NUCLEAR DNA-ANTIBODY SEQUENCING FOR JOINT PROFILING OF GENOTYPE AND PROTEIN IN SINGLE NUCLEI

NUCLEAR DNA-ANTIBODY SEQUENCING FOR JOINT PROFILING OF GENOTYPE AND PROTEIN IN SINGLE NUCLEI

Researchers at UCSF have developed a method for joint nucleic acid-protein profiling from single nuclei that can be applied to all manner of biological samples, including frozen tumor specimens or infectious disease samples.

Cellular heterogeneity is an aspect that drives disease progression and relapse in cancer, and because cancer cells are heterogeneous in genotype and phenotype, it is especially difficult to link genotypes to immunophenotypes. Single cell analysis thereby provides a powerful tool for characterizing this complexity. However, single-cell genotyping studies have yet to map DNA variants and cell surface phenotypes in the same cells, thereby precluding direct linkage of phenotypes to the genetic mutations that drive them. In cancer studies, it is particularly important to analyze mutations in intronic regions of the genome as well as other non-transcribed elements, such as transcription factor binding sites. Therefore, a high throughput single-cell methods for measuring genotype and phenotype with scalability and precision is necessary for the comprehensive and accurate marking of immune biomarkers.

Stage of Research

The inventors have developed “DAb-seq”; a method for joint profiling of DNA and surface proteins in single cells at high throughput. This approach directly sequences DNA for genotype and surface proteins for phenotype, both of which are gold standards in blood cancer studies. In addition, the inventors have addressed previous limitations in the published DAb-seq method requiring fresh samples with intact cells that limited analysis of frozen tumor specimens in retrospective tissue banks. The modified method is extendable to analyzing frozen specimens whereby DNA and protein information are obtained from single nuclei, and is therefore termed nuclear DAb-seq (nDAb-seq).

Applications

Isolation of nuclei and subsequent antibody staining.
High-throughput single-cell genotyping and phenotyping of the same cell.
The method can be extended to any biologic context (a cell line, a frozen tissue sample, a human or animal biopsy, a solid tumor sample, etc) where obtaining nucleic acid and protein information from single nuclei is valuable.

Advantages

nDAb-seq can be applied to all manner of biological samples including frozen tumor specimens and infections disease samples.
Applicability to frozen samples allows for analysis of bio banked samples for retrospective studies and does not solely rely on freshly-obtained samples or specimens.

Stage of Development

Research – in vitro

Publications

Demaree, B.*, Delley, C.L.*, Vasudevan, H.N., Peretz, C.A., Ruff, D., Smith, C.C. and Abate, A.R., Joint profiling of DNA and proteins in single cells to dissect genotype-phenotype associations in leukemia, Nature Communications, 2021. DOI: 10.1038/s41467-021-21810-3