10x Genomics
Visium Spatial Gene Expression

Space Ranger1.3, printed on 10/31/2024

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 or 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=/opt/refdata/GRCh38-2020-A \ #Path to Reference
                   --target-panel=/opt/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=/opt/refdata/GRCh38-2020-A \ #Path to Reference
                   --target-panel=/opt/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 /opt/spaceranger-1.3.0, then the target panel CSVs will be contained in /opt/spaceranger-1.3.0/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 from a Targeted Gene Expression analysis is generally similar to output from non-targeted analysis—see the table below for a comprehensive list of differences. The primary changes are:

• The addition of targeting metrics
• All secondary analysis for targeted data is performed after removing non-targeted genes from the count matrix
• The filtered feature-barcode matrix files and Loupe Browser file do not include non-targeted gene counts

The raw feature-barcode matrices and molecule info H5 file still provide information about all genes.

^* HDF5 files now contain indices of the targeted genes within the features HDF5 group.

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

• Browsing the summary HTML file in any supported web browser
• Opening the .cloupe file in Loupe Browser
• Referring to the Understanding Output section to explore the data by hand

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.