Doing more with less: comparative analysis of sample multiplexing methods for single-cell RNA-seq

Multiplexing of samples in single-cell RNA-seq studies allows a significant reduction of the experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or -lipids allow barcoding sample-specific cells, a process called “hashing.” Here, we compared the hashing performance of TotalSeq-A and -C anti-bodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and sin-gle-nucleus RNA-seq. We also compared TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMC and TotalSeq-B with different lipid hashes on primary mouse tissues. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines and mouse strains. Antibody hashing was further evaluated on clinical samples using PBMC from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable ap-proach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects.