Extracellular protein monitoring in the ResolveOME™ genomic and transcriptomic dual workflow to uncover cancer pathology mechanisms in single cells

T.V. Morozova1, V.J. Weigman1, J.G. Blackinton1, J. Croteau2, A. Fernandes2, K. Taylor2, Martin M Fabani3, J.A.A. West1, G.L. Harton1, J.S. Zawistowski1

1BioSkryb Genomics, Inc., Durham, NC 2BioLegend, San Diego, CA 3Singular Genomics Systems, Inc., San Diego, CA

Cancer is a disease of remarkable cell heterogeneity and is driven by complex, interconnected omic tiers. Single cell research has become an instrumental method in interrogating these multiple levels, as the bulk sequencing does not have enough resolution to reveal the true heterogeneity of the samples. The ResolveOME workflow aids understanding of cell-to-cell heterogeneity by providing unified genomics and transcriptomic information—including assessment of genome-wide single nucleotide variation, copy number changes, regulatory variants, splice isoform variation and cell state changes from the same single cell. Here, we have adapted the existing ResolveOME genomic/transcriptomic workflow to include the detection of cell-surface protein expression using the TotalSeq TM -A Human T-cell, B-cell, Natural Killer (TBNK) panel (BioLegend) of antibody-conjugated oligonucleotides. Primary peripheral blood mononuclear cells (PBMCs) were processed using the ResolveOME workflow which unifies template-switching single-cell RNAseq chemistry and Primary Template-directed Amplification (PTA) for whole genome amplification (WGA). We incorporated the TotalSeq TM-A antibody-oligo cocktail to this workflow, whereby both cellular mRNAs and antibody-derived oligos specifically bound to PBMC antigens anneal to oligo dT primers, followed by template switch-based reverse transcription. This resulted in the creation of first-strand cDNA molecules and antibody-derived tag molecules that could be affinity purified and pre-amplified following whole genome amplification by PTA. Separate protein+RNA and DNA fractions are processed as distinct library preparations at the end of the workflow. Sequencing was performed with the novel G4 Sequencing Platform from Singular Genomics.

Using this strategy with PBMC cells, we detected all nine barcode IDs corresponding to the specific antibodies in the TotalSeq TM -A panel. The total number of detected barcodes varied between antibodies as well as between single cells, whereby CD19, CD3, CD4, CD16 and CD56 showed the most significant variation between individual single cells. We are extending these analyses to other cellular systems as well as to primary cancer cells, using antibody-conjugated antibody panels tailored to the application.

Our study has devised a method for simultaneous detection of three omic tiers: whole genomic and transcriptomic signatures coupled to assessment of defined protein panels at the individual cell level, empowering insights into the interplay between the three tiers not possible in isolation.