Abstract 2070: Subcellular mapping of immune repertoire profiles with <i>in situ</i> sequencing on G4X

Abstract Detailed spatial analysis of immunology and pathology, such as immune repertoire profiling in tissue, requires in situ sequencing of variable gene regions. We introduce Direct-Seq, a novel technology using the G4X platform for in situ RNA sequencing at sub-cellular resolution, demonstrated by sequencing IgH and TCRB receptors in PBMCs and FFPE tissue. We developed a probe panel that targets the conserved V and J-regions that flank CDR3 on IgH transcripts and a second panel that targets the V and C-regions that flank CDR3 and J regions on TCRB transcripts. PBMCs were seeded onto a glass substrate, followed by fixation. The diverse gap sequence was reverse transcribed into the probes, which were then clonally amplified. Clonal amplification products were sequenced in situ using Singular Genomics’ SBS chemistry. We also applied the technique to tissue samples by transferring 5-µm thick FFPE tonsil sections to G4X slides, followed by deparaffinization and antigen retrieval. Amplicon preparation and sequencing were conducted in the same way as for PBMCs. Direct-Seq successfully profiled ∼ 60% of all B-cells (relative to CD19-positive fraction) and ∼ 20% of all T-cells (relative to CD3-positive fraction) in PBMC populations. We observed the expected high diversity of clonotypes, with 92-98% of B and T-cells having distinct CDR3 sequences. Comparing multiple 50 bp reads within the same cell, we calculate the sequencing accuracy to be ∼ 98%. For cells that had at least 1 read, we detected on average ∼ 6 reads/cell for B-cells and ∼ 2 reads/cell for T-cells. For FFPE tonsil samples we observed similar levels of diversity (94-99%) and accuracy (97-99%) as we did in PBMCs, but the detection efficiency was significantly lower, likely due to reduced RNA accessibility and degradation resulting from fixation. In conclusion, Direct-Seq has demonstrated its unique capability for in situ sequencing of variable regions of IgH and TCRB transcripts in both PBMCs and FFPE tissues. This technology enables detailed mapping of immune cell clonality and diversity, providing a powerful tool for advancing our understanding of immunity and disease. Citation Format: Michael J. Lawson, Tung T. Le, Zane Hiatt, Ryan Shultzaberger, Kenneth Gouin III, Daan Witters, Eli N. Subcellular mapping of immune repertoire profiles with in situ sequencing on G4X [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2070.