HKUST researchers develop a method for single cell analysis

25 February 2023

Single-cell genomics has become a mainstay technology used to dissect multicellular organisms and tissues that are composed of cells with diverse functions. The power of this approach has been demonstrated in comprehensive reference studies including cell atlases. Novel cell types have been discovered that further led to the elucidation of new mechanisms, complex cellular interactions and transitions associated with disease initiation or progression have been revealed, and cross-species analyses have shed light on evolutionary processes.

The use of single-cell technology in studying cancer is especially important. Regulatory mechanisms underlying drug resistance or immune evasion are elusive and complex, and tumor cell heterogeneity is a major contributing factor to this complexity, making it particularly challenging to dissect these mechanisms with bulk techniques. Single-cell technologies have greatly enhanced our understanding of tumor heterogeneity and accelerated mechanistic discovery.

Frozen tumors in biobanks represent most of the readily available clinical samples for cancer studies. Several single-cell methods that interrogate DNA and RNA simultaneously in the same cell have been developed but their applicability to frozen biobanked tissues is limited. Among these methods, many require physical separation of the nucleus from the cytosol. These methods are technically demanding and time-consuming and inevitably result in some sample loss. 

A team at the Hong Kong University of Science and Technology led by Dr Angela Wu has developed a single-cell whole genomic sequencing method that is versatile, easy to use, and compatible with single nuclei from frozen tissues.

scONE-seq amplifies the transcriptome and genome of a single cell or nucleus simultaneously in a one-pot reaction. During the coamplification, specifically designed DNA and RNA barcodes with unique molecular identifiers recognise each nucleic acid species allowing transcriptomic and genomic information to be distinguished after sequencing. scONE-seq has a simplified library construction workflow and is compatible with standard single-cell isolation methods such as fluorescence-activated cell sorting. It can be easily scaled up using liquid-handling robots and is still readily accessible for manual operation. The simplified workflow eliminates transferring steps that cause material loss, thereby ensuring good library quality; and because DNA and RNA can be coamplified, scONE-seq is applicable to nuclei from frozen samples.

The team benchmarked the technical performance of scONE-seq and showed that it is comparable to existing methods using various sample types, including cell lines and lymphocytes from the peripheral blood mononuclear cells of a healthy donor. scONE-seq allowed the discovery of novel disease-related phenotypes in a frozen mutant astrocytoma sample. 

The team used scONE-seq to integrate clonal and transcriptomic information and identified a unique normal-like tumor clone in this sample. Further analysis indicated that this subpopulation exhibits molecular phenotypes related to tumor-neuron synapse formation and immune repression regulation. This finding demonstrated the power of scONE-seq to identify unique cell subpopulations from true normal cells by harnessing both genome and transcriptome information.

The research was published in Science Advances.