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

Lukas M. Weber, Ariel A. Hippen, Peter F. Hickey, Kristofer C. Berrett, Jason Gertz, Jennifer Anne Doherty, Casey S. Greene, Stephanie C. Hicks

Research output: Contribution to journalArticlepeer-review


Background: 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, to our knowledge 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. Results: Here, we 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. Our 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. Conclusions: This strategy provides significant cost savings through pooled library preparation. To facilitate similar analyses at the experimental design phase, we provide freely accessible code and a reproducible Snakemake workflow built around the best-performing tools found in our in silico benchmark evaluations, available at

Original languageEnglish (US)
Article numbergiab062
Issue number9
StatePublished - Sep 1 2021


  • benchmarking
  • cancer
  • computational methods
  • genetic demultiplexing
  • high-grade serous ovarian cancer
  • lung adenocarcinoma
  • simulations
  • single-cell RNA sequencing
  • tumor mutational burden

ASJC Scopus subject areas

  • Health Informatics
  • Computer Science Applications


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