{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T18:46:06Z","timestamp":1790621166623,"version":"4.1.0"},"reference-count":30,"publisher":"Oxford University Press (OUP)","issue":"4","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":62,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,10,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Removing unwanted variation (RUV) is key for accurate biological interpretation in high-throughput sequencing studies. However, no standardized approach exists for pseudobulked single-cell RNA-sequencing (scRNA-seq) data. Improper implementation of RUV methods may remove biological information, jeopardizing power and false positive control in differential expression analysis. We evaluate the impact of three implementation strategies (\u2018trails\u2019) in three RUV methods (RUV2, RUVIII, RUV4) using simulated and real biological signals in pseudobulked scRNA-seq data. Effects of technical noise under confounding and model misspecification conditions are also considered. Additionally, we introduce a novel strategy, RUVIII PBPS, to remove unwanted variation in pseudobulk differential expression analyses with insufficient technical replicates or negative control genes. Our analysis demonstrates that removing unwanted variation per cell type with RUV2 or RUVIII extracts factors associated with technical noise and controls the false discovery rate (FDR), even in the presence of confounding. RUVIII PBPS successfully controls the FDR when other standard RUV methods cannot be used due to missing technical replicates, dependence between the factor of interest and the sources of unwanted variation, and lack of plausible negative control genes.<\/jats:p>","DOI":"10.1093\/nargab\/lqaf179","type":"journal-article","created":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T12:56:02Z","timestamp":1762606562000},"source":"Crossref","is-referenced-by-count":1,"title":["Removal of unwanted variation in pseudobulk analysis of single-cell RNA sequencing data and the leveraging of pseudoreplicates"],"prefix":"10.1093","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-5072-2411","authenticated-orcid":false,"given":"Sof\u00eda","family":"Prieto\u00a0Le\u00f3n","sequence":"first","affiliation":[{"name":"Data Science Institute and I-BioStat, Hasselt University , 3500 Hasselt ,","place":["Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2168-1373","authenticated-orcid":false,"given":"Ewoud","family":"De\u00a0Troyer","sequence":"additional","affiliation":[{"name":"Statistics and Decision Sciences, Johnson & Johnson Innovative Medicine , 2340 Beerse ,","place":["Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5347-9292","authenticated-orcid":false,"given":"Helena","family":"Geys","sequence":"additional","affiliation":[{"name":"Data Science Institute and I-BioStat, Hasselt University , 3500 Hasselt ,","place":["Belgium"]},{"name":"Statistics and Decision Sciences, Johnson & Johnson Innovative Medicine , 2340 Beerse ,","place":["Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1833-8478","authenticated-orcid":false,"given":"Koen","family":"Van\u00a0den\u00a0Berge","sequence":"additional","affiliation":[{"name":"Statistics and Decision Sciences, Johnson & Johnson Innovative Medicine , 2340 Beerse ,","place":["Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6442-4089","authenticated-orcid":false,"given":"Olivier","family":"Thas","sequence":"additional","affiliation":[{"name":"Data Science Institute and I-BioStat, Hasselt University , 3500 Hasselt ,","place":["Belgium"]},{"name":"Department of Mathematics, Computer Science and Statistics, Ghent University , 9000 Ghent ,","place":["Belgium"]},{"name":"National Institute for Applied Statistics Research Australia (NIASRA), University of Wollongong , 2522 Wollongong, New South Wales ,","place":["Australia"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,12,8]]},"reference":[{"key":"2025120810561534400_B1","doi-asserted-by":"publisher","first-page":"733","DOI":"10.1038\/nrg2825","article-title":"Tackling the widespread and critical impact of batch effects in high-throughput data","volume":"11","author":"Leek","year":"2010","journal-title":"Nat Rev Genet"},{"key":"2025120810561534400_B2","doi-asserted-by":"publisher","first-page":"lqaa078","DOI":"10.1093\/nargab\/lqaa078","article-title":"ComBat-seq: batch effect adjustment for RNA-seq count data","volume":"2","author":"Zhang","year":"2020","journal-title":"NAR Genom Bioinform"},{"key":"2025120810561534400_B3","doi-asserted-by":"publisher","first-page":"539","DOI":"10.1093\/biostatistics\/kxr034","article-title":"Using control genes to correct for unwanted variation in microarray data","volume":"13","author":"Gagnon-Bartsch","year":"2012","journal-title":"Biostatistics"},{"key":"2025120810561534400_B4","first-page":"1","article-title":"Removing unwanted variation from high dimensional data with negative controls","volume":"820","author":"Gagnon-Bartsch","year":"2013","journal-title":"Berkeley: Tech Rep Dep Stat Univ California"},{"key":"2025120810561534400_B5","doi-asserted-by":"publisher","first-page":"896","DOI":"10.1038\/nbt.2931","article-title":"Normalization of RNA-seq data using factor analysis of control genes or samples","volume":"32","author":"Risso","year":"2014","journal-title":"Nat Biotechnol"},{"key":"2025120810561534400_B6","first-page":"1145","article-title":"Unifying and generalizing methods for removing unwanted variation based on negative controls","volume":"31","author":"Gerard","year":"2021","journal-title":"Stat Sinica"},{"key":"2025120810561534400_B7","doi-asserted-by":"publisher","first-page":"6073","DOI":"10.1093\/nar\/gkz433","article-title":"A new normalization for Nanostring nCounter gene expression data","volume":"47","author":"Molania","year":"2019","journal-title":"Nucleic Acids Res"},{"key":"2025120810561534400_B8","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1038\/s41587-022-01440-w","article-title":"Removing unwanted variation from large-scale RNA sequencing data with PRPS","volume":"41","author":"Molania","year":"2023","journal-title":"Nat Biotechnol"},{"key":"2025120810561534400_B9","doi-asserted-by":"publisher","first-page":"1863","DOI":"10.1214\/16-AOS1511","article-title":"Confounder adjustment in multiple hypothesis testing","volume":"45","author":"Wang","year":"2017","journal-title":"Ann Stat"},{"key":"2025120810561534400_B10","doi-asserted-by":"publisher","first-page":"e96","DOI":"10.1093\/nar\/gkac486","article-title":"RUV-III-NB: Normalization of single cell RNA-seq Data","volume":"50","author":"Salim","year":"2022","journal-title":"Nucleic Acids Res"},{"key":"2025120810561534400_B11","doi-asserted-by":"publisher","first-page":"e161","DOI":"10.1371\/journal.pgen.0030161","article-title":"Capturing heterogeneity in gene expression studies by surrogate variable analysis","volume":"3","author":"Leek","year":"2007","journal-title":"PLoS Genet"},{"key":"2025120810561534400_B12","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1093\/biostatistics\/kxj037","article-title":"Adjusting batch effects in microarray expression data using empirical Bayes methods","volume":"8","author":"Johnson","year":"2007","journal-title":"Biostatistics"},{"key":"2025120810561534400_B13","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1186\/s12859-020-03559-6","article-title":"Simulating ComBat: how batch correction can lead to the systematic introduction of false positive results in DNA methylation microarray studies","volume":"21","author":"Zindler","year":"2020","journal-title":"BMC Bioinformatics"},{"key":"2025120810561534400_B14","doi-asserted-by":"publisher","first-page":"550","DOI":"10.1038\/s41576-023-00586-w","article-title":"Best practices for single-cell analysis across modalities","volume":"24","author":"Heumos","year":"2023","journal-title":"Nat Rev Genet"},{"key":"2025120810561534400_B15","doi-asserted-by":"publisher","first-page":"6077","DOI":"10.1038\/s41467-020-19894-4","article-title":"Muscat detects subpopulation-specific state transitions from multi-sample multi-condition single-cell transcriptomics data","volume":"11","author":"Crowell","year":"2020","journal-title":"Nat Commun"},{"key":"2025120810561534400_B16","doi-asserted-by":"publisher","first-page":"370","DOI":"10.2307\/2344614","article-title":"Generalized linear models","volume":"135","author":"Nelder","year":"1972","journal-title":"J Roy Stat Soc Ser A: Stat Soc"},{"key":"2025120810561534400_B17","doi-asserted-by":"publisher","first-page":"145","DOI":"10.2307\/621706","article-title":"The assumptions of the linear regression model","volume":"52","author":"Poole","year":"1971","journal-title":"T I Brit Geogr"},{"key":"2025120810561534400_B18","doi-asserted-by":"publisher","first-page":"2040004","DOI":"10.1142\/S0219720020400041","article-title":"Stably expressed genes in single-cell RNA sequencing","volume":"18","author":"Deeke","year":"2020","journal-title":"J Bioinform Comput Biol"},{"key":"2025120810561534400_B19","doi-asserted-by":"crossref","unstructured":"Lin Y, Ghazanfar S, Strbenac D \u00a0et al. \u00a0Evaluating stably expressed genes in single cells. GigaScience. 2019;8:giz106. 10.1093\/gigascience\/giz106.","DOI":"10.1093\/gigascience\/giz106"},{"key":"2025120810561534400_B20","article-title":"ruv: Detect and Remove Unwanted Variation using Negative Controls","volume-title":"R package version 0.9.7.1","author":"Gagnon-Bartsch","year":"2019"},{"key":"2025120810561534400_B21","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: a Bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2010","journal-title":"Bioinformatics"},{"key":"2025120810561534400_B22","doi-asserted-by":"publisher","first-page":"gkaf018","DOI":"10.1093\/nar\/gkaf018","article-title":"edgeR v4: powerful differential analysis of sequencing data with expanded functionality and improved support for small counts and larger datasets","volume":"53","author":"Chen","year":"2025","journal-title":"Nucleic Acids Res"},{"key":"2025120810561534400_B23","doi-asserted-by":"publisher","first-page":"7852","DOI":"10.1038\/s41467-022-35520-x","article-title":"Reply to: a balanced measure shows superior performance of pseudobulk methods in single-cell RNA-sequencing analysis","volume":"13","author":"Zimmerman","year":"2022","journal-title":"Nat Commun"},{"key":"2025120810561534400_B24","doi-asserted-by":"publisher","first-page":"eabf1970","DOI":"10.1126\/science.abf1970","article-title":"Single-cell RNA-seq reveals cell type\u2013specific molecular and genetic associations to lupus","volume":"376","author":"Perez","year":"2022","journal-title":"Science"},{"key":"2025120810561534400_B25","doi-asserted-by":"publisher","first-page":"D886","DOI":"10.1093\/nar\/gkae1142","article-title":"CZ CELLxGENE Discover: a single-cell data platform for scalable exploration, analysis and modeling of aggregated data","volume":"53","author":"Program","year":"2024","journal-title":"Nucleic Acids Res"},{"key":"2025120810561534400_B26","first-page":"9","article-title":"A study of effects of multicollinearity in the multivariable analysis","volume":"4","author":"Yoo","year":"2014","journal-title":"Int J Appl Sci Technol"},{"key":"2025120810561534400_B27","doi-asserted-by":"publisher","first-page":"1583","DOI":"10.1002\/art.38628","article-title":"Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures","volume":"66","author":"Chiche","year":"2014","journal-title":"Arthrit Rheumatol"},{"key":"2025120810561534400_B28","doi-asserted-by":"publisher","first-page":"1094","DOI":"10.1038\/s41590-020-0743-0","article-title":"Mapping systemic lupus erythematosus heterogeneity at the single-cell level","volume":"21","author":"Nehar-Belaid","year":"2020","journal-title":"Nat Immunol"},{"key":"2025120810561534400_B29","doi-asserted-by":"publisher","first-page":"e0191629","DOI":"10.1371\/journal.pone.0191629","article-title":"RLE plots: visualizing unwanted variation in high dimensional data","volume":"13","author":"Gandolfo","year":"2018","journal-title":"PloS One"},{"key":"2025120810561534400_B30","doi-asserted-by":"publisher","first-page":"9775","DOI":"10.1073\/pnas.1820006116","article-title":"scMerge leverages factor analysis, stable expression, and pseudoreplication to merge multiple single-cell RNA-seq datasets","volume":"116","author":"Lin","year":"2019","journal-title":"Proc Natl Acad Sci"}],"container-title":["NAR Genomics and Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nargab\/article-pdf\/7\/4\/lqaf179\/65801215\/lqaf179.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nargab\/article-pdf\/7\/4\/lqaf179\/65801215\/lqaf179.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T15:56:27Z","timestamp":1765209387000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nargab\/article\/doi\/10.1093\/nargab\/lqaf179\/8373957"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,7]]},"references-count":30,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2025,10,7]]}},"URL":"https:\/\/doi.org\/10.1093\/nargab\/lqaf179","relation":{},"ISSN":["2631-9268"],"issn-type":[{"value":"2631-9268","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2025,12]]},"published":{"date-parts":[[2025,10,7]]},"article-number":"lqaf179"}}