Cited by 1,644Single-cell and spatial transcriptomicsGenomics and Phylogenetic StudiesGene expression and cancer classification

The DOI matches its Crossref record; 32 of the 32 references it cites were found in a registry or an index; 15 of the 16 sources its claims cite were checked for support.

SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data

Open access
Young, Matthew D, Behjati, Sam·2020·GigaScience

Published version of a preprint

Abstract

Background: Droplet-based single-cell RNA sequence analyses assume that all acquired RNAs are endogenous to cells. However, any cell-free RNAs contained within the input solution are also captured by these assays. This sequencing of cell-free RNA constitutes a background contamination that confounds the biological interpretation of single-cell transcriptomic data. Results: We demonstrate that contamination from this "soup" of cell-free RNAs is ubiquitous, with experiment-specific variations in composition and magnitude. We present a method, SoupX, for quantifying the extent of the contamination and estimating "background-corrected" cell expression profiles that seamlessly integrate with existing downstream analysis tools. Applying this method to several datasets using multiple droplet sequencing technologies, we demonstrate that its application improves biological interpretation of otherwise misleading data, as well as improving quality control metrics. Conclusions: We present SoupX, a tool for removing ambient RNA contamination from droplet-based single-cell RNA sequencing experiments. This tool has broad applicability, and its application can improve the biological utility of existing and future datasets.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

SoupX is a method and R package that estimates and removes ambient cell-free RNA contamination from droplet-based scRNA-seq by using empty droplets to define a background profile and estimating a contamination fraction. Across species-mixing, PBMC, kidney tumour, and fetal liver datasets, correction improved marker specificity and biological interpretation.

Key findings

  • Ambient RNA contamination was ubiquitous: in 10X species-mixing data, ∼1% of transcripts were cross-species contamination, providing a lower bound, with true contamination later estimated at ∼2%; background profiles correlated with cell aggregates at Pearson 0.71–0.96 (median 0.86).
  • SoupX reduced cross-species contamination by a factor of ≥2 and usually an order of magnitude, while correct-species expression was effectively unchanged.
  • In PBMCs, automated contamination estimation gave 6% and an IG-gene-based estimate gave ∼5%; correction increased marker fold-change contrast and improved LYZ specificity for mononuclear phagocytes.
  • In kidney tumour data, correction removed haemoglobin expression from non-red-blood cells, identified collagen expression in T/MNP clusters as contamination, increased cross-batch entropy, and yielded 2 fewer clusters; in fetal liver, HBB was confirmed as erythroid-specific and GYPA expression in EI macrophages as genuine.

Methods

  • SoupX has three steps: estimate the ambient mRNA profile from empty droplets with <Nemp UMIs (best correlation at Nemp < 10), estimate or manually set the contamination fraction using genes/cells assumed to have no endogenous expression or an automated marker-based procedure, and correct counts via a multinomial likelihood, optionally redistributing cluster-level corrected counts.
  • Datasets included 10X and DropSeq species-mixing experiments, PBMC data, a kidney tumour dataset (7 tumours across 10 channels in the Results; the Introduction describes 12 kidney tumour biopsies), and 40 channels of human fetal liver data.
  • Analyses compared corrected and uncorrected data using Seurat tSNE representations, cluster membership, marker gene fold changes, marker sensitivity, and cross-batch entropy.

Limitations

  • The paper states that best results require a user-specified set of genes and cells where no endogenous expression can be assumed; when this is not possible, the automated method requires clustering and may not always have an appropriate gene set.
  • The paper notes that a constant contamination fraction within a channel is assumed in most experiments, and that manual contamination setting can be used if estimates are deemed inaccurate or overcorrection is desired.
  • Evident from the text: the method assumes empty-droplet profiles represent the background and that background composition is shared across cells; evaluation is limited to the datasets analysed, and the paper has no formal limitations section.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1093/gigascience/giaa151

Cites

32 of 32 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

15 of 16 cited sources checked · 10 supported · 0 flagged · accuracy not yet measured

Show all 63 claims

  • It may state two separate claims as one. 11
  • Probably not a substantial claim. 7
18 of 18
  • The paper relies on this without support.

    Ambient mRNA contamination is increased in necrotic or complex samples.

    “with increased contamination in necrotic or complex samples”
  • The authors state this as an assumption.

    Droplets with fewer than Nemp UMIs unambiguously do not contain cells.

    “To estimate the background expression profile we consider all droplets with <Nemp UMIs, which we assume unambiguously do not contain cells.”
  • The authors state this as an assumption.

    The relative abundance of genes that make up the background does not differ between cells.

    “We assume that the relative abundance of genes that make up the background does not differ between cells”
  • The authors state this as an assumption.

    There is a combination of genes and cells for which mg,c = 0 exists.

    “To proceed, we assume that there is a combination of genes and cells for which mg, c = 0 exists.”
  • The authors state this as an assumption.

    If it can be shown for any cell c in cluster P that mg,c > 0, then mg,c > 0 for all c ∈ P.

    “If it can be shown for any cell c in cluster P that mg, c > 0, then it is assumed that mg, c > 0 for all c ∈ P (see Supplementary Fig. S1).”
  • The authors state this as an assumption.

    For each strong marker, mg,c = 0 for all cells in clusters where the gene is not a marker.

    “For each strong marker, it is assumed that mg, c = 0 for all cells in clusters where the gene is not a marker and the contamination fraction is estimated (Supplementary Fig. S1).”
  • The authors state this as an assumption.

    Inaccurate estimates of the contamination fraction will have no preferred value while true estimates will cluster around the true value.

    “To obtain a final value, it is assumed that inaccurate estimates will have no preferred value while true estimates will cluster around the true value.”
  • The authors state this as an assumption.

    Droplets with ≤10 UMIs contain only ambient mRNAs.

    “which we assumed to contain only ambient mRNAs”
  • The authors state this as an assumption.

    For species-mixing genes/cells (human transcripts in mouse cells and vice versa), mg,c = 0.

    “For these genes/cells it is assumed that mg, c = 0 and the contamination fraction is calculated using Equation 4.”
  • The authors state this as an assumption.

    SoupX assumes a constant contamination fraction within a channel.

    “SoupX assumes a constant contamination fraction within a channel.”
  • The paper relies on this without support.

    In most experiments there is less power to determine cell-specific contamination fractions.

    “In most experiments there is less power to determine cell-specific contamination fractions and so SoupX assumes a constant contamination fraction within a channel.”
  • The authors state this as an assumption.

    Some genes can be assumed to be unexpressed in some cells (mg,c = 0).

    “To confirm the accuracy of this estimate, we also calculated the background contamination rate using a set of genes that could be assumed to be unexpressed in some cells (i.e., where mg, c = 0).”
  • The paper relies on this without support.

    Haemoglobin genes are highly specific to red blood cells.

    “together with the knowledge that haemoglobin genes are highly specific to red blood cells.”
  • The paper relies on this without support.

    The ambient mRNA expression profile is experiment specific.

    “Because the ambient mRNA expression profile is experiment specific, we reasoned that background contamination likely creates batch effects.”
  • The paper relies on this without support.

    Red blood cells are the only cells that produce haemoglobin under normal physiological conditions.

    “Furthermore, red blood cells are the only cells that produce haemoglobin under normal physiological conditions, so for the set of haemoglobin genes, it is safe to assume that there is no cell endogenous expression for cells that are not red blood cells.”
  • The authors state this as an assumption.

    For haemoglobin genes, there is no cell endogenous expression for cells that are not red blood cells.

    “Furthermore, red blood cells are the only cells that produce haemoglobin under normal physiological conditions, so for the set of haemoglobin genes, it is safe to assume that there is no cell endogenous expression for cells that are not red blood cells.”
  • The authors state this as an assumption.

    Inaccurate estimates of the contamination fraction are not strongly correlated (i.e., there is no preferred, incorrect estimate).

    “By using all high-quality marker genes identified in the data to independently estimate the contamination fraction, this method estimates the true contamination fraction by assuming that inaccurate estimates of the contamination fraction are not strongly correlated (i.e., there is no preferred, incorrect estimate).”
  • The authors state this as an assumption.

    For a chosen set of genes and cells, the only source of expression for these genes is background contamination.

    “The best case for applying SoupX occurs when the user can specify a set of genes and cells where there is no cell endogenous expression, i.e., a set of genes and cells where it is safe to assume that the only source of expression for these genes is from background contamination.”
18 of 18

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Citations

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Research works printed but not linked: 1

  1. Gayoso, 2017, DoubletDetection: Identifying technical error in single-cell RNA-sequencing data

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Metadata

Authors
Young, M. D., & Behjati, S.
Affiliations
  • Wellcome Sanger Institute (GB)
  • University of Cambridge (GB)
Corresponding author
Young, Matthew D
Venue
GigaScience
Published
2020-12
Volume
9
Issue
12
ISSN
2047-217X (ISSN-L)
OpenAlex
W3114078084
Cited by
1,619
References
30
Citations by year
2026: 358 · 2025: 388 · 2024: 347 · 2023: 211 · 2022: 149 · 2021: 86 · 2020: 54
FWCI
35.00
Type
journal article
Language
en
Open access
Locations
  • GigaScience (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
Funders
Wellcome Trust
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
Keywords
cell-free RNA, background correction, single-cell transcriptomics, quality control, contamination quantification
MeSH terms
Base Sequence, RNA, Sequence Analysis, RNA, Gene Expression Profiling, Single-Cell Analysis, Transcriptome

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