PARTITIONING CELLS FOR HIGH-THROUGHPUT SINGLE-CELL SEQUENCING

PARTITIONING CELLS FOR HIGH-THROUGHPUT SINGLE-CELL SEQUENCING

Researchers at UCSF have developed a method for high throughput single-cell multi-omic sequencing that is rapid, simple to operate, and offers key innovations over existing technologies.

Single-cell sequencing technologies refer to the methods to obtain genomics, transcriptomics or multi-omics information of individual cells. Traditional sequencing methods work with samples of many cells, reveal average signals of the pooled samples, and are thereby unable to resolve cellular heterogeneity. Several single-cell sequencing methods exist; however these face several limitations. For example, microfluidics-based techniques are technologically challenging to perform and can require expensive equipment to operate. Well plate-based methods are limited in throughput. Finally, most available methods target transcriptome sequencing, yet single-cell genome sequencing and other single-cell multi-omic technologies are not well established. Thus, there remain unmet needs in the field of high throughput single-cell sequencing.

Stage of Research

The inventors have developed methods and compositions for high throughput single-cell, multi-omic sequencing that is simple to operate and uses hydrogel-based single-cell partitioning techniques. This method comprises encapsulating single cells into hydrogel particles or fixating cells as permeable, single-cell particles, barcoding the cells, and amplifying the nucleic acids using one of several possible methods of amplification. The method can be performed with or without the adaptation of a commercially available microfluidic device. The inventors have also developed EASI-seq, Easily Accessible Single microbe sequencing, for sequencing single microbe genomic DNA by adopting commercially available single cell sequencing platforms, which requires no custom-build microfluidic devices.

Applications

Whole genome sequencing using hydrogel-based compartmentalization.
Single-cell RNA sequencing using fixed cells as compartmentalization.
High-throughput single microbe whole genome sequencing on commercially available microfluidics platforms.

Advantages

Single cells are isolated and encapsulated in hydrogel microbeads which allows for fast processing of millions of cells without any complex instrumentation, and at a throughput that surpasses other currently-available methods.
Because the hydrogel materials allow free diffusion of molecules with diameters smaller than the gel pore size, while still sterically trapping genomic DNA, multistep washing and buffer exchange reactions can be performed which are not easily performed in other systems.
The technology is compatible with single-cell multi-omics sequencing, allowing for protein epitope profiling, mRNA profiling or gDNA sequencing to be performed.
The technique is applicable to bacteria and fungi as well as mammalian cells.

Stage of Development

Research – in vitro

Publications

WO2022/251509