ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets

Ye Zheng, Daniel P. Caron, Ju Yeong Kim, Seong-Hwan Jun, Yuan Tian, Florian Mair, Kenneth D. Stuart, Peter A. Sims and Raphael Gottardo ()
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Ye Zheng: University of Texas MD Anderson Cancer Center
Daniel P. Caron: Columbia University
Ju Yeong Kim: Fred Hutchinson Cancer Center
Seong-Hwan Jun: Fred Hutchinson Cancer Center
Yuan Tian: Fred Hutchinson Cancer Center
Florian Mair: ETH Zürich
Kenneth D. Stuart: University of Washington and Center for Global Infectious Disease Research, Seattle Children’s Research Institute
Peter A. Sims: Columbia University
Raphael Gottardo: Fred Hutchinson Cancer Center

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) enables paired measurement of surface protein and mRNA expression in single cells using antibodies conjugated to oligonucleotide tags. Due to the high copy number of surface protein molecules, sequencing antibody-derived tags (ADTs) allows for robust protein detection, improving cell-type identification. However, variability in antibody staining leads to batch effects in the ADT expression, obscuring biological variation, reducing interpretability, and obstructing cross-study analyses. Here, we present ADTnorm, a normalization and integration method designed explicitly for ADT abundance. Benchmarking against 14 existing scaling and normalization methods, we show that ADTnorm accurately aligns populations with negative- and positive-expression of surface protein markers across 13 public datasets, effectively removing technical variation across batches and improving cell-type separation. ADTnorm enables efficient integration of public CITE-seq datasets, each with unique experimental designs, paving the way for atlas-level analyses. Beyond normalization, ADTnorm includes built-in utilities to aid in automated threshold-gating as well as assessment of antibody staining quality for titration optimization and antibody panel selection. Applying ADTnorm to an antibody titration study, a published COVID-19 CITE-seq dataset, and a human hematopoietic progenitors study allowed for identifying previously undetected phenotype-associated markers, illustrating a broad utility in biological applications.

Date: 2025
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DOI: 10.1038/s41467-025-61023-6

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