Comparative analysis of antibody- and lipid-based multiplexing methods for single-cell RNA-seq

Viacheslav Mylka, Irina Matetovici, Suresh Poovathingal, Jeroen Aerts, Niels Vandamme, Ruth Seurinck, Kevin Verstaen, Gert Hulselmans, Silvie Van den Hoecke, Isabelle Scheyltjens, Kiavash Movahedi, Hans Wils, Joke Reumers, Jeroen Van Houdt, Stein Aerts, Yvan Saeys

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)
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BACKGROUND: 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."

RESULTS: Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. We also compare TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMCs and TotalSeq-B with different lipids 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 PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects.

CONCLUSIONS: Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. On nuclei datasets, lipid hashing delivers the best results. Lipid hashing also outperforms antibodies on cells isolated from mouse brain. However, antibodies demonstrate better results on tissues like spleen or lung.

Original languageEnglish
Article number55
Number of pages21
JournalGenome Biology
Issue number1
Publication statusPublished - Dec 2022

Bibliographical note

© 2022. The Author(s).


  • Animals
  • Antibodies/chemistry
  • COVID-19/blood
  • Case-Control Studies
  • Cell Line, Tumor
  • Cell Nucleus/chemistry
  • Humans
  • Lipids/chemistry
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Neutrophils/chemistry
  • Sequence Analysis, RNA/methods
  • Single-Cell Analysis/methods


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