Space Ranger outputs specific files, along with modifications to the GEX outputs, for PEX analysis.

• Feature-Barcode Matrices (MEX & HDF5 formats): All Protein Capture counts become new features in addition to the standard per-gene features, and are output alongside gene counts in the feature-barcode matrices. For every row in the feature_reference.csv file, there will be a corresponding row in the feature-barcode matrix and the rowname will correspond to the value in the id column in the Feature Reference file for that feature.

• Molecule Info (HDF5 format): The per-molecule information file (molecule_info.h5) captures the protein-associated feature information in the feature reference HDF5 group.

• BAM: New tags are added to the BAM file to capture the quality, sequence and feature id for the protein Captured reads.

• Run Metrics: The metrics_summary.csv file includes additional protein-specific metrics in addition to the Gene Expression metrics and are tabulated in the Protein Expression Metrics section below.

• Web Summary: The web_summary.html (learn more) captures protein-specific metrics. The secondary analysis results, including histograms on UMI counts and a treemap plot of the protein distribution, are displayed.

• Secondary Analysis: protein-specific secondary analysis are included. The k-means and graph-based clustering for protein counts are run on PCA-reduced space similar to the Gene Expression. Both t-distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation and Projection (UMAP) are generated using raw protein counts. This contrasts the t-SNE and UMAP projections for gene counts which are run on PCA-reduced space. The spatial enrichment file spatial_enrichment.csv also includes the value for Antibodies and an additional column for the secondary name if included.

• Isotype Correlation: The isotype_correlations.csv file is an protein-specific output. It includes the correlation values of the antibodies in the panel against the four included isotype controls. This file is helpful is determining the background signal.

Space Ranger v2.1 supports protein expression analysis with built-in isotype normalization to correct for technical noise that is inherent in antibody-based assays. The filtered feature barcode matrices contain the normalized counts while the raw feature barcode matrices contain raw UMI counts. For convenience, the normalization factor is provided in the outputs.

The isotype_normalization_factors.csv has the following columns: