Toti-n-seq Breakthrough Enables Universal And Scalable Single-cell Profiling

Background

The accelerated advancement of single-cell and single-nucleus RNA sequencing (sc/snRNA-seq) has opened unprecedented windows into cellular diversity, yet existing methods for multiplexing samples struggle pinch scalability and accuracy. Traditional techniques relying connected antibodies aliases lipid-based barcodes often neglect to uniformly explanation cells crossed different types aliases species, peculiarly successful analyzable objective samples. These limitations—cell-type bias, cross-contamination risks, and nonaccomplishment of uncommon compartment populations—hinder large-scale studies and objective translation. To flooded these challenges, a squad led by Professor Yiwei Li astatine Huazhong University of Science and Technology (HUST) has pioneered Toti-N-Seq, a groundbreaking exertion that harnesses nan cosmopolitan beingness of N-glycans connected compartment and atomic surfaces. Published arsenic a screen communicative successful Research (2025, DOI: 10.34133/research.0678), this invention redefines really researchers attack high-throughput cellular profiling.

Research progress

At nan bosom of Toti-N-Seq lies an engineered protein, Stv-Fg, derived from modifying nan earthy glycan-binding macromolecule Fbs1. This fusion macromolecule binds non-selectively to each N-glycan types, enabling cosmopolitan tagging of cells and nuclei. By attaching DNA barcodes to Stv-Fg, nan squad achieved precise sample multiplexing without cell-type aliases type restrictions. Experimental validations underscored its robustness: travel cytometry revealed labeling efficiencies arsenic debased arsenic 37.5 pM for compartment membranes and 75.0 pM for nuclei, pinch cross-contamination beneath 2% moreover aft prolonged sample mixing.

In applicable applications, Toti-N-Seq demonstrated exceptional accuracy. When applied to single-nucleus sequencing, it achieved an wide classification accuracy (OCA) of 0.987, outperforming accepted antibody- aliases lipid-based methods. Notably, nan exertion preserved uncommon compartment populations, specified arsenic nan 0.5% plasmacytoid dendritic cells (pDCs) successful quality peripheral humor samples, while reducing doublet rates to 0.04% for azygous cells and 0.02% for nuclei. These capabilities were further validated successful 12-plex experiments, wherever sample ratio deviations remained nether 4%, proving its reliability for large-scale studies.

Future prospects

Looking ahead, nan Toti-N-Seq level is group to toggle shape some basal and applied research. The squad plans to grow its multiplexing capacity to 24-plex aliases higher, facilitating eager projects for illustration cross-organ compartment atlases and high-throughput supplier screening. Integration pinch epigenetic and proteomic devices will alteration multi-dimensional single-cell analyses, shedding ray connected analyzable regulatory networks.

Clinically, Toti-N-Seq's expertise to clasp uncommon compartment subsets positions it arsenic a powerful instrumentality for dissecting tumor microenvironments and predicting immunotherapy responses. Upcoming multi-center studies will research its diagnostic imaginable successful crab diligent cohorts. Beyond academia, nan technology's compatibility pinch platforms for illustration MobiNova microfluidics promises to streamline business workflows, accelerating supplier improvement and toxicity testing done standardized, reproducible protocols.

Conclusion

Toti-N-Seq represents a leap guardant successful single-cell genomics, addressing long-standing bottlenecks successful multiplexing accuracy and scalability. By leveraging nan ubiquity of N-glycans, Professor Li's squad has created a versatile instrumentality that bridges type and compartment types while preserving biologic nuance. As nan exertion moves toward objective and business adoption, it holds nan imaginable to democratize high-resolution cellular profiling, empowering discoveries from developmental biology to personalized medicine.