GSE279255 Processing Pipeline

Publication

Nature (2025) — PMID 39843748

Dataset

Tissue-resident memory CD8 T Cell Diversity is Spatiotemporally Imprinted

1. 1

   BCL files were demultiplexed into FASTQ files using spaceranger mkfastq followed by spaceranger count to generate the spatial representation of gene counts by matrix at 2 and 8 mm resolution binning

   Space Ranger vInferred with models/gemini-2.5-flash

   $ Bash example

   # Install Space Ranger (example, adjust for actual installation method)
   # For example, download from 10x Genomics website and add to PATH:
   # wget https://cf.10xgenomics.com/releases/spatial-gene-expression/spaceranger-X.Y.Z.tar.gz
   # tar -xzf spaceranger-X.Y.Z.tar.gz
   # export PATH=/path/to/spaceranger-X.Y.Z:$PATH
   
   # Define variables (replace with actual paths and IDs)
   BCL_DIR="/path/to/bcl_files_directory" # Directory containing BCL files (e.g., from an Illumina sequencer)
   SAMPLE_SHEET="/path/to/sample_sheet.csv" # CSV file describing the sequencing run, including sample and index information
   FASTQ_RUN_ID="sequencing_run_id" # A unique identifier for the mkfastq output directory
   SAMPLE_ID="sample_name" # Unique identifier for your specific sample (as defined in the sample sheet)
   REFERENCE_PATH="/path/to/10x_genomics_refdata/refdata-gex-GRCh38-2020-A" # Path to a 10x Genomics pre-built transcriptome reference (e.g., for Human GRCh38)
   SLIDE_ID="V12A01-001" # The ID of the Visium slide (e.g., from the slide sticker)
   AREA_ID="A1" # The capture area on the slide (e.g., A1, B1, C1, D1)
   IMAGE_PATH="/path/to/brightfield_or_fluorescence_image.tif" # Path to the brightfield or fluorescence image of the tissue section
   
   # Step 1: Demultiplex BCL files into FASTQ files
   # This command processes raw BCL data from an Illumina sequencer to generate FASTQ files.
   # The --run parameter points to the directory containing the BCL files.
   # The --csv parameter specifies the sample sheet for demultiplexing.
   # The --id parameter names the output directory where FASTQ files will be stored.
   spaceranger mkfastq \
       --id="${FASTQ_RUN_ID}" \
       --run="${BCL_DIR}" \
       --csv="${SAMPLE_SHEET}"
   
   # Step 2: Generate spatial gene counts matrix
   # This command takes FASTQ files, aligns them to a reference transcriptome, and maps reads to spatial spots.
   # It requires the path to the FASTQ files (generated by mkfastq), a reference transcriptome, and image data.
   # The "2 and 8 mm resolution binning" mentioned in the description likely refers to a post-processing analysis
   # or a specific configuration for Visium HD, as standard spaceranger count processes data at the native spot resolution.
   # For Visium HD, additional parameters like --probe-set might be used to specify resolution.
   spaceranger count \
       --id="${SAMPLE_ID}_spatial_analysis" \
       --transcriptome="${REFERENCE_PATH}" \
       --fastqs="${FASTQ_RUN_ID}/HXXXXX" \
       --sample="${SAMPLE_ID}" \
       --image="${IMAGE_PATH}" \
       --slide="${SLIDE_ID}" \
       --area="${AREA_ID}" \
       --output-dir="${SAMPLE_ID}_spatial_output"
   

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2. 2

   Library strategy: VisiumHD spatial transcriptomics

   Spatial transcriptomics v2.2.0 (Inferred with models/gemini-2.5-flash)

   $ Bash example

   # Download and extract Space Ranger software (replace with actual version and path)
   # wget https://cf.10xgenomics.com/releases/spatial-transcriptomics/spaceranger-2.2.0.tar.gz
   # tar -xzf spaceranger-2.2.0.tar.gz
   # export PATH=/path/to/spaceranger-2.2.0:$PATH
   
   # Download 10x Genomics reference data (e.g., human GRCh38)
   # wget https://cf.10xgenomics.com/supp/spatial-transcriptomics/refdata-gex-GRCh38-2020-A.tar.gz
   # tar -xzf refdata-gex-GRCh38-2020-A.tar.gz
   
   # Placeholder for VisiumHD probe set file (download from 10x Genomics support site or create if custom)
   # wget https://cf.10xgenomics.com/supp/spatial-transcriptomics/VisiumHD_probe_set_v1.0_GRCh38-2020-A.csv
   
   # Run Space Ranger for VisiumHD data processing
   # Replace placeholders with actual paths and sample-specific information
   spaceranger count \
       --id=visiumhd_sample1_output \
       --transcriptome=/path/to/refdata-gex-GRCh38-2020-A \
       --fastqs=/path/to/your/fastq_files \
       --image=/path/to/your/high_resolution_image.tif \
       --slide=V12345678 \
       --area=A1 \
       --probe-set=/path/to/VisiumHD_probe_set_v1.0_GRCh38-2020-A.csv \
       --localcores=8 \
       --localmem=64

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Tools Used