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Running spaceranger count (FFPE)

In this tutorial, you will learn how to run the spaceranger count pipeline on Visium Spatial Gene Expression data derived from a formalin fixed paraffin embedded (FFPE) mouse brain section.

To successfully run this tutorial, you should:

  • Be comfortable in the Linux environment
  • Have familiarity running command line tools
  • Choose a compute platform
  • Have access to a system that meets the minimum system requirements

Visium Spatial Gene Expression data from FFPE tissues are analyzed using the spaceranger count pipeline. The pipeline inputs a microscope image of the Visium slide (in TIFF or JPEG format), a reference, and FASTQ files, and performs alignment, tissue and fiducial detection, and barcode/UMI counting. Outputs include the feature-spot matrices, clustering and differential gene expression (DGE) which can be further analyzed and visualized in Loupe Browser.

In this tutorial, we will run the spaceranger count pipeline on a mouse brain FFPE section.

Key dataset features are:

  • Tissue section of 5 µm thickness
  • Section Orientation: Coronal
  • H&E image
  • Sequencing Depth: 28,826 reads per spot
  • Sequencing Coverage: Read 1 - 28 bp (includes 16 bp Spatial Barcode, 12 bp UMI); Read 2 - 120 bp (transcript); i7 sample index - 10 bp; i5 sample index - 10 bp
  • Visium Slide: V11J26-127
  • Capture Area: B1

The following commands will be run in the working directory (spaceranger_tutorial) that was used to install spaceranger on a compatible compute platform.

Example FFPE dataset

Download with curl command:

# Create datasets folder mkdir datasets # Download FASTQ to datasets folder curl https://cf.10xgenomics.com/samples/spatial-exp/1.3.0/Visium_FFPE_Mouse_Brain/Visium_FFPE_Mouse_Brain_fastqs.tar -o datasets/Visium_FFPE_Mouse_Brain_fastqs.tar # Download image file to datasets folder curl https://cf.10xgenomics.com/samples/spatial-exp/1.3.0/Visium_FFPE_Mouse_Brain/Visium_FFPE_Mouse_Brain_image.jpg -o datasets/Visium_FFPE_Mouse_Brain_image.jpg # Expected output % Total % Received % Xferd Average Speed Time Time Time Current Dload Upload Total Spent Left Speed 5 4154M 5 218M 0 0 32.7M 0 0:02:06 0:00:06 0:02:00 33.0M

Alternatively, download with the wget command:

# Create datasets folder mkdir datasets # Download FASTQ to datasets folder wget -P datasets/ https://cf.10xgenomics.com/samples/spatial-exp/1.3.0/Visium_FFPE_Mouse_Brain/Visium_FFPE_Mouse_Brain_fastqs.tar # Download image file to datasets folder wget -P datasets/ https://cf.10xgenomics.com/samples/spatial-exp/1.3.0/Visium_FFPE_Mouse_Brain/Visium_FFPE_Mouse_Brain_image.jpg # Expected output Resolving cf.10xgenomics.com (cf.10xgenomics.com)... 104.18.0.173, 104.18.1.173, 2606:4700::6812:1ad, ... Connecting to cf.10xgenomics.com (cf.10xgenomics.com)|104.18.0.173|:443... connected. HTTP request sent, awaiting response... 200 OK Length: 4356188160 (4.1G) [application/x-tar] Saving to: ‘datasets/Visium_FFPE_Mouse_Brain_fastqs.tar’ 37% [=======================> ] 1,649,129,803 207MB/s eta 13s

Reference data

Download the latest version of the mouse transcriptome reference from the Downloads page.

# Download mouse reference curl -O https://cf.10xgenomics.com/supp/spatial-exp/refdata-gex-mm10-2020-A.tar.gz # Expected output % Total % Received % Xferd Average Speed Time Time Time Current Dload Upload Total Spent Left Speed 1 9835M 1 158M 0 0 34.1M 0 0:04:48 0:00:04 0:04:44 34.1M

Probe set reference

Since this is a FFPE tissue sample, the assay uses a pair of oligonucleotide probes targeting protein-coding genes. In addition to the reference transcriptome, spaceranger count also requires the species-specific probe set reference file in CSV format. You can either download the probe set reference from the 10x support website or use the probe set references pre-bundled in Space Ranger.

# Method 1 # Download mouse probe set reference from support website curl -O https://cf.10xgenomics.com/supp/spatial-exp/probeset/Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv # Method 2 # Space Ranger 2.0 comes bundled with probe set files ## Source mouse probe set reference ~/spaceranger_tutorial/spaceranger-2.0.0/probe_sets/Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv # Expected output % Total % Received % Xferd Average Speed Time Time Time Current Dload Upload Total Spent Left Speed 100 2257k 100 2257k 0 0 5579k 0 --:--:-- --:--:-- --:--:-- 5587k

In this tutorial, we will use the path associated with the first option.

Extract files

After downloading the required files, the contents of the tar files need to be extracted.

# Extract sample FASTQ files tar -xvf datasets/Visium_FFPE_Mouse_Brain_fastqs.tar -C datasets/ && rm datasets/Visium_FFPE_Mouse_Brain_fastqs.tar # Extract mouse reference transcriptome tar -xzvf refdata-gex-mm10-2020-A.tar.gz && rm refdata-gex-mm10-2020-A.tar.gz # Expected output # Sample FASTQ files Visium_FFPE_Mouse_Brain_fastqs/ Visium_FFPE_Mouse_Brain_fastqs/Visium_FFPE_Mouse_Brain_S3_L002_R1_001.fastq.gz Visium_FFPE_Mouse_Brain_fastqs/Visium_FFPE_Mouse_Brain_S3_L001_I2_001.fastq.gz Visium_FFPE_Mouse_Brain_fastqs/Visium_FFPE_Mouse_Brain_S3_L002_R2_001.fastq.gz ... # Reference mouse transcriptome refdata-gex-mm10-2020-A/ refdata-gex-mm10-2020-A/fasta/ refdata-gex-mm10-2020-A/fasta/genome.fa ...

Successful extraction will create two additional folders, highlighted in yellow, within the working directory.

1spaceranger_tutorial 2├── datasets 3│   ├── Visium_FFPE_Mouse_Brain 4│   └── Visium_FFPE_Mouse_Brain_image.jpg 5├── refdata-gex-mm10-2020-A 6├── Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv 7└── spaceranger-2.0.0

You can now build the spaceranger count command to run from your working directory (spaceranger_tutorial). If running from a different directory, amend the paths accordingly to avoid any errors.

spaceranger count --id="Visium_FFPE_Mouse_Brain" \ --transcriptome=refdata-gex-mm10-2020-A \ --probe-set=Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv \ --fastqs=datasets/Visium_FFPE_Mouse_Brain_fastqs \ --image=datasets/Visium_FFPE_Mouse_Brain_image.jpg \ --slide=V11J26-127 \ --area=B1 \ --reorient-images=true \ --localcores=16 \ --localmem=128

Below are brief descriptions of the command line options:

OptionDescription
--idThe id must be a unique string and will be used to name the resulting folder with all of the pipeline outputs.
--transcriptomeThe path to the species-specific pre-compiled transcriptome files. You can provide the relative or absolute path to this folder.
--probe-setThe absolute or relative path to the species-specific probe set reference file in CSV format.
--fastqsThe path to the folder containing sample sequencing files in FASTQ format.
--imageThe path to a single brightfield image with H&E staining in either TIFF or JPEG formats.
--slideThe Visium slide serial number.
--areaThe Capture Area identifier on the visium slide. It can be one of four values: A1, B1, C1 or D1.
--reorient-imagesOption to choose whether spaceranger should rotate and mirror the image to find the best fiducial alignment. Acceptable values are true or false with default being true. Useful to set to false when you are absolutely certain the fiducial corners in the input image are in the canonical positions (hourglass in top left corner) similar to included image Visium_FFPE_Mouse_Brain_image.jpg. Setting this to false will reduce pipeline runtime and prevent the pipeline from finding a fiducial alignment where the image is rotated/mirrored.
--localcoresThe number of CPU cores available to run the spaceranger count pipeline.
--localmemThe max memory in GB available to run the spaceranger count pipeline.

At the start of the run, you should see the preflight checks printed to the command line.

# Run spaceranger count spaceranger count --id="Visium_FFPE_Mouse_Brain" \ --description="Adult Mouse Brain (FFPE) using Mouse WTA Probe Set" \ --transcriptome=refdata-gex-mm10-2020-A \ --probe-set=Visium_Mouse_Transcriptome_Probe_Set_v1.0_mm10-2020-A.csv \ --fastqs=datasets/Visium_FFPE_Mouse_Brain_fastqs \ --image=datasets/Visium_FFPE_Mouse_Brain_image.jpg \ --slide=V11J26-127 \ --area=B1 \ --reorient-images=true \ --localcores=16 \ --localmem=128 # Expected output Martian Runtime - v4.0.5 Running preflight checks (please wait)... Checking sample info... Checking FASTQ folder... Checking reference... Checking reference_path... Checking optional arguments... ...

Successful completion of the pipeline is indicated by a list of output files.

Outputs: - Run summary HTML: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/web_summary.html - Outputs of spatial pipeline: aligned_fiducials: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/aligned_fiducials.jpg detected_tissue_image: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/detected_tissue_image.jpg scalefactors_json: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/scalefactors_json.json tissue_hires_image: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/tissue_hires_image.png tissue_lowres_image: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/tissue_lowres_image.png cytassist_image: null aligned_tissue_image: null tissue_positions: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/tissue_positions.csv spatial_enrichment: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/spatial/spatial_enrichment.csv barcode_fluorescence_intensity: null - Run summary CSV: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/metrics_summary.csv - Correlation values between isotypes and Antibody features: null - BAM: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/possorted_genome_bam.bam - BAM BAI index: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/possorted_genome_bam.bam.bai - BAM CSI index: null - Filtered feature-barcode matrices MEX: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/filtered_feature_bc_matrix - Filtered feature-barcode matrices HDF5: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/filtered_feature_bc_matrix.h5 - Unfiltered feature-barcode matrices MEX: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/raw_feature_bc_matrix - Unfiltered feature-barcode matrices HDF5: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/raw_feature_bc_matrix.h5 - Secondary analysis output CSV: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/analysis - Per-molecule read information: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/molecule_info.h5 - Loupe Browser file: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/cloupe.cloupe - Feature Reference: null - Target Panel file: null - Probe Set file: /spaceranger_tutorial/Visium_FFPE_Mouse_Brain/outs/probe_set.csv Waiting 6 seconds for UI to do final refresh. Pipestance completed successfully!

After the run is completed, the working directory will have a new folder named V1_Adult_Mouse_Brain (value provided to --id argument) that contains all the metadata and outputs generated from the spaceranger count pipeline. We will highlight some key components of this folder:

Visium_FFPE_Mouse_Brain ├── _cmdline ├── _filelist ├── _finalstate ├── _invocation ├── _jobmode ├── _log ├── _mrosource ├── outs ├── _perf ├── _sitecheck ├── SPATIAL_RNA_COUNTER_CS ├── _tags ├── _timestamp ├── _uuid ├── Visium_FFPE_Mouse_Brain.mri.tgz ├── _vdrkill └── _versions
  • Visium_FFPE_Mouse_Brain.mri.tgz contains diagnostic information helpful to 10x Genomics support to resolve any errors
  • _sitecheck captures the system configuration similar to sitecheck subcommand
  • _timestamp contains information on pipeline runtimes. The runtime for the example dataset with the above configuration was 50:58
  • _cmdline captures the count command provided to run the pipeline
  • _versions contains both the spaceranger and Martian versions used in the run
  • outs contains all the final pipeline generated outputs:

You can further explore and understand these results by

  • Browsing the web_summary.html file.
  • Opening the .cloupe file in [Loupe Browser](/support/software/loupe-browser/latest
  • Referring to the Understanding Outputs page to explore individual files