186 Direct-Seq: de novo sequencing of IgH and TCRB RNA at sub-cellular resolution on the G4X
Year: 2024
Type: conference-abstract
Abstract: Background Spatial multi-omics represents an emerging frontier in life sciences poised to enhance our core understanding of biology and diseases, expedite drug development, and revolutionize pathology. Although considerable progress has been made in spatial transcriptomics, there remains a compelling need for de novo, in situ sequencing of transcripts of single cells in blood samples and solid tissues. Sequencing transcripts in situ would benefit immune repertoire profiling by revealing the spatial distributions of B and T cell clonotypes. Here, we introduce Direct-Seq, a novel technology leveraging the G4X platform that enables in situ, de novo sequencing of RNA molecules at sub-cellular resolution. We demonstrate its application by sequencing diverse regions of IgH/TCRB antigen receptors in human cell lines and peripheral blood mononuclear cells (PBMCs). Methods To demonstrate this ability, two populations of cells were prepared: a mixture of Ramos, Jurkat, and Sup-T1 cell lines, for which we know the sequence of the variable region, and a sample of human PBMCs. Both populations were seeded onto G4X slides at a density of ~ 0.25 M cells/lane (~ 1 M cells per flow cell). Seeding was followed by fixation and assembly of the G4X flow cell. Padlock probes were designed to bind known sequences flanking the unknown gap sequence: complementary IgH RNA sequences in Ramos and PBMC B-cells, and complementary TCRB RNA sequences in Jurkat and Sup-T1 cells. Prior to ligation, the unknown gap sequence was incorporated into the probe, which was then amplified by rolling circle amplification (RCA). Clonal amplification products were sequenced in situ using Singular Genomics’ SBS chemistry on the G4X. Results High-accuracy 50 bp reads of the cell lines mixture showed that ~ 70% of the cells revealed the expected on-target reads with an average of ~ 20 reads/cell for IgH in Ramos, and 3-4 reads/cell for TCRB in Jurkat and Sup-T1 cells. IgH profiling of PBMCs was successfully sequenced in more than 60% of all B cells (as confirmed by CD19-positive fraction provided by the vendor), with an average on-target specific read count of ~ 5 reads/cell for 50-100 bp reads. This result was obtained for sequencing the J-sequence and the highly diverse CDR3 region. Conclusions In conclusion, we have demonstrated the unique capability of Direct-Seq to perform de novo sequencing of IgH/TCRB transcripts in cell line mixtures and B-cells from PBMC samples, highlighting its significant potential in elucidating the distributions of B and T cell in cells and tissue.
Authors: Tung Le, Zane Hiatt, Yeoan Youn, Ryan Shultzaberger, Kenneth Gouin, Sabrina Shore, Maria Gallina, Michael Lawson, Eli Glezer, Daan Witters
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
OpenAlex ID: W4404065597