Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Single-nucleus RNA sequencing (snRNA-seq) is crucial for studying complex tissues and advancing human genetics.
• Current snRNA-seq applications are hindered by batch effects, processing challenges, and high costs.

Purpose of the Study:

• To develop a cost-effective multiplexing method for snRNA-seq using sample-barcoded antibodies.
• To create a computational tool for accurate sample assignment and multiplet detection in multiplexed snRNA-seq data.

Main Methods:

• Nucleus hashing with sample-barcoded antibodies to label nuclei from distinct samples.
• Development and validation of the DemuxEM computational tool for demultiplexing snRNA-seq data.
• Comparison of snRNA-seq profiles with and without hashing antibodies on human brain cortex samples.

Main Results:

• Nucleus hashing did not significantly alter recovered gene expression profiles.
• DemuxEM accurately assigned singlets to their sample of origin and detected inter-sample multiplets.
• Validation using sex-specific gene expression, species-mixing, and natural genetic variation confirmed DemuxEM's accuracy.

Conclusions:

• Nucleus hashing is a viable strategy to multiplex snRNA-seq, overcoming limitations of batch effects and cost.
• The DemuxEM tool enables accurate demultiplexing, facilitating large-scale studies.
• This approach supports the creation of comprehensive tissue atlases and large-scale perturbation screens.