B-T.34: Order Matters: Ambient RNA correction affects doublet detection in single-cell
Authors
George Howitt
PeterMac Callum Cancer Centre
Calandra Grima
PeterMac Callum Cancer Centre
Hamish King
WEHI
Jovana Maksimovic
PeterMac Callum Cancer Centre
Alicia Oshlack
PeterMac Callum Cancer Centre
Keywords
scRNA-seq, ambient RNA, doublets, workflow
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A common technical artifact in droplet-based single-cell RNA sequencing (scRNA-seq) is the capture of two cells in the same droplet, termed doublets. Doublets may present as hybrid cell types, and their removal is a critical step during quality control. Another form of contamination is ambient RNA, which originates from other cells in suspension. Like doublets, the presence of ambient RNA results in unexpected gene expression patterns. Despite similarities between ambient contamination and doublets, no formal investigation on how doublet detection is affected by ambient RNA correction has been conducted.
Here, we evaluate the impact of ambient RNA correction on the performance of doublet detection using a probe-based, multiplexed dataset. We apply three of the most widely-used methods for removing ambient RNA (decontX, soupX, cellbender), in conjunction with six doublet detection methods (scDblFinder, scds, bccds, cxds scrublet, DoubletDetection). We find that, with the exception of cellbender, more true doublets are detected when doublet detection is performed on data without ambient RNA correction. Particularly for decontX, ambient RNA correction leads to an increased proportion of doublets retained in the dataset. We also find that decontX removes proportionally more ambient RNA from doublets that are retained after decontX processing, suggesting that ambient RNA correction may partially mask doublet signal, leading to reduced doublet detection performance.
Our results show that doublet detection is impacted by the choice of the ambient RNA correction tool and we suggest that in many settings doublet detection should be performed prior to ambient RNA correction.
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