R2 sequence is limited to the first 50 bases.
--lanesOptional. Lanes associated with this sample.
--localcoresRecommended when run in localmode. Restricts spaceranger to use specified number of cores to execute pipeline stages. By default, spaceranger will use all of the cores available on your system.
--localmemRecommended when run in localmode. Restricts spaceranger to use specified amount of memory (in GB) to execute pipeline stages. By default, spaceranger will use 90% of the memory available on your system.