ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets
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-deri...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61023-6 |
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| author | Ye Zheng Daniel P. Caron Ju Yeong Kim Seong-Hwan Jun Yuan Tian Florian Mair Kenneth D. Stuart Peter A. Sims Raphael Gottardo |
| author_facet | Ye Zheng Daniel P. Caron Ju Yeong Kim Seong-Hwan Jun Yuan Tian Florian Mair Kenneth D. Stuart Peter A. Sims Raphael Gottardo |
| author_sort | Ye Zheng |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-37bb22c42fd74fa68d3616dd60364340 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-37bb22c42fd74fa68d3616dd603643402025-08-20T03:45:31ZengNature PortfolioNature Communications2041-17232025-07-0116111310.1038/s41467-025-61023-6ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasetsYe Zheng0Daniel P. Caron1Ju Yeong Kim2Seong-Hwan Jun3Yuan Tian4Florian Mair5Kenneth D. Stuart6Peter A. Sims7Raphael Gottardo8Department of Bioinformatics and Computational Biology, Department of Systems Biology, University of Texas MD Anderson Cancer CenterDepartment of Microbiology and Immunology, Columbia UniversityVaccine and Infectious Disease Division, Fred Hutchinson Cancer CenterVaccine and Infectious Disease Division, Fred Hutchinson Cancer CenterVaccine and Infectious Disease Division, Fred Hutchinson Cancer CenterFlow Cytometry Core Facility, ETH ZürichDepartment of Pediatrics, University of Washington and Center for Global Infectious Disease Research, Seattle Children’s Research InstituteDepartment of Systems Biology, Columbia UniversityVaccine and Infectious Disease Division, Fred Hutchinson Cancer CenterAbstract 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.https://doi.org/10.1038/s41467-025-61023-6 |
| spellingShingle | Ye Zheng Daniel P. Caron Ju Yeong Kim Seong-Hwan Jun Yuan Tian Florian Mair Kenneth D. Stuart Peter A. Sims Raphael Gottardo ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets Nature Communications |
| title | ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets |
| title_full | ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets |
| title_fullStr | ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets |
| title_full_unstemmed | ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets |
| title_short | ADTnorm: robust integration of single-cell protein measurement across CITE-seq datasets |
| title_sort | adtnorm robust integration of single cell protein measurement across cite seq datasets |
| url | https://doi.org/10.1038/s41467-025-61023-6 |
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