10x Genomics
Visium Spatial Gene Expression

Space Ranger2.0, printed on 04/19/2024

Targeted Gene Expression Analysis

Table of Contents

• Input files
• Run spaceranger count
• Target panel CSV
• Output files
• Targeted analysis relevant command-line arguments

Space Ranger provides the same interface and features for both Targeted and Whole Transcriptome Analysis (WTA) Gene Expression data, as described in Single-Library Analysis with spaceranger count. The workflow diagram shows all the required inputs and the corresponding spaceranger count flags for Targeted Gene Expression analysis:

Input files

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

• The FASTQ files for a given capture area (--fastqs)
  • If starting with Illumina BCL output folder, follow the instructions on running spaceranger mkfastq to generate FASTQ files
  • For help on which arguments to use to target a particular set of FASTQs, consult Specifying Input FASTQ Files for 10x Pipelines
• The corresponding capture area image in either TIFF, QPTIFFor JPEG format:
  • --image for brightfield image
  • --darkimage for dark background fluorescence image
  • --colorizedimage for composite colored fluorescence image
• The slide layout GPR file specified by:
  • --slide & --area if spaceranger has access to internet
  • --slidefile, --slide & --area if spaceranger has no access to internet. The slide layout file is directly downloaded
  • --unknown-slide if visium slide details are unknown
• The reference transcriptome for the species from which the sample was obtained (--transcriptome)
• The relevant Target Panel CSV file specified using the --target-panel flag

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.

• Automatic Alignment
• Manual Alignment

$ cd /home/jdoe/runs
$ spaceranger count --id=sample345 \ #Output directory
                   --transcriptome=/home/jdoe/refdata/GRCh38-2020-A \ #Path to Reference
                   --target-panel=/home/jdoe/spaceranger-1.3.0/target_panels/immunology_v1.0_GRCh38-2020-A.target_panel.csv \ #Path to the Target Panel CSV
                   --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

$ cd /home/jdoe/runs
$ spaceranger count --id=sample345 \ #Output directory
                   --transcriptome=/home/jdoe/refdata/GRCh38-2020-A \ #Path to Reference
                   --target-panel=/home/jdoe/spaceranger-1.3.0/target_panels/immunology_v1.0_GRCh38-2020-A.target_panel.csv \ #Path to the Target Panel CSV
                   --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

Target panel CSV

Target panel CSV files corresponding to predesigned gene panels can be found within the target_panels directory in the Space Ranger package. Spatial Gene Expression does not include support for fully custom panels.

target_panels/
├── gene_signature_v1.0_GRCh38-2020-A.target_panel.csv
├── immunology_v1.0_GRCh38-2020-A.target_panel.csv
├── neuroscience_v1.0_GRCh38-2020-A.target_panel.csv
└── pan_cancer_v1.0_GRCh38-2020-A.target_panel.csv

For example, if you installed Space Ranger to /home/jdoe/spaceranger-2.0.0, then the target panel CSVs will be contained in /home/jdoe/spaceranger-2.0.0/target_panels.

For the target panel CSV files, go to:

• The Panel Selection page for information about each panel as well as direct downloads of all the accompanying files.

• The Panel File Descriptions page for detailed documentation on all the file formats used in targeted analysis.

Output files

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 outputs.

Targeted analysis relevant command-line arguments

The list of all the relevant arguments are described in Single-Library Analysis with spaceranger count or run spaceranger count --help.