{
  "abstract": "Background Spatial multiomics is an emerging field with the potential to transform our understanding of biology and disease, accelerate drug development, and redefine pathology. While spatial transcriptomics has advanced significantly, there remains a critical need for in situ sequencing of variable transcript regions at single-cell resolution in both blood and tissue samples. This is particularly relevant for immune repertoire profiling, where spatial mapping of B and T cell clonotypes offers valuable biological insight.Here, we present Direct-Seq™, a novel technology built on the G4X™ platform that enables in situ sequencing of RNA at subcellular resolution. We applied Direct-Seq to sequence the diverse IgH and TCRβ antigen receptor regions in peripheral blood mononuclear cells (PBMCs), formalin-fixed paraffin-embedded (FFPE) tissues, and fresh frozen (FF) human tonsils.Methods Using oligonucleotide probes designed to target the variable (V) and joining (J) regions flanking the highly diverse CDR3 region of IgH and TCRβ transcripts, we performed in situ reverse transcription and clonal amplification on fixed non-stimulated PBMCs. Sequencing was conducted using Singular Genomics’ sequencing-by-synthesis (SBS) chemistry. For tissue applications, 5-µm FFPE tonsil sections were transferred to G4X slides, deparaffinized, and subjected to antigen retrieval. Similarly, 10-µm FF tonsil sections were permeabilized and processed using the same Direct-Seq workflow.Results Direct-Seq successfully profiled up to 30% of B cells (relative to CD19+) and T cells (relative to CD3+) in PBMCs. In FFPE tonsil tissue, up to 9% of B cells were captured, while in FF tissue, we detected approximately 20% of both B and T cells. In all sample types, we observed extensive CDR3 sequence diversity. Notably, clonally expanded B cells with identical CDR3 sequences were spatially clustered within germinal centers of the tonsil, demonstrating the high-resolution clonotyping capabilities of Direct-Seq.Furthermore, Direct-seq was combined with multiplexed protein detection using immune-specific markers in the same tissue sections. The results confirmed the spatial correlation between transcript identity and phenotypic protein expression.Conclusions In summary, Direct-Seq enables direct, spatially resolved sequencing of IgH and TCRβ transcripts in PBMCs, FFPE and FF tissue samples, uncovering immune cell clonality and distribution. When integrated with high-plex proteomic data, Direct-Seq offers a powerful tool for advancing spatial immunology and disease research.",
  "authors": [
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Tung Le"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Ryan Shultzaberger"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Ashley Tsue"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Zane Hiatt"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Nathan Ing"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Kenneth H Gouin"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Michael Lawson"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Daan Witters"
    },
    {
      "affiliations": [
        "Singular Genomics, San Diego, CA, USA"
      ],
      "name": "Eli Glezer"
    }
  ],
  "title": "160 Spatially resolved in situ sequencing of immune receptors in single cells and tissue microenvironments",
  "uid": "942ccb1a-69c2-5902-9f47-51330d970f0e"
}