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Bioinform."],"abstract":"<jats:p>Gene expression profiling of single cells using single-cell and single-nucleus RNA sequencing (sxRNA-seq) enables researchers to characterize cellular heterogeneity and unraveling complex biological processes at unprecedented resolution. However, sxRNA-seq faces challenges due to the presence of ambient RNA, extraneous RNA molecules not originating from the cells of interest. Sample preparation is a major source of ambient RNA, where harsh conditions can lead to cell lysis and the release of intracellular RNA. This inescapable inclusion of ambient RNA can cause erroneous results and hinder downstream analyses. To address this issue, various methodologies have been developed to identify, quantify, and remove ambient RNA. Here, we rigorously evaluate 7 state-of-the-art methodologies for ambient RNA removal using simulated datasets, species-mixing experiments of varying complexities, and genotype-mixing experiments. We find that no single method performs the best across all datasets and metrics, but CellBender, DecontX and SoupX generally perform well.<\/jats:p>","DOI":"10.3389\/fbinf.2026.1844838","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T05:44:26Z","timestamp":1787118266000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Benchmarking computational decontamination of ambient RNA"],"prefix":"10.3389","volume":"6","author":[{"given":"Cecilie B\u00f8gh","family":"Cargnelli","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Southern Denmark","place":["Odense, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jakob Vennike","family":"Nielsen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Southern Denmark","place":["Odense, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jesper Grud Skat","family":"Madsen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Southern Denmark","place":["Odense, Denmark"]},{"name":"Center for Functional Genomics and Tissue Plasticity (ATLAS)","place":["Odense, Denmark"]},{"name":"The Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard","place":["Cambridge, MA, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"B1","volume-title":"GRCh38 and mm10 Reference Genome (2020-A)","year":"2020"},{"key":"B2","doi-asserted-by":"publisher","first-page":"821","DOI":"10.1002\/hep4.1854","article-title":"Single\u2010cell, single\u2010nucleus, and spatial RNA sequencing of the human liver identifies cholangiocyte and mesenchymal heterogeneity","volume":"6","author":"Andrews","year":"2022","journal-title":"Hepatol. 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