biostudies-literatureHeaton HBritish Heart FoundationMedical Research CouncilNational Institute for Health Research (NIHR)Wellcome Trust615-620https://www.ebi.ac.uk/biostudies/studies/S-EPMC7617080biostudies-literatureUnknown17(6)Methods to deconvolve single-cell RNA-sequencing (scRNA-seq) data are necessary for samples containing a mixture of genotypes, whether they are natural or experimentally combined. Multiplexing across donors is a popular experimental design that can avoid batch effects, reduce costs and improve doublet detection. By using variants detected in scRNA-seq reads, it is possible to assign cells to their donor of origin and identify cross-genotype doublets that may have highly similar transcriptional profiles, precluding detection by transcriptional profile. More subtle cross-genotype variant contamination can be used to estimate the amount of ambient RNA. Ambient RNA is caused by cell lysis before droplet partitioning and is an important confounder of scRNA-seq analysis. Here we develop souporcell, a method to cluster cells using the genetic variants detected within the scRNA-seq reads. We show that it achieves high accuracy on genotype clustering, doublet detection and ambient RNA estimation, as demonstrated across a range of challenging scenarios.Nature methodsSouporcell: robust clustering of single-cell RNA-seq data by genotype without reference genotypes.PMC7617080206194207492RG/18/13/33946HDR-9004WT098051G1100339MR/L003120/1WT098503RG/13/13/30194206194/Z/17/ZWT207492RG/13/13/30194; RG/18/13/33946098051Talman AMHeaton HImaz MGaffney DJLawniczak MKNKnights AHemberg MDurbin RfalseSouporcell: robust clustering of single-cell RNA-seq data by genotype without reference genotypes.Methods to deconvolve single-cell RNA-sequencing (scRNA-seq) data are necessary for samples containing a mixture of genotypes, whether they are natural or experimentally combined. Multiplexing across donors is a popular experimental design that can avoid batch effects, reduce costs and improve doublet detection. By using variants detected in scRNA-seq reads, it is possible to assign cells to their donor of origin and identify cross-genotype doublets that may have highly similar transcriptional profiles, precluding detection by transcriptional profile. More subtle cross-genotype variant contamination can be used to estimate the amount of ambient RNA. Ambient RNA is caused by cell lysis before droplet partitioning and is an important confounder of scRNA-seq analysis. Here we develop souporcell, a method to cluster cells using the genetic variants detected within the scRNA-seq reads. We show that it achieves high accuracy on genotype clustering, doublet detection and ambient RNA estimation, as demonstrated across a range of challenging scenarios.2020-01-01T00:00:00Z2020 Jun2026-06-02T22:02:36.449Z2025-04-06T22:40:48.731ZS-EPMC76170803236698910.1038/s41592-020-0820-1