Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design

Pooling cells from multiple biological samples prior to library preparation within the same single-cell RNA sequencing experiment provides several advantages, including lower library preparation costs and reduced unwanted technological variation, such as batch effects. Computational demultiplexing tools based on natural genetic variation between individuals provide a simple approach to demultiplex samples, which does not require complex additional experimental procedures. However, these tools have not been evaluated in cancer, where somatic variants, which could differ between cells from the same sample, may obscure the signal in natural genetic variation.

A team led by researcher’s at the Johns Hopkins Bloomberg School of Public Health performed in silico benchmark evaluations by combining raw sequencing reads from multiple single-cell samples in high-grade serous ovarian cancer, which has a high copy number burden, and lung adenocarcinoma, which has a high tumor mutational burden. The results confirm that genetic demultiplexing tools can be effectively deployed on cancer tissue using a pooled experimental design, although high proportions of ambient RNA from cell debris reduce performance.

Schematic illustrating the steps in the Snakemake workflow


The workflow is designed to be modular, allowing users to substitute alternative tools. The Snakemake workflow runs a complete analysis for one dataset (HGSOC) and doublets simulation scenario (20% doublets). The main benchmark evaluations include a second dataset (lung adenocarcinoma) and additional doublet simulation scenarios (30% doublets, no doublets).

Availability – To facilitate similar analyses at the experimental design phase, the devlopers provide freely accessible code and a reproducible Snakemake workflow built around the best-performing tools found in our in silico benchmark evaluations, available at

Weber LM, Hippen AA, Hickey PF, Berrett KC, Gertz J, Doherty JA, Greene CS, Hicks SC. (2021) Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design. bioRXiv [Epub ahead of print]. [abstract]

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